Sulfonyl-triazoles useful as covalent activators of the glycolytic enzyme PFKL for t cell activation

Abstract

A family of sulfonyl-azole compounds are described which include small molecule activators of phosphofructokinase-1, liver isoform (PFKL). The small molecule activators selectively form covalent adducts with PFKL, including particularly lysine 677 of the human PFKL. Also provided are methods for using the small molecule activators to activate PFKL, e.g., to provide increased glycolysis and / or T cell activation, to treat cancers and / or tumors, and / or to enhance fructose-1,6-bisphosphate (FBP) content in cells.

Description

[0001] DESCRIPTION

[0002] SULFONYL-TRIAZOLES USEFUL AS COVALENT ACTIVATORS OF THE

[0003] GLYCOLYTIC ENZYME PFKL FOR T CELL ACTIVATION

[0004] CROSS REFRENCE TO RELATED APPLICATION

[0005] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 665,894, filed June 28, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0006] REFERENCE TO SEQUENCE LISTING XML SUBMITTED ELECTRONICALLY

[0007] The content of the Sequence Listing XML filed using Patent Center as an XML file (Name: 3436_4_PCT_ST26.xml; Size: 71,718 bytes; and Date of Creation: June 29, 2025) is incorporated herein by reference in its entirety.

[0008] GOVERNMENT INTEREST

[0009] This invention was made with government support under Grant no. R01 CA272490, R35 GM152218, R01 GM144472, R01 AI169412, and DA043571 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0010] TECHNICAL FIELD

[0011] The presently disclosed subject matter relates to sulfonyl-triazole compounds and their use as activators for the liver isoform of phosphofructokinase- 1 (PFKL). The presently disclosed subject matter further relates to the use of the sulfonyl-triazole PFKL activators in increasing glycolytic flux and reactivating tumor infdtrating T cells, e.g., for use as cancer immunotherapy agents and agents for treating other diseases.

[0012] BACKGROUND

[0013] Cell metabolism is essential for life by supplying fuel, signaling molecules, and building blocks required for growth, survival, and adaptation to environmental fluctuations (DeBerardinis & Thompson, 2012). Dysregulation of metabolic pathways has emerged as a hallmark of various diseases, including cancer (Pavlova & Thompson, 2016), diabetes (Taylor, 2012), neurodegenerative disorders, (Lin & Beal, 2006) and metabolic syndromes (Saltiel & Kahn, 2001). Notably, many cancer cells adopt a metabolic phenotype with increased dependence on aerobic glycolysis despite availability of oxygen. This metabolic reprogramming, also known as the Warburg effect (Warburg, 1956), drives increased glucose uptake and lactate production, supporting rapid cell proliferation and adaptation to nutrient- deprived or hypoxic environments (Vander Heiden et al., 2009).

[0014] While glycolytic rewiring facilitates tumor growth and survival, it concurrently creates vulnerabilities that can be exploited therapeutically (Vander Heiden, 2011). Beyond catabolism, altered glucose metabolism generates key metabolic intermediates that serve as signaling molecules, orchestrating diverse cellular processes including gene expression, epigenetic modifications, and redox homeostasis (Gomes & Blenis, 2015; Ho et al., 2015; Hicks et al., 2023; Wu et al., 2023). Excessive accumulation of glycolytic intermediates in cancer cells has been reported to induce imbalances in bioenergetics and impair tumor growth (Li et al., 2023; Snaebjomsson et al., 2025). Alterations in tumor microenvironment (e.g. pH) or intracellular enzymes (e.g., GAPDH) differentially regulated during the Warburg effect have been targeted as anticancer strategies (Gerwec & Seetharaman, 1996; Liberti et al., 2017). Nevertheless, inhibiting the upregulation of glycolysis in cancer cells remains a challenge due to their inherent metabolic plasticity, including resistance through utilization of alternative carbon sources such as amino acids to support growth (Stine et al., 2022).

[0015] Glycolytic flux is regulated principally by phosphofructokinase- 1 (PFK1) activity. PFK1 catalyzes the ATP-dependent phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6- bisphosphate (FBP; see Webb et al., 2015). PFK1 is the rate-limiting enzyme in glycolysis and tightly regulated by: (i) cellular energy status, where a low ATP / ADP ratio activates PFK1 and a high ratio is inhibitory; (ii) post-translational modifications (PTMs) such as phosphorylation (Lee et al., 2018), acetylation (Zhao et al., 2010), and glycosylation (Yi et al., 2012); and (iii) allosteric modulators that stabilize its tetrameric conformational states (Lynch et al., 2024). In mammals, PFK1 is expressed as three isoforms (PFKL, PFKM, PFKP) with high sequence homology but distinct allosteric regulation (Fernandes et al., 2020) and moonlighting functions (Meng et al., 2024; Guo et al., 2025). Genetic mutations resulting in increased or decreased activity of individual PFK1 isoforms has been implicated in cancer cells (Tauri et al., 1965; Webb et al., 2015). Collectively, PFK1 activity could be exploited for anticancer applications but requires selective targeting modalities that can address the metabolic plasticity of cancer cells.

[0016] Immunotherapy through blockade of inhibitory receptors on T cells has shown great efficacy in the clinic, but only benefits a small population of cancer patients. When effective, these immune checkpoint inhibitors can produce durable responses in tumors with preexisting tumor-infiltrating T cells (i.e., TIL) that are immunosuppressed (Pardoll, 2012; Herbst et al., 2014; Tumeh et al., 2014). The discovery of additional targets to bypass immunosuppression in tumors and broaden the efficacy of TILs has the potential to transform cancer therapy.

[0017] Accordingly, there is an ongoing need in the art for additional targets and agents for activating T cells and for use in cancer immunotherapy.

[0018] SUMMARY

[0019] This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

[0020] In some embodiments, the presently disclosed subject matter provides a compound having a structure of Formula (I): wherein: X, Y, and Z are independently CH or N, subject to the proviso that at least one of X, Y, and Z is N; Ri is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; Li is -(CH2)n- or -C(=O)-(CH2)n-i-, wherein n is an integer selected from 3, 4, and 5; and R’ is selected from H, wherein: — is a double or single bond; A is selected from methylene, NH, O, and S; Ai is selected from CH and N; A2is selected from C, CH, and N, subject to the proviso that when — is a single bond, A2is N or CH, and when — is a double bond, A2is C; R2and R, are each selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and R4, R5, Re, and R7 are each independently selected from H and alkyl; or a pharmaceutically acceptable salt thereof. In some embodiments, two of X, Y, and Z are N, optionally wherein Z and Y are each N and X is CH.

[0021] In some embodiments, Ri is selected from lower alkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, wherein substituted aryl and substituted heteroaryl are aryl or heteroaryl groups substituted with one or more substituent selected from the group consisting of halo, cyano, alkyl, alkoxy, haloalkyl, haloalkoxy, perhaloalkyl, perhaloalkoxy, cycloalkyl, aralkyl, aryl, amino, and amido, or wherein two substituents together form a divalent group, optionally alkylene, halo-substituted alkylene, alkylenedioxy, or halo-substituted alkylenedioxy group. In some embodiments, Ri is selected from substituted phenyl or substituted pyridinyl, optionally wherein said substituted phenyl or substituted pyridinyl is phenyl or pyridinyl substituted with at least one alkoxy or haloalkoxy group or wherein the substituted phenyl is a phenyl group substituted with two substituents that together form a divalent group, optionally an alkylenedioxy group or halo-substituted alkylenedioxy group, further optionally - O(CH2)O-, -O(CF2)O-, or -O(CH2)2O-.

[0022] In some embodiments, n is 4 and Li is butylene.

[0023] In some embodiments, R’ is H or optionally wherein A is methylene, further optionally wherein R4, Rs, Re, and R7 are each H.

[0024] In some embodiments, the compound of Formula (I) has a structure of Formula (II) or Formula (III): wherein: — is a double or single bond; X, Y, Z, Li, Ai, A2, and R1-R7 are as defined for Formula (I); or a pharmaceutically acceptable salt thereof. In some embodiments, R2 and / or R3 is substituted aryl or substituted heteroaryl, wherein said substituted aryl or substituted heteroaryl is aryl or heteroaryl substituted with one or more substituents selected from halo, nitro, cyano, carboxyl, ester, formyl, alkyl, halo-substituted alkyl, alkoxy, haloalkoxy, aryl, and heteroaryl. In some embodiments, R2 and / or R3 is halo-substituted phenyl, optionally fluoro-substituted phenyl, further optionally mono- or di-fluoro- substituted phenyl. In some embodiments, R4, Rs, IC, and R7 are each independently H or methyl, optionally wherein two, three, or four of R4, Rs, IC, and R7 are H.

[0025] In some embodiments, the compound of Formula (I) has a structure of Formula (IV): wherein: X, Y, and Z are independently CH or N, subject to the proviso that at least one of X, Y, and Z is N, optionally wherein Y and Z are N and X is CH; Ri is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, optionally substituted aryl or substituted heteroaryl; Li is - (CH2)n-, wherein n is an integer selected from 3, 4, and 5, optionally 4; R2 and R3 are each selected from aryl, substituted aryl, heteroaryl, and substituted heteroaryl, optionally fluoro-substituted phenyl; and R4, Rs, FC. and R7 are each independently selected from the group consisting of H and alkyl, optionally H or methyl; or a pharmaceutically acceptable salt thereof.

[0026] In some embodiments, the compound is selected from the group consisting of:

[0027]

[0028] XJ-4-5 XJ-4-7 or a pharmaceutically acceptable salt thereof.

[0029] In some embodiments, the presently disclosed subject matter provides a pharmaceutical composition comprising a compound of Formula (I), (II), (III), or (IV), or a pharmaceutically acceptable salt thereof.

[0030] In some embodiments, the presently disclosed subject matter provides a method of selectively activating the liver isoform of phosphofructokinase (PFKL), wherein the method comprises contacting a sample comprising PFKL with a compound of Formula (I), (II), (III), or (IV), or a pharmaceutical composition thereof. In some embodiments, the sample comprising PFKL is selected from a cell extract, a biological fluid, a cell, a tissue, an organ, or an organism. In some embodiments, the sample comprising PFKL is a mammal, optionally a human, and activating PFKL in the sample provides increased glycolysis and / or T cell activation, optionally tumor-infiltrating T cell (TIL) reactivation.

[0031] In some embodiments, the presently disclosed subject matter provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of Formula (I), (II), (III), or (IV), or a pharmaceutical composition thereof. In some embodiments, the compound is selected from the group consisting of XJ-3-41, XJ-4-27, XJ-3- 129, XJ-3-71, XJ-4-65, XJ-4-85, XJ-4-99, XJ-4-89, XJ-3-9, XJ-3-65, and pharmaceutically acceptable salts thereof. In some embodiments, the compound is XJ-4-85.

[0032] In some embodiments, the cancer is selected from breast cancer, liver cancer and colorectal cancer.

[0033] In some embodiments, the presently disclosed subject matter also provides methods for enhancing fructose- 1,6-bisphosphate (FBP) content in cells. In some embodiments, the methods comprise contacting a cell with an effective amount of a compound as disclosed herein or a pharmaceutical composition comprising the same. In some embodiments, the compound is selected from the group consisting of XJ-3-41, XJ-4-27, XJ-3-129, XJ-3-71, XJ-4-65, XJ-4-85, XJ-4-89, XJ- 4-97, XJ-3-9, XJ-3-65, and pharmaceutically acceptable salts thereof. In some embodiments, the compound is XJ-4-85. In some embodiments, the cell is a tumor cell and / or a cancer cell, or an endothelial cell associated with a tumor and / or a cancer. In some embodiments, the cell is from a solid tumor and / or cancer selected from the group consisting of breast tumor and / or cancer, a liver tumor and / or cancer, and / or a colorectal tumor and / or cancer. In some embodiments, the tumor cell and / or the cancer cell is a leukemia cell, optionally monocytic leukemia cell, a T-cell leukemia cell, and / or an acute myeloid leukemia cell; a hepatocellular carcinoma cell, a lung adenocarcinoma cell, a melanoma cell, a triple-negative breast cancer cell, and / or a neuroblastoma cell.

[0034] In some embodiments, the presently disclosed subject matter also provides uses of the compounds disclosed herein and / or pharmaceutical compositions comprising the same for treating a cancer and / or a tumor in a subject in need thereof, for inhibiting growth of a cell associated therewith, or for enhancing fructose- 1,6-bisphosphate (FBP) content in a cell of cancer and / or the tumor. In some embodiments, the compound is selected from the group consisting of XJ-3-41, XJ-4-27, XJ-3-129, XJ- 3-71, XJ-4-65, XJ-4-85, XJ-4-89, XJ-4-97, XJ-3-9, XJ-3-65, and pharmaceutically acceptable salts thereof. In some embodiments, the compound is XJ-4-85. In some embodiments, the cancer and / or the tumor is selected from a breast cancer and / or tumor, optionally a triple-negative breast cancer and / or tumor; a liver cancer and / or tumor, a colorectal cancer and / or tumor, a leukemia, optionally a monocytic leukemia, a T-cell leukemia, and / or an acute myeloid leukemia; a hepatocellular carcinoma, a lung adenocarcinoma, a melanoma, and / or a neuroblastoma.

[0035] The presently disclosed subject matter also provides in some embodiments compounds for use in treating cancers and / or tumors in subjects in need thereof, for inhibiting growth of cells associated therewith, and / or for enhancing fructose- 1,6-bisphosphate (FBP) content in cells of cancers and / or tumors. In some embodiments, the compound is selected from the group consisting of XJ-3-41, XJ-4- 27, XJ-3-129, XJ-3-71, XJ-4-65, XJ-4-85, XJ-4-89, XJ-4-97, XJ-3-9, XJ-3-65, and pharmaceutically acceptable salts thereof. In some embodiments, the compound is XJ-4-85. In some embodiments, the cancer and / or the tumor is selected from a breast cancer and / or tumor, optionally a triple-negative breast cancer and / or tumor; a liver cancer and / or tumor, a colorectal cancer and / or tumor, a leukemia, optionally a monocytic leukemia, a T-cell leukemia, and / or an acute myeloid leukemia; a hepatocellular carcinoma, a lung adenocarcinoma, a melanoma, and / or a neuroblastoma.

[0036] Accordingly, it is an object of the presently disclosed subject matter to provide compounds of Formula (I), as well as to provide related pharmaceutical compositions and methods of activating PFKL and treating cancer. This and other objects are achieved in whole or in part by the presently disclosed subject matter. Further, an object of the presently disclosed subject matter having been stated above, other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, Figures, and EXAMPLES.

[0037] BRIEF DESCRIPTIONS OF THE FIGURES

[0038] Figure 1 is a schematic diagram showing the role of phosphofructokinase- 1, liver isoform (PFKL) in glycolysis and the use of small molecule PFKL activators in cancer immunotherapy, e.g., in reactivating tumor-infiltrating T cells (TIL) to improve TIL function.

[0039] Figure 2A is a schematic diagram showing the structure of NA- 11 , a previously described small molecule activator of phosphofructokinase- 1, liver isoform (PFKL). Figure 2B is a schematic diagram showing the structure of TH211, a previously described sulfonyl-triazole probe used for identifying covalent binding sites in proteins, including kinases, via sulfonyl -triazole exchange (SuTEx) chemistry. The RF001 recognition moiety that can provide target specificity is enclosed in the dotted square. TH211 also includes a terminal alkyne group that can be modified with a detectable group, e.g., desthiobiotin, via Click chemistry after TH211 reacts with a nucleophilic group in a protein or peptide to form a covalent adduct.

[0040] Figure 2C is a schematic diagram showing the structure of TH220, a previously described sulfonyl-triazole exchange (SuTEx) compound that binds to the phosphofructokinase- 1, liver isoform (PFKL) in cells.

[0041] Figure 3A is a schematic diagram showing the structures of a series of exemplary sulfonyl- triazole compounds of the presently disclosed subject matter that activate phosphofructokinase- 1, liver isoform (PFKL).

[0042] Figure 3B is a graph showing the results of a bioluminescence activation assay for phosphofructokinase- 1, liver isoform (PFKL) treated with the compounds shown in Figure 3 A. The half-maximal effective concentration (EC50) for each of the compounds is shown in the upper left: 4.9 micromolar (pM) for XJ-3-9 (circles); 2.8 pM for XJ-3-41 (triangles), and 3.0 pM for XJ-3-65 (diamonds). Error bars are ± standard deviation (SD).

[0043] Figure 4A are schematic diagrams showing chemical structures of additional exemplary sulfonyl triazole compounds of the presently disclosed subject matter.

[0044] Figure 4B is a graph showing the results of a bioluminescence assay for the activation of phosphofructokinase- 1, liver isoform (PFKL) using different small molecules, including exemplary sulfonyl-triazole compounds shown in Figure 4B. Compounds were tested at two different concentrations (10 micromolar (pM) and 2 pM) as indicated on the x-axis. For comparison, results with medium, dimethyl sulfoxide (DMSO), and 1 pM NA- 11 are also shown. Error bars are ± standard deviation (SD).

[0045] Figure 5 is a schematic diagram showing the formation of a covalent adduct via reaction of an exemplary sulfonyl-triazole compound of the presently disclosed subject matter (XH-3-71) and a lysine (K) residue in histidine-tagged phosphofructokinase- 1, liver isoform (His-PFKL). Mass spectroscopy studies of the covalent adduct indicated that the site of adduct formation is lysine 677 (see Accession No. NP_002617.3 of the GENBANK® biosequence database (SEQ ID NO: 5) for amino acid position numbering employed throughout this disclosure).

[0046] Figure 6 is a schematic diagram of the chemical structure of an exemplary sulfonyl-triazole compound of the presently disclosed subject matter referred to as XJ-4-65 (left) and a graph showing the concentration dependent activation of wild-type (WT) phosphofructokinase- 1, liver isotype (PFKL) with XJ-4-65 (right). The half-maximal effective concentration (EC50) of XJ-4-65 for wild-type PFKL (PFKL WT) was 712.4 nM. Error bars are ± standard deviation (SD). Figure 7 a graph showing the concentration dependent activation of wild-type (WT, circles) phosphofructokinase- 1, liver isotype (PFKL) and of a mutant PFKL wherein the asparagine at residue 702 is replaced with threonine (N702T, triangles) with XJ-4-65. The asparagine at residue 702 in the PFKL regulatory domain is involved in mediating filament assembly and is close to the lysine at residue 677. Error bars are ± standard deviation (SD).

[0047] Figure 8A is a schematic diagram showing the ribbon structure of an exemplary phosphofructokinase- 1, liver isoform (PFKL) indicating the catalytic domain (lower left, brown), the regulatory domain (upper right, light blue), and the positions of the lysines at residues 677 (K677), 715 (K715), and 315 (K315) within the PFKL protein.

[0048] Figure 8B is a bar graph showing the concentration dependence of activation of the wild-type phosphofructokinase- 1, liver isoform (PFKL), i.e., WT (bars with black outline) and a mutant PFKL where the lysine at residue 315 is replaced with an arginine (K315R, bars outlined in purple) using an exemplary sulfonyl -triazole compound of the presently disclosed subject matter referred to as XJ-4-65. Error bars are ± standard deviation (SD).

[0049] Figure 9A is a schematic drawing showing the chemical structure of an exemplary sulfonyl triazole compound of the presently disclosed subject matter referred to herein as XJ-4-85.

[0050] Figure 9B is a portion of a gel showing the concentration dependent phosphofructokinase- 1, liver isoform (PFKL) binding competition of XJ-4-85 with the PFKL-binding probe compound TH211.

[0051] Figure 9C is a graph showing the concentration dependence of activation of the phosphofructokinase- 1, liver isoform (PFKL) protein with XJ-4-85. The half maximal effective concentration of XJ-4-85 was 746.3 nM. Error bars are ± standard deviation (SD).

[0052] Figure 9D is a graph showing the concentration-dependent decrease in probe labeling of the phosphofructokinase- 1, liver isoform (PFKL) protein based on the concentration of XJ-4-85 used to pre-treat PFKL prior to labeling. Error bars are ± standard deviation (SD).

[0053] Figure 10A is a schematic drawing showing the overlapped ribbon structures of three different isoforms of phosphofructokinase- 1 (PFK1): liver isoform (PFKL, blue), muscle isoform (PFKM, pink), and platelet isoform (PFKP, yellow).

[0054] Figure 1 OB is a schematic drawing showing the covalent reaction of a lysine side chain group in a phosphofructokinase- 1, liver isoform (PFKL) protein with exemplary sulfonyl -triazole compound XJ-4-85. The structure of a PFKL-inactive negative control compound, XJ-4-97, is also shown.

[0055] Figure 10C is a bar graph showing the concentration dependence of activation of the wild-type phosphofructokinase- 1, liver isoform (PFKL) protein with XJ-4-85. For comparison, results with medium, dimethyl sulfoxide (as negative controls,) and 1 micromolar NA- 11 (as a positive control) are also shown. All data are from studies using 0.1 millimolar (mM) ATP. Error bars are ± standard deviation (SD). Figure 10D is a bar graph showing the results of an activation assay for the wild-type phosphofructokinase- 1, muscle isoform (PFKM) protein with XJ-4-85. For comparison, results with medium, dimethyl sulfoxide (DMSO), and 1 micromolar NA- 11 (as a positive control) are also shown. All data with XJ-4-85 and NA-11 were from studies using 0.1 millimolar (mM) ATP. Results using DMSO and 1.0 mM ATP are also shown. Error bars are ± standard deviation (SD).

[0056] Figure 10E is a bar graph showing the results of an activation assay for the wild-type phosphofructokinase- 1, platelet isoform (PFKP) protein with XJ-4-85. For comparison, results with medium, dimethyl sulfoxide (DMSO), and 1 micromolar NA- 11 (as a positive control) are also shown. All data with XJ-4-85 and NA-11 were from studies using 0.5 millimolar (mM) ATP. Results using DMSO and 0. 1 mM ATP are also shown. Error bars are ± standard deviation (SD).

[0057] Figure 11A is a graph showing the proteome-wide selectivity of exemplary sulfonyl-triazole compound XJ-4-85 in compound treated HEK293T cells.

[0058] Figure 1 IB is a graph showing the proteome-wide selectivity of exemplary sulfonyl-triazole compound XJ-4-85 in compound treated THP-1 macrophages.

[0059] Figure 11C is a graph showing the proteome-wide selectivity of exemplary sulfonyl-triazole compound XJ-4-85 in compound treated Jurkat T cells.

[0060] Figure 12A is a series of graphs showing verification of the activation of phosphofructokinase- 1 metabolism with exemplary sulfonyl-triazole XJ-4-85, but not XJ-4-97 using a metabolic sensor. The graphs and schematic on the left show the design of a fluorescent sensor assay for verification of phosphofructokinase- 1 (PFK1) metabolism using a fructose- 1,6-bisphosphate (FBP) biosensor. The graph on the right shows the results of the assay with XJ-4-85 and XJ-4-97.

[0061] Figure 12B is a graph showing increased glycolytic flux in THP-1 cells treated with an exemplary sulfonyl-triazole (XJ-4-85; gray circles) as measured by Seahorse assay. Results are also shown for a second compound, XJ-4-97 (black circles). Error bars are ± standard deviation (SD).

[0062] Figure 12C is a pair of graphs and a composite gel image showing that exemplary sulfonyl triazole compound XJ-4-85 increased immune cell signaling. The graphs on the top row show that XJ- 4-85, but not XJ-4-97, potentiates TNF-a (left) and IL- ip (right) signaling in LPS-stimulated THP-1 macrophages. The gel image at the bottom shows that XJ-4-85 treatment potentiated CD3 / CD28- stimulated TCR signaling in Jurkat cells as determined by phospho-ERK western blot analysis. The reported NA-11 reversible PFKL activator did not show comparable activity as XJ-4-85. Error bars are ± standard deviation (SD).

[0063] Figure 13 is a graph showing the concentration dependent effect of treatment with exemplary sulfonyl triazole phosphofructokinase- 1, liver isoform (PFKL) activator XJ-4-85 on cell viability of human liver cancer (HepG2) cells (circles), breast cancer (MCF-7) cells (squares), and colorectal cancer (Colo-205) cells (triangles). Error bars are ± standard deviation (SD). Figure 14 is a bar graph showing the effect of treatment with an exemplary sulfonyl -triazole phosphofructokinase- 1, liver isoform (PFKL) activator, XJ-4-65, on formation of reactive oxygen species (ROS). Error bars are ± standard deviation (SD).

[0064] Figure 15A is a series of schematic drawings showing the chemical structures of select exemplary Ri groups for the compounds of Formula (I).

[0065] Figure 15B is a series of schematic drawings showing the structures of select exemplary alternative R’ groups for the compounds of Formula (I).

[0066] Figure 16 is a graph showing further results of a bioluminescence assay for the activation of phosphofructokinase- 1, liver isoform (PFKL) using different small molecules, including exemplary sulfonyl-triazole compounds, including previously described sulfonyl-triazoles TH220 and TH207, and sulfonyl-triazoles of the presently disclosed subject matter, XJ-3-23, XJ-3-9, and XJ-3-17. The chemical structures of some of the compounds tested are also shown. For comparison, results with a known PFKL activator, NA-11, are also shown.

[0067] Figure 17 is a series of schematic drawings showing the chemical structures of exemplary compounds of the presently disclosed subject matter.

[0068] Figure 18 is a graph showing the results of a bioluminescence assay for the activation of phosphofructokinase- 1, liver isoform (PFKL) using different exemplary compounds of the presently disclosed subject matter. For comparison, results with a known PFKL activator, NA-11, are also shown. Error bars are ± standard deviation (SD).

[0069] Figure 19 is a graph showing the results of a bioluminescence assay for the activation of phosphofructokinase- 1, liver isoform (PFKL) using exemplary compounds of the presently disclosed subject matter. For comparison, results with a known PFKL activator, NA-11, are also shown. Error bars are ± standard deviation (SD).

[0070] Figure 20 is a graph showing the results of a bioluminescence assay for the activation of phosphofructokinase- 1, liver isoform (PFKL) using different small molecules, including exemplary compounds of the presently disclosed subject matter, e.g., XJ-4-17. For comparison, results with a known PFKL activator, NA-11, are also shown. Error bars are ± standard deviation (SD).

[0071] Figure 21 is a graph showing the results of a bioluminescence assay for the activation of phosphofructokinase- 1, liver isoform (PFKL) using exemplary compounds of the presently disclosed subject matter. For comparison, results with a known PFKL activator, NA-11, are also shown. Error bars are ± standard deviation (SD).

[0072] Figure 22 is a graph showing the results of a bioluminescence assay for the activation of phosphofructokinase- 1, liver isoform (PFKL) using exemplary compounds of the presently disclosed subject matter. For comparison, results with a known PFKL activator, NA-11, are also shown. Error bars are ± standard deviation (SD). Figure 23 is a graph showing several exemplary sulfonyl triazole compounds of the presently disclosed subject matter, including XJ-3-129 and XJ-4-65 XJ-4-85 increase immune cell signaling, as indicated by potentiation of TNF-a.

[0073] Figure 24 is a schematic drawing showing an exemplary workflow for in situ gel-based activitybased protein profding (ABPP) analysis.

[0074] Figure 25 is a schematic drawing showing an exemplary workflow for competitive activitybased protein profding (ABPP) stable isotope labeling by amino acids in cell culture (SILAC) studies to detect liganded sites with sulfonyl-triazole exchange (SuTEx) compounds.

[0075] Figure 26 is a schematic drawing showing an exemplary workflow for tandem mass tag (TMT)- based quantitative chemical proteomics with the presently disclosed sulfonyl-triazole compounds.

[0076] Figures 27A-27I. Discovery of a site-specific, proteome wide-selective covalent PFKL activator. (Figure 27A) Chemical structures of XJ-4-85, XJ-4-97 (negative control) and XJ-4-119 (Leaving group). (Figure 27B) Dose- and time-dependent biochemical activation of PFKL (WT) by XJ- 4-85 compared to XJ-4-97 and XJ-4-119 using a substrate assay. (Figure 27C) Gel-based competitive ABPP evaluation of XJ-4-85 binding activity in HEK293T cells. Representative data from n = 4 replicates are shown. (Figures 27D-27I) Competitive TMT-ABPP analysis of proteomes from XJ-4- 85-treated cells (5 pM, 1 h) to demonstrate PFKL site specificity (K677) and proteome-wide selectivity. Data shown are representative of n = 3 biological replicates.

[0077] Figures 28A-28H. Cryo-EM structure of XJ-4-85 bound to PFKL. (Figure 28A) Cryo-EM structure of a PFKL tetramer bound to XJ-4-85, colored by monomer. Monomer-a is colored by domain. (Figure 28B) Cryo-EM structure of a PFKL monomer bound to XJ-4-85 (PDB: 9P0J). Inset shows an expanded view of the XJ-4-85 adduct and leaving group binding sites. (Figure 28C) Overlay of XJ-4- 85 with ADP (PDB: 8W2G) and NA-11 (PDB: 7LW1) in the allosteric activating site. (Figure 28D and 28E) Catalytic domain dimer from the XJ-4-85-bound PFKL structure compared with PFKL in the R- state (PDB: 8W2G, PDB: 7LW1) and T-state (PDB: 8W2H). Structures are aligned on subunit-a. F6P and ADP are shown in the active sites of the XJ-4-85-bound structure. PFKL bound to XJ-4-85 is in the R-state conformation, which involves a 7o rotation between catalytic domains relative to the T-state conformation. (Figure 28F) Monomers of XJ-4-85-bound and T-state PFKL aligned on their regulatory domains. Transition to the T-state involves a 7o rotation between the catalytic and regulatory domains, which is sterically inhibited by the XJ-4-85 leaving group. Orange circles in the expanded view highlight residues that would clash with the XJ-4-85 leaving group in the T-state conformation. (Figure 28G) Washout experiment demonstrated the irreversible binding of XJ-4-85. (Figure 28H) PFK1 isoform specificity of XJ-4-85 using a substrate assay.

[0078] Figures 29A-29H. XJ-4-85 treatment induces rapid alterations in glycolytic metabolism and signaling. (Figure 29A) Schematic of glycolytic pathway. Image was created with BioRender. Quantification of the HYlight sensor ratio for THP-1 (Figure 29B), Jurkat (Figure 29C) and MOLM-14 (Figure 29D) cells treated with DMSO, XJ-4-85 or XJ-4-97. (Figure 29E) Left - a representative image in SH-SY5Y illustrates the sensor's response to fructose- 1,6-bisphosphate (FBP) detection following 2 hours of treatment with either DMSO vehicle or XJ-4-85. Right - the fluorescence ratios (488 / 405 nm) were statistically analyzed across the same fields. Data are presented as mean ± SEM, n = 24. (Figure 29F) Upregulation of glycolysis in THP-1 cells by Seahorse assay. (Figure 29G) Volcano plot showing metabolomic changes in Jurkat cells treated with XJ-4-85 compared to DMSO vehicle (2 h). Down- or up-regulated metabolites are shown in blue (log2(fold change) < -1) or red (log2(fold change) > 1), respectively. Significance was calculated using a two-tailed Student’s t-test (n = 4 biologically independent replicates). (Figure 29H) Enhanced expression of proinflammatory cytokine TNF-a and IL- 1 P in THP-1 MO cells pretreated with DMSO vehicle, NA-11 or XJ-4-85 (5 pM, 2 h) followed by 4 h stimulation with LPS (n = 3 biologically independent samples).

[0079] Figures 30A-30G. XJ-4-85 blocks tumor growth in vivo. (Figure 30A) Dose-response curves for a panel of cancer cells treated with increasing concentrations of XJ-4-85 (48 h). The noncancerous HEK293T cell line was included for comparison. Cell viability was performed as described in the Materials and Methods for the EXAMPLES section below. Data points are normalized relative to vehicle-treated controls for each respective treatment and shown as mean ± SD (n = 4 replicates). (Figure 30B) Basal FBP levels in B16-F10-Luc2, MOLM-14, THP-1, SH-SY5Y cells. (Figure 30C) Cell viability after 48 h in B10-F10-Luc2 cells pretreated with 10 pM of XJ-4-85 or XJ-4-119 for 2 h (n = 10 biological replicates). (Figure 30D) Schematic of electrophile -drug conjugate (EDC) concept. (Figure 30E) Treatment paradigm for evaluating compound treatments on B16-F10-Luc2 tumor outgrowth. Scheme was created with BioRender (BioRender, Toronto, Canada). (Figure 30F) C57BL / 6 mice bearing B16-F10-Luc2 tumors were treated daily with XJ-4-85, XJ-4-97, XJ-4-119 or vehicle (intraperitoneal injections) at the indicated dose. Tumor measurements were performed daily after tumors became palpable (indicated numbers of mice per group are shown). Data are shown as mean ± SEM. (Figure 30G) Representative in vivo bioluminescence imaging at day 8 and day 14 in B16-F10- Luc2 -implanted male mice treated with 50 mg / kg of XJ-4-85 or vehicle.

[0080] Figures 31A-31D. XJ-4-85 destabilizes cancer cell glycolysis and signaling. (Figure 31A) KEGG pathway analysis of untargeted LC-MS / MS metabolomics profiling showing metabolic pathways significantly enriched following the treatment of XJ-4-85 (5 pM, 2h) in B16-F10-Luc2 cells. Size of circles denotes the number of metabolites in each respective category. Coloring of circles shows the degree of significance in changes from XJ-4-85 treatments (enrichment P values). (Figure 3 IB) Alterations in glycolysis-related metabolites following XJ-4-85 treatment in B16-F10-Luc2 cells. Data are shown as mean ± SEM, n = 3 biological replicates. (Figure 31C) Waterfall plot showing protein abundance changes in B16-Fl-Luc2 cells treated with XJ-4-85 (5 pM, 2 h) relative to DMSO vehicle, n = 3 biologically independent replicates. (Figure 3 ID) Volcano plot showing phosphorylated sites that were significantly up- or down-regulated in B16-F10-Luc2 cells treated with XJ-4-85 (5 pM, 2 h) relative to DMSO vehicle, n = 3 biologically independent replicates. Phosphorylation sites with a P value of < 0.05 and |Log2(fold change)| > 0.5 relative to DMSO vehicle treatment are highlighted in color.

[0081] Figure 32. The chemical structures and designated names of SuTEx ligands screened for PFKL activation.

[0082] Figures 33A-33E. Identification of sulfonyl-triazole ligands that covalently bind a conserved lysine on PFKL in live cells. (Figure 33A) Gel-based competitive ABPP screening in HEK293T cells using TH211 probe. HEK293T cells were treated with DMSO vehicle or SuTEx ligands (1 pM) for 1 h followed by treatment with TH211 probe (25 pM, 2 hours). (Figure 33B) Competitive TMT-ABPP analysis. The normalized TMT reporter ion intensities are shown in bar plot (top), n = 4 biological replicates. K677 is highlighted (blue) in the protein structure of PFKL (bottom). (Figure 33C) The chemical structure of TH220 (top) and its dose-dependent binding activity in cells as determined by gel-based competitive ABPP analysis (bottom). HEK293T cells were treated with DMSO vehicle or varying concentrations of TH220 for 1 hour followed by treatment with TH211 probe (25 pM, 2 hours). (Figure 33D) The integrated band intensities from gel-based studies were quantified in Image Lab. Data shown are mean ± SD, n = 4 biological replicates. (Figure 33E) Selectivity of TH220 in HEK293T cells as determined by competitive TMT-ABPP analysis.

[0083] Figure 34. Exemplary workflow for competitive TMT-ABPP analysis. Chemical structure of the SuTEx probe TH211 used for chemical proteomics is shown.

[0084] Figure 35. MS2 annotation of the PFKL (K677) TH211 -modified tryptic peptide. Left panel: Probe-modified peptide sequence and MS2 fragment ion annotation of NYGTK*LGVK (SEQ ID NO: 14; residues 673-681 of SEQ ID NO: 5) peptide from human PFKL. Covalent reaction of TH211 with K677 results in a modified lysine (K*) with the addition of +635.2737 Da. Fragmentation of the desthiobiotin-containing tag is also shown. Right panel: predicted MS2 b- and y-fragment ions from CID as determined using Protein Prospector software. Bottom panel: annotation of the MS2 spectrum for the TH211 -modified PFKL K677 tryptic peptide including fragment ions containing the probe modified lysine site. Data shown are representative of n = 4 biologically independent experiments.

[0085] Figures 36A-36D. Structure and activity relationship (SAR) study for PFKL activators tested. (Figure 36A) Schematic of PFKL biochemical assay using ADP-GloTM. (Figure 36B) Dose-dependent activation of PFKL by TH220 after incubation for 30 minutes. (Figure 36C) Dose-response activation of PFKL by XJ-3-9, XJ-3-41, and XJ-3-65 after incubation for 30 minutes. (Figure 36D) Biochemical screening results for PFKL activation using substrate assay.

[0086] Figures 37A-37F. TMT proteomics analysis of protein expression changes in HEK293T (Figure 37A), Jurkat (Figure 37B), THP-1 macrophages (Figure 37C), MOLM-14 (Figure 37D), SH-SY5Y (Figure 37E), and B16-F10-Luc2 (Figure 37F) cells treated with DMSO vehicle or XJ-4-85 (5 pM, 1 hour) followed by treatment with TH211 probe (5 pM, 2 hour). PFKL is highlighted in green. Figure 38. Exemplary cryo-EM data processing workflow for PFKL bound to XJ-4-85.

[0087] Figures 39A and 39B. Cryo-EM structure of PFKL bound to XJ-4-85. (Figure 39A) Noise- substituted corrected FSC curves (lighter grey lines) and FSCref curves after density modification (darker gray lines to the right of the lighter gray lines) and corresponding resolution estimates for the PFKL-XJ-4-85 monomer and tetramer structures. (Figure 39B) PFKL-XJ-4-85 cryo-EM structure, with ADP and F6P highlighted in the active site (rectangle in top left panel) and FBP highlighted in the allosteric sugar-binding site (circle in top right panel).

[0088] Figures 40A-40C. (Figure 40A) Multiple sequence alignment of PFK1 isoforms. Included are the catalytic (SEQ ID NO: 15) and regulatory (SEQ ID NO: 16) domains of the human phosphofructokinase- 1, liver isoform (PFKL; amino acids 310-323 and 672-682 of SEQ ID NO: 5, respectively); the catalytic (SEQ ID NO: 17) and regulatory (SEQ ID NO: 18) domains of the human phosphofructokinase- 1, muscle isoform (PFKM; amino acids 310-323 and 673-683 of SEQ ID NO: 21, respectively); and the catalytic (SEQ ID NO: 19) and regulatory (SEQ ID NO: 20) domains of the human phosphofructokinase- 1, platelet isoform (PFKP; amino acids 319-332 and 683-693 of SEQ ID NO: 22, respectively). (Figure 40B) Location of K315 and K677, mapped onto the cryo-EM structure of human PFKL (PDB ID: 7LW1). Catalytic and regulatory domains are colored pale cyan and light purple, respectively in color versions of Figure 40B. (Figure 40C) Comparison of biochemical activity of PFKL WT (circles) and the K315R mutant (triangles) after treatment with XJ-4-85 for 30 min.

[0089] Figure 41. Glycolysis analysis (extracellular acidification rate; ECAR) using a Seahorse XF analyzer revealed no difference in glycolytic capacity in THP- 1 cells treated with 5 pM XJ-4-97 (lighter circles; lower trace) compared to DMSO vehicle (darker circles; upper trace). See Materials and Methods for the EXAMPLES section below for details of assay. Statistical significance was determined by an unpaired Student’s t-test. Data shown are mean ± SD and representative of n = 3 biologically independent experiments.

[0090] Figures 42A and 42B. Assessment of body weight changes during in vivo compound treatments. (Figure 42A) Pilot study that identified a tolerated dose range for XJ-4-85 treatments in mice. (Figure 42B) Plot showing body weights of mice over time in response to treatment with vehicle (black diamonds), 50 mg / kg XJ-4-85 (circles), 25 mg / kg XJ-4-85 (asterisks), 25 mg / kg XJ-4-119 (inverted triangles), or 100 mg / kg XJ-4-97 (gray diamonds). The numbers of mice per group are shown. Data are shown as mean ± SEM.

[0091] Figure 43. In vivo bioluminescence imaging of B16-F10-Luc2-engrafted male mice treated with vehicle, 50 mg / kg XJ-4-85, 25 mg / kg XJ-4-85, 25 mg / kg XJ-4-119, or 100 mg / kg XJ-4-97. The numbers of mice per group are shown.

[0092] Figure 44. Luminescent intensity of photons emitted from each male mouse in the images from Figure 43 were quantified at day 8 and 14. Figure 45. Gel-based competitive ABPP analysis in situ. HEK293T cells were treated with DMSO vehicle or the indicated compounds for 1 hour, followed by treatment with TH211 probe (25 pM, 2 hours), n = 4 biological replicates. Related to Figure 33A.

[0093] Figure 46. Concentration dependent blockade of probe labeling in HEK293T cells treated with TH220 as measured by gel-based competitive ABPP. HEK293T cells were treated with DMSO or TH220 (5-1000 nM, 1 hour) followed by treatment with TH211 probe (25 pM, 2 hours), n = 4 biological replicates. Related to Figures 33C and 33D.

[0094] Figure 47. Dose-dependent blockade of probe labeling in XJ-4-85-treated HEK293T cells as determined by gel-based competitive ABPP. HEK293T cells were treated with DMSO vehicle or XJ- 4-85 (2-1000 nM, 1 hour) followed by treatment with TH211 probe (25 pM, 2 hours), n = 4 biological replicates. Related to Figure 27C.

[0095] DETAILED DESCRIPTION

[0096] The presently disclosed subject matter provides novel sulfonyl-heterocycle compounds useful, for example, in sulfonyl-triazole exchange (SuTEx) chemistry, and methods of using sulfonylheterocycle compounds as covalent modulators of protein activity, e.g., to covalently modify and modulate the activity of intracellular checkpoints of T cell metabolism for selectively reactivating TILs. For instance, the presently disclosed subject matter provides sulfonyl-heterocycle compounds that covalently modify and activate phosphofructokinase- 1, liver isoform (PFKL) to target glycolytic flux in T cells to provide a new druggable and therapeutic checkpoint for cancer immunotherapy and to gain a mechanistic understanding of metabolic regulation of effector versus tumor T cells to guide the design of better and more effective immunotherapy drugs.

[0097] Boosting glycolysis also has applicability for patients with Parkinson’s Disease, as supported by ongoing clinical trials. Currently, however, this is achieved through an off-target effect. The presently disclosed subject matter provides novel activators for glycolysis with unprecedented selectivity, which can be used to study how regulation of glycolysis conveys neuroprotection.

[0098] The presently disclosed subject matter will now be described more fully. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein below and in the accompanying Examples. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.

[0099] All references listed herein, including but not limited to all patents, patent applications and publications thereof, and scientific journal articles, are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and / or compositions employed herein. Throughout the specification and claims, a given chemical formula or name shall encompass all optical and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist, unless as otherwise specifically indicated.

[0100] L Definitions

[0101] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.

[0102] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0103] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0104] In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefits and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.

[0105] Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the presently disclosed and claimed subject matter.

[0106] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including in the claims. For example, the phrase “an antibody” refers to one or more antibodies, including a plurality of the same antibody. Similarly, the phrase “at least one”, when employed herein to refer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of that entity, including but not limited to whole number values between 1 and 100 and greater than 100.

[0107] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. The term “about”, as used herein when referring to a measurable value such as an amount of mass, weight, time, volume, concentration, or percentage, is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1 % from the specified amount, as such variations are appropriate to perform the disclosed methods and / or employ the disclosed compositions. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0108] A disease or disorder is “alleviated” if the severity of a symptom of the disease, condition, or disorder, or the frequency at which such a symptom is experienced by a subject, or both, are reduced.

[0109] As used herein, the term “and / or” when used in the context of a list of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and / or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

[0110] The terms “additional therapeutically active compound” and “additional therapeutic agent”, as used in the context of the presently disclosed subject matter, refers to the use or administration of a compound for an additional therapeutic use for a particular injury, disease, or disorder being treated. Such a compound, for example, could include one being used to treat an unrelated disease or disorder, or a disease or disorder which may not be responsive to the primary treatment for the injury, disease, or disorder being treated.

[0111] As used herein, the term “adjuvant” refers to a substance that elicits an enhanced immune response when used in combination with a specific antigen.

[0112] As use herein, the terms “administration of’ and / or “administering” a compound should be understood to refer to providing a compound of the presently disclosed subject matter to a subject in need of treatment.

[0113] The term “comprising”, which is synonymous with “including” “containing”, or “characterized by”, is inclusive or open-ended and does not exclude additional, unrecited elements and / or method steps. “Comprising” is a term of art that means that the named elements and / or steps are present, but that other elements and / or steps can be added and still fall within the scope of the relevant subject matter.

[0114] As used herein, the phrase “consisting essentially of’ limits the scope of the related disclosure or claim to the specified materials and / or steps, plus those that do not materially affect the basic and novel characteristic(s) of the disclosed and / or claimed subject matter. For example, a pharmaceutical composition can “consist essentially of’ a pharmaceutically active agent or a plurality of pharmaceutically active agents, which means that the recited pharmaceutically active agent(s) is / are the only pharmaceutically active agent(s) present in the pharmaceutical composition. It is noted, however, that carriers, excipients, and / or other inactive agents can and likely would be present in such a pharmaceutical composition and are encompassed within the nature of the phrase “consisting essentially of’.

[0115] As used herein, the phrase “consisting of’ excludes any element, step, or ingredient not specifically recited. It is noted that, when the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0116] With respect to the terms “comprising”, “consisting of’, and “consisting essentially of’, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms. For example, a composition that in some embodiments comprises a given active agent also in some embodiments can consist essentially of that same active agent, and indeed can in some embodiments consist of that same active agent.

[0117] The term “aqueous solution” as used herein can include other ingredients commonly used, such as sodium bicarbonate described herein, and further includes any acid or base solution used to adjust the pH of the aqueous solution while solubilizing a peptide.

[0118] The term “binding” refers to the adherence of molecules to one another, such as, but not limited to, enzymes to substrates, ligands to receptors, antibodies to antigens, DNA binding domains of proteins to DNA, and DNA or RNA strands to complementary strands.

[0119] “Binding partner”, as used herein, refers to a molecule capable of binding to another molecule.

[0120] The term “biocompatible”, as used herein, refers to a material that does not elicit a substantial detrimental response in the host.

[0121] As used herein, the terms “biologically active fragment” and “bioactive fragment” of a peptide encompass natural and synthetic portions of a longer peptide or protein that are capable of specific binding to their natural ligand and / or of performing a desired function of a protein, for example, a fragment of a protein of larger peptide which still contains the epitope of interest and is immunogenic.

[0122] The term “biological sample”, as used herein, refers to samples obtained from a subject, including but not limited to skin, hair, tissue, blood, plasma, cells, sweat, and urine.

[0123] A “coding region” of a gene comprises the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.

[0124] “Complementary” as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids (e.g., two DNA molecules). When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other at a given position, the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (in some embodiments at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides that can base pair with each other (e.g., A:T and G:C nucleotide pairs). Thus, it is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. By way of example and not limitation, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, in some embodiments at least about 50%, in some embodiments at least about 75%, in some embodiments at least about 90%, and in some embodiments at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In some embodiments, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[0125] A “control” cell, tissue, sample, or subject is a cell, tissue, sample, or subject of the same type as a test cell, tissue, sample, or subject. The control may, for example, be examined at precisely or nearly the same time as the test cell, tissue, sample, or subject is examined. The control may also, for example, be examined at a time distant from the time at which the test cell, tissue, sample, or subject is examined, and the results of the examination of the control may be recorded so that the recorded results may be compared with results obtained by examination of a test cell, tissue, sample, or subject. The control may also be obtained from another source or similar source other than the test group or a test subject, where the test sample is obtained from a subject suspected of having a condition, disease, or disorder for which the test is being performed.

[0126] A “test” cell is a cell being examined.

[0127] A “pathoindicative” cell is a cell that, when present in a tissue, is an indication that the animal in which the tissue is located (or from which the tissue was obtained) is afflicted with a condition, disease, or disorder.

[0128] A “pathogenic” cell is a cell that, when present in a tissue, causes or contributes to a condition, disease, or disorder in the animal in which the tissue is located (or from which the tissue was obtained).

[0129] A tissue “normally comprises” a cell if one or more of the cell are present in the tissue in an animal not afflicted with a condition, disease, or disorder.

[0130] As used herein, the terms “condition”, “disease condition”, “disease”, “disease state”, and “disorder” refer to physiological states in which diseased cells or cells of interest can be targeted with the compositions of the presently disclosed subject matter. In some embodiments, a disease is leukemia, which in some embodiments is Acute Myeloid Leukemia (AML).

[0131] As used herein, the term “diagnosis” refers to detecting a risk or propensity to a condition, disease, or disorder. In any method of diagnosis there exist false positives and false negatives. Any one method of diagnosis does not provide 100% accuracy. A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0132] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0133] As used herein, an “effective amount” or “therapeutically effective amount” refers to an amount of a compound or composition sufficient to produce a selected effect, such as but not limited to alleviating symptoms of a condition, disease, or disorder. In the context of administering compounds in the form of a combination, such as multiple compounds, the amount of each compound, when administered in combination with one or more other compounds, may be different from when that compound is administered alone. Thus, an effective amount of a combination of compounds refers collectively to the combination as a whole, although the actual amounts of each compound may vary. The term “more effective” means that the selected effect occurs to a greater extent by one treatment relative to the second treatment to which it is being compared.

[0134] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of an mRNA corresponding to or derived from that gene produces the protein in a cell or other biological system and / or an in vitro or ex vivo system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence (with the exception of uracil bases presented in the latter) and is usually provided in Sequence Listing, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0135] As used herein, an “essentially pure” preparation of a particular protein or peptide is a preparation wherein in some embodiments at least about 95% and in some embodiments at least about 99%, by weight, of the protein or peptide in the preparation is the particular protein or peptide.

[0136] In some embodiments, the terms “fragment”, “segment”, or “subsequence” refer to a portion of an amino acid sequence, comprising at least one amino acid, or a portion of a nucleic acid sequence comprising at least one nucleotide. The terms “fragment”, “segment”, and “subsequence” can be used interchangeably herein.

[0137] As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property by which it can be characterized. A functional enzyme, for example, is one that exhibits the characteristic catalytic activity by which the enzyme can be characterized. The terms “high throughput protein identification,” “proteomics” and other related terms are used herein to refer to the processes of identification of a large number or (in some cases, all) proteins in a certain protein complement. Post-translational protein modifications and quantitative information can also be assessed by such methods. One example of “high throughput protein identification” is a gelbased process that includes the pre-fractionation and purification of proteins by one-dimensional protein gel electrophoresis. The gel can then be fractionated into several molecular weight fractions to reduce sample complexity, and proteins can be in-gel digested with trypsin. The tryptic peptides are extracted from the gel, further fractionated by liquid chromatography and analyzed by mass spectrometry. In another approach, a sample can be fractionated without using the gels, for example, by protein extraction followed by liquid chromatography. The proteins can then be digested in-solution, and the proteolytic fragments further fractionated by liquid chromatography and analyzed by mass spectrometry.

[0138] As used herein “injecting”, “applying”, and administering” include administration of a compound of the presently disclosed subject matter by any number of routes and modes including, but not limited to, topical, oral, buccal, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, ophthalmic, pulmonary, vaginal, and rectal approaches.

[0139] The term “Sulfonyl-Triazole Exchange” (or “SuTEx”) as used herein can refer to a synthetic compound that includes a sulfonyl group directly attached to a nitrogen atom of a substituted or unsubstituted triazine moiety or other azole (e.g., imidazole) moiety. Typically, a SuTEx compound can undergo SuTEx chemistry, e.g., in which the sulfonyl group of the SuTEx compound acts as an electrophile in a covalent reaction between the SuTEx compound and a reactive nucleophilic group of another compound, such as a reactive nucleophilic amino acid residue in a protein or peptide. SuTEx compounds described herein can include SuTEx “ligands” and SuTEx “probes.”

[0140] As used herein, the terms “SuTEx probe” or “probe” can refer to a SuTEx compound that is broadly reactive and can be used to detect sites amenable to covalent reactions with SuTEx compounds. SuTEx probes can include a tag for detection. The tag for detection can be a moiety that can be directly used for detection (e.g., a fluorophore, biotin or another affinity label, a radioisotope, etc.) or a moiety (e.g., an alkyne group) that can be chemically modified to incorporate a detectable group (e.g., biotin) after the SuTEx probe has undergone a covalent reaction with a SuTEx reactive site. In contrast, the terms “SuTEx ligand” or “ligand”, as used herein, can refer to a SuTEx compound that does not include a tag for detection and / or that has been tailored to undergo covalent reactions more selectively with a particular reactive site and / or protein and / or peptide of interest. For example, the selectivity of a SuTEx ligand can be tailored via the addition of substituents that can alter the reactivity of the SuTEx compound for a particular target via steric or electronic effects. In some embodiments, the SuTEx ligands can be used to covalently modify and modulate the activity of one or more particular biological molecules of interest.

[0141] Thus, as used herein, in some embodiments, “ligand” can refer to a synthetic molecule (e.g., a SuTEx compound) that binds to a target compound or molecule, such as a reactive nucleophilic amino acid residue in a peptide or protein, e.g., PFKL. In some embodiments, as can be determined by one of ordinary skill in the art based on context, the term “ligand” as used herein can be used more generally to refer to any entity (e.g., a molecule) that specifically or selectively binds to a target compound or molecule. A ligand “specifically binds to” or “is specifically reactive with” a compound when the ligand functions in a binding reaction which is determinative of the presence of the compound in a sample of heterogeneous compounds. In some embodiments, a ligand can modulate (increase or decrease) a biological activity of biological target, e.g. a protein or peptide.

[0142] As used herein, the term “linkage” refers to a connection between two groups. The connection can be either covalent or non-covalent, including but not limited to ionic bonds, hydrogen bonding, and hydrophobic / hydrophilic interactions .

[0143] As used herein, the term “linker” refers to a molecule that joins two other molecules either covalently or noncovalently, such as but not limited to through ionic or hydrogen bonds or van der Waals interactions.

[0144] As used herein, the term “mass spectrometry” (MS) refers to a technique for the identification and / or quantitation of molecules in a sample. MS includes ionizing the molecules in a sample, forming charged molecules; separating the charged molecules according to their mass-to-charge ratio; and detecting the charged molecules. MS allows for both the qualitative and quantitative detection of molecules in a sample. The molecules can be ionized and detected by any suitable approach known to one of skill in the art. Some examples of mass spectrometry are “tandem mass spectrometry” or “MS / MS,” which are the techniques wherein multiple rounds of mass spectrometry occur, either simultaneously using more than one mass analyzer or sequentially using a single mass analyzer. The term “mass spectrometry” can refer to the application of mass spectrometry to protein analysis. In some embodiments, electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI) can be used in this context. In some embodiments, intact protein molecules can be ionized by the above techniques and then introduced to a mass analyzer. Alternatively, protein molecules can be broken down into smaller peptides, for example, by enzymatic digestion by a protease, such as trypsin. Subsequently, the peptides are introduced into the mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry.

[0145] As used herein, the term “mass spectrometer” is used to refer an apparatus for performing mass spectrometry that includes a component for ionizing molecules and detecting charged molecules. Various types of mass spectrometers can be employed in the methods of the presently disclosed subject matter. For example, whole protein mass spectroscopy analysis can be conducted using time-of-flight (TOF) or Fourier transform ion cyclotron resonance (FT-ICR) instruments. For peptide mass analysis, MALDI time-of-flight instruments can be employed, as they permit the acquisition of peptide mass fingerprints (PMFs) at high pace. Multiple stage quadrupole-time-of-flight and the quadrupole ion trap instruments can also be used.

[0146] The terms “measuring the level of expression” and “determining the level of expression” as used herein refer to any measure or assay which can be used to correlate the results of the assay with the level of expression of a gene or protein of interest. Such assays include measuring the level of mRNA, protein levels, etc. and can be performed by assays such as northern and western blot analyses, binding assays, immunoblots, etc. The level of expression can include rates of expression and can be measured in terms of the actual amount of an mRNA or protein present. Such assays are coupled with processes or systems to store and process information and to help quantify levels, signals, etc. and to digitize the information for use in comparing levels.

[0147] The term “otherwise identical sample”, as used herein, refers to a sample similar to a first sample, that is, it is obtained in the same manner from the same subject from the same tissue or fluid, or it refers a similar sample obtained from a different subject. The term “otherwise identical sample from an unaffected subject” refers to a sample obtained from a subject not known to have the disease or disorder being examined. The sample may of course be a standard sample. By analogy, the term “otherwise identical” can also be used regarding regions or tissues in a subject or in an unaffected subject.

[0148] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.

[0149] The term “pharmaceutical composition” refers to a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

[0150] “Pharmaceutically acceptable” means physiologically tolerable, for either human or veterinary application. Similarly, “pharmaceutical compositions” include formulations for human and veterinary use. As used herein, the term “pharmaceutically acceptable carrier” means a chemical composition with which an appropriate compound or derivative can be combined and which, following the combination, can be used to administer the appropriate compound to a subject.

[0151] As used herein the term “phosphofructokinase- 1, liver isoform (PFKL)”, also referred to as the “liver isoform of phosphofructokinase” or the “ATP-dependent 6-phosphofructokinase, liver type”, refers to a genetic locus that is found on human chromosome 21 at nucleotides 44,300,053-44,327,373 of Accession NO. NC_000021.9 of the GENBANK® biosequence database (SEQ ID NO: 1) as well as all transcription and translation products derived therefrom. In particular, the human PFKL locus encodes several isoforms, two of which are isoform a, which corresponds to Accession No. NM_001002021.3 of the GENBANK® biosequence database, and isoform b, which corresponds to Accession No. NM_002626.6 of the GENBANK® biosequence database. Accession No. NM_001002021.3 of the GENBANK® biosequence database encodes a protein with the amino acid sequence disclosed as Accession No. NP_001002021.2 of the GENBANK® biosequence database, and Accession No. NM_002626.6 of the GENBANK® biosequence database encodes a protein with the amino acid sequence disclosed as Accession No. NP_002617.3 of the GENBANK® biosequence database (SEQ ID NO: 5).

[0152] It is understood that the human sequences disclosed herein are representative only, and that orthologs from other species including but not limited to mouse (Accession Nos. NM_008826.5 and NP_032852.2 of the GENBANK® biosequence database; (SEQ ID NOs: 6 and 7, respectively), rat (Accession Nos. NM_013190.4 and NP_037322.1 of the GENBANK® biosequence database; (SEQ ID NOs: 8 and 9, respectively), dog (Accession Nos. XM_038443637.1 and XP_038299565.1 of the GENBANK® biosequence database; (SEQ ID NOs: 10 and 11, respectively), cat (Accession Nos. XM_02323865I.2 and XP_023094419.1 of the GENBANK® biosequence database; (SEQ ID NOs: 12 and 13, respectively), and others.

[0153] As used herein, the term “physiologically acceptable” ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.

[0154] “Plurality” means at least two.

[0155] “Polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof.

[0156] “Synthetic peptides or polypeptides” refers to non-naturally occurring peptides or polypeptides. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods are known to those of skill in the art.

[0157] As used herein, the term “prevents” means to stop something from happening, or taking advance measures against something possible or probable from happening. In the context of medicine, “prevention” generally refers to action taken to decrease the chance of getting a disease or condition. It is noted that “prevention” need not be absolute and thus can occur as a matter of degree.

[0158] A “preventive” or “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs, or exhibits only early signs, of a condition, disease, or disorder. A prophylactic or preventative treatment is administered for the purpose of decreasing the risk of developing pathology associated with developing the condition, disease, or disorder.

[0159] The term “protein” typically refers to large polypeptides. Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl -terminus.

[0160] As used herein, the term “purified” and like terms relate to an enrichment of a molecule or compound relative to other components normally associated with the molecule or compound in a native environment. The term “purified” does not necessarily indicate that complete purity of the particular molecule has been achieved during the process.

[0161] A “highly purified” compound as used herein refers to a compound that is in some embodiments greater than 90% pure, that is in some embodiments greater than 95% pure, and that is in some embodiments greater than 98% pure.

[0162] As used herein, the term “mammal” refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

[0163] A “sample”, as used herein, refers in some embodiments to a biological sample from a subject, including, but not limited to, normal tissue samples, diseased tissue samples, biopsies, blood, saliva, feces, semen, tears, and urine. A sample can also be any other source of material obtained from a subject which contains cells, tissues, or fluids of interest. A sample can also be obtained from cell or tissue culture.

[0164] The expression “stable isotope labeling by amino acids in cell culture” (SILAC) is used herein to refer to an approach for incorporation of a label into proteins for mass spectrometry (MS)-based quantitative proteomics. SILAC comprises metabolic incorporation of a given “light” or “heavy” form of the amino acid into the proteins. For example, SILAC comprises the incorporation of amino acids with substituted stable isotopic nuclei (e.g. deuterium,13C,15N). In an illustrative SILAC experiment, two cell populations are grown in culture media that are identical, except that one of them contains a “light” and the other a “heavy” form of a particular amino acid (for example,12C and13C labeled L- lysine, respectively). When the labeled analog of an amino acid is supplied to cells in culture instead of the natural amino acid, it is incorporated into all newly synthesized proteins. After a number of cell divisions, each instance of the amino acid is replaced by its isotope-labeled analog. Since there is little chemical difference between the labeled amino acid and the natural amino acid isotopes, the cells behave substantially similarly to the control cell population grown in the presence of a normal amino acid.

[0165] The term “standard”, as used herein, refers to something used for comparison. For example, it can be a known standard agent or compound which is administered and used for comparing results when administering a test compound, or it can be a standard parameter or function which is measured to obtain a control value when measuring an effect of an agent or compound on a parameter or function. Standard can also refer to an “internal standard”, such as an agent or compound which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured. Internal standards are often a purified marker of interest which has been labeled, such as with a radioactive isotope, allowing it to be distinguished from an endogenous marker.

[0166] The term “subject” as used herein can refer to a member of a species for whom analysis, diagnosis, and / or treatment of a disease or disorder using the compositions and methods of the presently disclosed subject matter can be desirable. Accordingly, the term “subject” is intended to encompass in some embodiments any member of the Kingdom Animalia including, but not limited to the phylum Chordata (e.g., members of Classes Osteichthyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals), and all Orders and Families encompassed therein.

[0167] The compositions and methods of the presently disclosed subject matter are particularly useful for warm-blooded vertebrates. Thus, in some embodiments the presently disclosed subject matter concerns mammals and birds. More particularly provided are compositions and methods derived from and / or for use in mammals such as humans and other primates, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans) and / or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), rodents (such as mice, rats, and rabbits), marsupials, and horses. Also provided is the use of the disclosed methods and compositions on birds, including those kinds of birds that are endangered, kept in zoos, as well as fowl, and more particularly domesticated fowl, e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, also provided is the use of the disclosed methods and compositions on livestock, including but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.

[0168] As used herein, a “subject in need thereof’ is a patient, animal, mammal, or human, who will benefit from the method of this presently disclosed subject matter. The term “substantially pure” describes a compound, e.g., a protein or polypeptide, which has been separated from components which naturally accompany it. Typically, a compound is substantially pure when in some embodiments at least 10%, in some embodiments at least 20%, in some embodiments at least 50%, in some embodiments at least 60%, in some embodiments at least 75%, in some embodiments at least 90%, and in some embodiments at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, gel electrophoresis, or HPLC analysis. A compound, e.g., a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state.

[0169] The term “symptom”, as used herein, refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. In contrast, a “sign” is objective evidence of disease. For example, a bloody nose is a sign. It is evident to the patient, doctor, nurse, and other observers.

[0170] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.

[0171] A “therapeutically effective amount” of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.

[0172] As used herein, the phrase “therapeutic agent” refers to an agent that is used to, for example, treat, inhibit, prevent, mitigate the effects of, reduce the severity of, reduce the likelihood of developing, slow the progression of, and / or cure, a disease or disorder.

[0173] The terms “treatment” and “treating” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, and / or lower the chances of the individual developing a condition, disease, or disorder, even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have or predisposed to having a condition, disease, or disorder, or those in whom the condition is to be prevented.

[0174] As used herein, the terms “vector”, “cloning vector”, and “expression vector” refer to a vehicle by which a polynucleotide sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transduce and / or transform the host cell in order to promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.

[0175] As used herein, the term “Western blot,” which can be also referred to as “immunoblot”, and related terms refer to an analytical technique used to detect specific proteins in a sample. The technique uses gel electrophoresis to separate the proteins, which are then transferred from the gel to a membrane (typically nitrocellulose or PVDF) and stained, in membrane, with antibodies specific to the target protein.

[0176] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs and / or orthologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only and is not to be interpreted as a limitation unless the context in which it appears clearly indicates.

[0177] As used herein the term “alkyl” refers to C1-20 inclusive, linear (z.e., “straight-chain”), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (z.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. In some embodiments, the alkyl group is “lower alkyl.” “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (z.e., a Ci-s alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In some embodiments, the alkyl is “higher alkyl.” “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, “alkyl” refers, in particular, to Ci-s straight-chain alkyls. In other embodiments, “alkyl” refers, in particular, to Ci-s branched-chain alkyls.

[0178] Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different. The term “alkyl group substituent” includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), or aryl.

[0179] Thus, as used herein, the term “substituted alkyl” includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

[0180] The term “aryl” is used herein to refer to an aromatic moiety that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenylamine. The term “aryl” specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others. In some embodiments, the term “aryl” means a cyclic aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.

[0181] The aryl group can be optionally substituted (a “substituted aryl”) with one or more aryl group substituents, which can be the same or different, wherein “aryl group substituent” includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, carbonyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR’R”, wherein R’ and R’ ’ can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

[0182] Thus, as used herein, the term “substituted aryl” includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

[0183] Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, triazole, pyrazine, triazine, tetrazole, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.

[0184] The term “heteroaryl” refers to aryl groups wherein at least one atom of the backbone of the aromatic ring or rings is an atom other than carbon. Thus, heteroaryl groups have one or more noncarbon atoms selected from the group including, but not limited to, nitrogen, oxygen, and sulfur. The term “N-heteroaryl” refers to heteroaryl groups comprising one or more nitrogen atoms, such as, but not limited to, pyrazole, imidazole, tetrazole, and triazole.

[0185] As used herein, the term “acyl” refers to an organic carboxylic acid group wherein the -OH of the carboxyl group has been replaced with another substituent (i.e., as represented by RCO — , wherein R is an alkyl or an aryl group as defined herein). As such, the term “acyl” specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group. Specific examples of acyl groups include acetyl and benzoyl.

[0186] “Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group also can be optionally substituted with an alkyl group substituent as defined herein, oxo, and / or alkylene. There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thus providing a heterocyclic group. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.

[0187] The terms “heterocycle” or “heterocyclic” refer to cycloalkyl groups (i.e., non-aromatic, cyclic groups as described hereinabove) wherein one or more of the backbone carbon atoms of a cyclic ring is replaced by a heteroatom (e.g., nitrogen, sulfur, or oxygen). Examples of heterocycles include, but are not limited to, tetrahydrofuran, tetrahydropyran, morpholine, dioxane, piperidine, piperazine, and pyrrolidine.

[0188] “Alkylene” refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and / or substituted with one or more “alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (-CH2-); ethylene (-CH2-CH2-); propylene (-(CIDs-); cyclohexylene (-CeHio-); -CH=CH — 04=04-; -04=04-042-; -(CH2)q- N(R)-(CH2)r-, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (-O-CH2-O-); and ethylenedioxyl (-O-(CH2)2-O-). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons.

[0189] “Alkoxy!” or “alkoxy” refers to an alkyl-O- group wherein alkyl is as previously described. The term “alkoxy!” as used herein can refer to, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, / -butoxy I. and pentoxyl. The term “oxyalkyl” can be used interchangeably with “alkoxy!”.

[0190] “Aryloxy” or “aryloxyl” refer to an aryl-O- group, where aryl is as previously described. Exemplary aryloxy groups include phenoxy.

[0191] “Aralkyl” refers to an aryl-alkyl- group wherein aryl and alkyl are as previously described and include substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

[0192] The term “amino” refers to the -NR’R” group, wherein R’ and R” are each independently selected from the group including H and substituted and unsubstituted alkyl, cycloalkyl, aralkyl, and aryl. In some embodiments, the amino group is -NH2. In some embodiments, R’ and R”, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 atoms (i.e., R’ and R” together form an alkylene group, wherein optionally one or more carbon atoms of the alkylene group are replaced by an oxygen, sulfur or NH group). Amino groups can be primary (where R’ and R” are each H), secondary (where one of R’ and R” is H and the other is \substituted or unsubstituted alkyl, cycloalkyl, aralkyl, or aryl), or tertiary (where both R’ and R” are independently substituted or unsubstituted alkyl, cycloalkyl, aralkyl, or aryl), and in cationic form, may be quaternary (-+NH1(R’)(R”)). Examples of amino groups include, but are not limited to, -NH2, -NHCH3, - NHC(CH3)2, -N(CH3)2, -N(CH2CH3)2, and -NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidine, piperidine, piperazine, morpholino, and thiomorpholino.

[0193] The term “carbonyl” refers to the -(C=O)- or a double bonded oxygen substituent attached to a carbon atom of a previously named parent group.

[0194] The terms “carboxyl” and “carboxylic acid” refer to the -COOH group. The term “carboxylate” can refer to the -COO" group, i.e., to a deprotonated carboxylic acid group.

[0195] The terms “halo”, “halide”, or “halogen” as used herein refer to fluoro, chloro, bromo, and iodo groups.

[0196] The term “haloalkyl” can be used to refer to an alkyl group wherein one or more hydrogen atoms have been replaced by halo groups.

[0197] The term “perhaloalkyl” refers to an alkyl group wherein all of the hydrogen atoms are replaced by halo. Thus, for example, perhaloalkyl can refer to a “perfluroalkyl” group wherein all of the hydrogen atoms of the alkyl group are replaced by fluoro. Perhaloalkyl groups include, but are not limited to, - CF3.

[0198] The terms “hydroxyl” and “hydroxy” refer to the -OH group.

[0199] The term “oxo” refers to a compound described previously herein wherein a carbon atom is replaced by an oxygen atom.

[0200] The term “cyano” refers to the -CN group.

[0201] The term “nitro” refers to the -NO2group.

[0202] The term “azido” refers to the -N3group.

[0203] The term ester refers to the -C(=O)OR group, wherein R is selected from alkyl, aralkyl, cycloalkyl, and aryl. Examples of ester groups include, but are not limited to, -C(=O)OCH, - C(=O)OCH2CH3, -C(=O)OC(CH3)3, and -C(=O)OPh.

[0204] The term “amido” as used refer refers to a -C(=O)NR R , wherein R and R are independently selected from H, alkyl, aralkyl, cycloalkyl and aryl, or wherein R’ and R” together with the nitrogen to which they are attached from a form a heterocyclic ring having from 4 to 8 atoms (i.e., R and R” together form an alkylene group, wherein optionally one or more carbon atoms of the alkylene group are replaced by an oxygen, sulfur or NH group). Examples of amido groups include, but are not limited to, - C(=O)NH2, -C(=O)NHCH3, -C(=O)N(CH3)2, -C(=O)NHCH2CH3, and -C(=O)N(CH2CH3)2, as well as amido groups in which R and R , together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl. Amido groups can also be referred to as carbamoyl.

[0205] The term “sulfonyl” refers to the -S(=O)2R group, wherein R is alkyl, substituted alkyl, aralkyl, aryl, or substituted aryl. A dashed line representing a bond in a chemical formula indicates that the bond can be either present or absent. For example, the chemical structure: refers to compounds wherein Ci and C2 can be joined by either a single or double bond.

[0206] A line crossed by a wavy line, e.g., in the structure: indicates the site where a substituent can bond to another group.

[0207] II. General Considerations

[0208] Glycolysis fuels vital cellular functions and dysregulation is implicated in cancer, neurodegeneration, antibiotic resistance and diabetes. The glycolytic dependency of cancer, known as the Warburg effect, presents a key vulnerability for developing targeted anticancer agents but remains challenging due to metabolic heterogeneity and resistance. Here, we developed a first-in-class covalent phosphofructokinase- 1 liver type (PFKL) activator that induces metabolic imbalance coupled to delivery of a cytotoxic payload to cancer cells in vitro and in vivo. The electrophile-drug conjugate (EDC) site-specifically and proteome-wide selectively modifies K677 in the allosteric effector site to stabilize the R-state tetramer of PFKL and destabilize cell metabolism. We introduce EDCs as a new delivery mechanism analogous to antibody-drug conjugates but differentiated by payload delivery triggered by covalent engagement of an intracellular protein.

[0209] Beyond inhibitory receptors, additional mechanisms exist in the tumor environment that can prevent effective tumor clearance by T cells, including intracellular signaling and metabolism. Deficient enolase activity or phosphoenolpyruvate (PEP) metabolite have been attributed to defective tumorinfiltrating T cell (TIL) function and overexpression of glycolytic enzymes in TILs can enhance antitumor T cell responses. Translation of these findings could be achieved through pharmacological activation of glycolysis in TILs, but to date, this hypothesis has not been tested because of the lack of effective small molecule agents. According to one aspect, the presently disclosed subject matter addresses this gap by studying whether small molecule activators of PFKL (e.g., sulfonyl-triazole activators of PFKL) can restore defective TIL function, establishing this enzymatic pathway as a metabolic checkpoint for cancer immunotherapy. Importantly, the data described hereinbelow suggest that, paradoxically, tumor cells treated with PFKL activators result in blockade of proliferation, indicating that the presently disclosed PFKL activators can be used in the development of anti-cancer agents.

[0210] Parkinson’s Disease (PD) is a progressive and devastating brain disorder that affects over 6 million people with growing numbers. There is no cure. Although a limited number of therapies can temporarily treat some of the symptoms, no currently known treatment can stop neurodegeneration. As disclosed herein, increasing glycolysis by use of a PFKL covalent activator thus represents the first approach with potential to halt neuronal loss and alleviate both motor and cognitive decline.

[0211] Accordingly, in some embodiments, the presently disclosed subject matter is based on glycolytic activation as a new immunotherapy strategy in cancer treatment using small molecule PFKL activators displaying unprecedented proteome-wide selectivity.

[0212] ILA, Glycolytic Flux and T Cells

[0213] T cells destroy tumor cells through activation and expansion of antigen (Ag)-specific populations expressing T-cell receptors (TCRs; see Wucherpfennig et al., 2010) that recognize peptides (bound to major histocompatibility complex (MHC) molecules) derived from proteins synthesized by diseased cells (Chen & Davis, 2005). Ag-specific T cells home to tumor sites and eradicate malignant cells by producing biomolecules necessary for cytotoxicity (e.g. cytokines, perforin, granzyme B; see Wherry, 2011). However, TILs often present a dysfunctional phenotype characterized by poor proliferation, susceptibility to apoptosis, and compromised cytotoxic functions (Wherry, 2011). Strategies to restore full functional activity to TILs have used antibodies that block inhibitory receptors expressed on T cells (clinical examples include CTLA-4 (Leach et al., 1996; Sharma et al., 2011) and PD-1 (Wong et al., 2007; Heger, 2012)) to overcome immunosuppressive mechanisms that persist in the tumor microenvironment (TME). Despite remarkable clinical efficacy, only 20-30% of patients respond to inhibitor checkpoint therapies, highlighting the need for additional strategies and new molecular targets to reactivate TILs for immunotherapy.

[0214] The massive expansion in T cells after TCR (Wucherpfennig et al., 2010) engagement involves substantial alterations to their metabolism to support proliferation and differentiation. This includes the induction of glycolysis and an increase in oxidative phosphorylation (OXPHOS). Glycolysis is activated by either AKT (helper T cells; see Rathmell et al., 2003) or Erk-mediated regulation of c-Myc (cytotoxic T cells; see Wang et al., 2011). The key kinase mTORCl controls metabolism via its regulation of p70-S6 kinase and eIF4E binding protein to affect transcription and translation, as well as the SREBP fatty acid synthesis regulators (Zeng & Chi, 2017). Additionally, mTORCl regulates the transcription factor HIF-1 to promote the sustained expression of glucose transporters and the major enzymes of glycolysis, as well as many genes that are reflected in the “effector” activities of T cells such as the expression of lytic granules and cytokines (Finlay et al., 2012). Conversely, liver kinase Bl (LKB1) functions with AMP-activated kinase al (AMPKal) to sense nutrient availability and can attenuate mTORCl and arrest glycolysis once nutrients and ATP become limiting, enhancing OXPHOS and stimulating a greater use of fatty acids rather than glucose as the T cells transition into a more quiescent state (Tamas et al., 2006; Tamas et al., 2010). Thus, glycolysis is important for the effector functions of T cells, and its induction and sustained activation is regulated by kinases at critical points.

[0215] The first committed step of glycolysis is regulated by phosphofructokinase- 1 (PFK1), which catalyzes the ATP-dependent conversion of fructose 6-phosphate to fructose 1,6-bisphosphate (FBP) (Mor et al., 2011; Webb et al., 2015; Campos & Albrecht, 2023). See Figure 1. As the gatekeeper of glycolysis, PFK1 is tightly regulated through multiple mechanisms including (i) energy state of the cell (low energy = PFK1 activation; high energy = PFK1 inhibition), (ii) post-translational modifications including phosphorylation, acetylation, glycosylation (Campos & Albrecht, 2023), and (iii) allosteric ligands that promote or disrupt the active tetramer state (Webb et al., 2017; Lynch et al., 2024). In mammalian systems, PFK1 is expressed as three isoforms: platelet isoform (PFKP), muscle isoform (PFKM), and liver isoform (PFKL). These isoforms have high sequence homology (-70%; see Figure 10A; see also Campos & Albrecht, 2023) and distinct regulation and tissue-specific expression profiles. Of note, PFKL is expressed in T cells despite the liver nomenclature (Amara et al., 2021). The high sequence homology makes it challenging to target a specific isoform, which is important in the context of drug discovery given that disruption of individual PFK1 proteins can produce side effects (e.g., PFKM deficiency can lead to Tarui disease; see Tarui et al., 1965).

[0216] The presently disclosed subject matter is based in part on the hypothesis that selective activation of PFKL can restore a deficient T cell glycolytic state to overcome immunosuppression of TIL activity. Previous studies have linked TIL dysfunction to decreased glycolysis via downregulation of enzyme activity (Gemta et al., 2019) or glucose competition in tumors (Leone & Powell, 2020). The tumor microenvironment (TME) is competitive for nutrients to fuel both cancer and immune cell proliferation (Maclver et al., 2013; Sukumar et al., 2015). Akin to tumor cells, activated T cells switch from the more energy efficient oxidative phosphorylation (OXPHOS) to aerobic glycolysis despite sufficient oxygen (Warburg effect; Warburg, 1956) to meet the metabolic demands for rapid proliferation (Pearce & Pearce, 2013; Chang & Pearce, 2016; Buck et al., 2017). A shift towards glycolysis supplies ATP, albeit less efficiently than OXPHOS, as well as biosynthetic precursors including lipids, amino acids, and nucleotides to support energy and biosynthesis for rapid T cell proliferation. Glycolysis can also regulate cytokine signaling of activated T cells via IFN-g expression regulated by GAPDH binding to IFN-g mRNA (Chang et al., 2013). Limiting glucose availability or downregulating glycolytic enzyme activity can suppress TIL function in the TME (Ho et al., 2015; Gemta et al., 2019). Metabolic immunosuppression has been reversed through recombinant overexpression of glycolytic enzymes in TILs (PCK1; Ho et al., 2015) or through supplementation of glycolytic metabolites (e.g., pyruvate; Gemta et al., 2019).

[0217] A small molecule, NA-11, has been previously described that selectively activates PFKL to suppress excessive NOX2-dependent oxidative burst and NETosis (Amara et al., 2021). The structure of NA-11 is shown in Figure 2A. Besides neutrophils, T cells and hepatocytes also express PFKL. However, no previous compounds have been reported to target PFKL on these cells.

[0218] II. B, Sulfonyl -Triazole Exchange (SuTEx) Compounds for Targeting PFKL

[0219] Small molecules can serve as versatile tools for perturbing the functions of proteins in biological systems. Recently, sulfonyl-triazoles have emerged as anew class of reactive compounds for covalent modification of tyrosine and / or lysine sites on proteins through sulfur-triazole exchange (SuTEx) chemistry. See PCT International Publication No. WO 2020 / 214336 to Hsu et al., published on October 22, 2020, the disclosure of which is incorporated by reference in its entirety. For example, Scheme 1, below, shows the reaction of a SuTEx compound (e.g., a SuTEx ligand or a SuTEx probe) with a protein having a reactive tyrosine (Y) or lysine (K).

[0220] Scheme 1. SuTEx Reactions with Proteins with Reactive Tyrosines or Lysines

[0221] The SuTEx compound comprises a sulfur electrophile, i.e., a sulfonyl group directed attached to a nitrogen atom of a nitrogen-containing heteroaryl group. The nitrogen-containing heteroaryl group acts as a leaving group in the reaction of the compound with the nucleophilic phenol or amine of the tyrosine or lysine, resulting in a modified protein where a modified tyrosine or lysine residue is covalently attached to the SuTEx compound sulfonyl group, which is itself directly attached to an adduct group (AG) from the original SuTEx compound. AGs of SuTEx ligands can include a variety of optionally substituted alkyl, cycloalkyl (including heterocyclic), aryl (including heteroaryl), and aralkyl groups, while SuTEx “probes” can contain AG groups that comprise an alkyne group, a fluorophore moiety, a detectable moiety, or a combination thereof. For instance, the alkyne group of a SuTEx probe can be used as the site of reaction of a protein modified by the probe with a detectable moiety. While the nitrogen-containing heteroaryl group shown in the SuTEx compound of Scheme 1 is a 1,2,4-triazole or a 1,2,3-triazole substituted by an R group (i.e., H or an aryl group substituent), SuTEx compounds can also include other nitrogen-containing heteroaryl groups as the leaving group, e.g., pyrazole, imidazole, or tetrazole, each of which can be optionally substituted by one or more aryl group substituents.

[0222] Thus, sulfonyl-triazoles exchange (SuTEx) chemistry provides sulfonyl-triazole and related sulfonyl-heterocycle compounds that act as electrophiles for covalently targeting functional tyrosine and lysine sites on proteins (Borne et al., 2021; Grams & Hsu, 2022; Brulet et al., 2023). An alkyne- modified SuTEx probe, referred to herein as TH211 and whose structure is shown in Figure 2B, has been previously described. See PCT International Publication No. WO 2020 / 214336 to Hsu et al., published on October 22, 2020, the disclosure of which is incorporated by reference in its entirety. The structure of TH211 contains a kinase fragment binding element (“RF001”; Franks et al., 2017) for chemical proteomic profding of the kinome in live cells (Huang et al., 2021). Using the TH211 scaffold to design more targeted ligands, an analog, referred to as TH220 (see Figure 2C) was developed that blocked TH211 labeling ofa ~85 kDa protein inHEK293T live cells. See PCT International Publication No. WO 2023 / 023664, published February 23, 2023, the disclosure of which is incorporated by reference in its entirety.

[0223] To date, SuTEx compounds have principally been provided as inhibitors of protein function (Hahm et al., 2020; Toroitich et al., 2021). However, as described herein, a series of novel SuTEx compounds (e.g., XJ-4-85) that potently and selectively activate PFKL biochemical activity and glycolysis through covalent modification of the lysine at residue 677 (K677) on the glycolytic enzyme PFKL. In particular, an exemplary covalent PFKL activator XJ-4-85 more potently activates immune cell signaling compared with the only other known PFKL activator, NA-11 (see Figure 2A) that operates through reversible binding (Amara et al., 2021). Thus, the presently disclosed subject matter provides covalent PFKL activators with potential to reverse metabolic and functional defects observed in TILs of the TME.

[0224] III. Exemplary Compounds

[0225] The presently disclosed subject matter relates, in one aspect, to the further development of SuTEx ligands, e.g., to study modulation of PFKL and / or other glycolytic enzymes. In some embodiments, the presently disclosed subject matter provides a compound having a structure of formula (I): wherein: X, Y, and Z are independently CH or N, subject to the proviso that at least one of X, Y, and Z is N; Ri is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; Li is -(CH2)n- or -C(=O)-(CH2)n i-, wherein n is an integer selected from 3, 4, and 5; and R’ is selected from H, wherein: — is a double or single bond; A is selected from methylene, NH, O, and S; Ai is selected from CH and N; A2is selected from C, CH, and N, subject to the proviso that when — is a single bond, A2is N or CH, and when — is a double bond, A2is C; R2and FC are each selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and R4, R5, Re, and R7 are each independently selected from the group consisting of H and alkyl; or a pharmaceutically acceptable salt thereof.

[0226] In some embodiments, the compound of Formula (I) comprises a sulfonyl -triazole or -sulfonylimidazole group, i.e., where two of X, Y, and Z are N or where one of X, Y, and Z (e.g., Z) is N. In some embodiments, two of X, Y, and Z are N. In some embodiments, Z and Y are each N and X is CH (i.e., the compound comprises a 1,2,3-triazole group). In some embodiments, X and Z are each N and Y is CH (i.e., the compound comprises a 1,2,4-triazole group).

[0227] In some embodiments, Ri is selected from lower alkyl (e.g., C1-C6 alkyl), aryl, substituted aryl, heteroaryl, and substituted heteroaryl. In some embodiments, Ri is selected from aryl (e.g., phenyl or naphthyl), substituted aryl (e.g., substituted phenyl), heteroaryl (e.g., pyridinyl, thiazolinyl, quinolinyl, etc.), and substituted heteroaryl (e.g., substituted pyridinyl). In some embodiments, the substituted aryl and substituted heteroaryl are aryl or heteroaryl groups substituted with one or more substituent selected from the group consisting of halo, cyano, alkyl, alkoxy, haloalkyl, haloalkoxy, perhaloalkyl, perhaloalkoxy, cycloalkyl, aralkyl, aryl, amino, and amido, or wherein two substituents together form a divalent group (e.g., forming a ring structure fused to the substituted aryl or heteroaryl group). In some embodiments, the divalent group is substituted or unsubstituted alkylene (e.g., C3-C5 alkylene). In some embodiments, wherein one or more carbon atoms in the alkylene group is replaced by O, N, NH, or S. In some embodiments, the alkylene group can include an alkene bond. In some embodiments, one of the carbon atoms in the alkylene group can be a carbonyl (i.e., -C(=O)-) group. For example, in some embodiments, two substituents together are -CH=CH-C(=O)-O. In some embodiments, a carbon atom in the alkylene group can unsubstituted alkylene (e.g., propylene or butylene), halo-substituted alkylene, alkyleneoxy (i.e., -CH2CH2-O-), alkylenedioxy (e.g., -O-CH2-O- or -O-CH2CH2-O-), or a halo-substituted alkylenedioxy group (e.g., -O-C(F)2-O-). In some embodiments, Ri is selected from substituted phenyl or substituted pyridinyl. In some embodiments, the substituted phenyl or substituted pyridinyl is phenyl or pyridinyl substituted with at least one alkoxy or haloalkoxy group or wherein the substituted phenyl is a phenyl group substituted with two substituents that together form a divalent group, such as an alkylenedioxy group or halo-substituted alkylenedioxy group. In some embodiments, the divalent group is -O(CH2)O-, -O(CF2)O-, or -O(CH2)2O-.

[0228] In some embodiments, Li is -(CH2)n-. In some embodiments, n is 4 or 5. In some embodiments, n is 4 and Li is butylene.

[0229] In some embodiments, R’ is H or

[0230] In some embodiments, A is methylene (i.e., -CH2-), In some embodiments, R4, R5, Re, and R7 are each H.

[0231] In some embodiments, the compound of Formula (I) has a structure of Formula (II) or Formula (III): wherein: — is a double or single bond; X, Y, Z, Li, Ai, A2, and R1-R7 are as defined for Formula (I); or a pharmaceutically acceptable salt thereof. In some embodiments, R2and / or R3 is substituted aryl or substituted heteroaryl, wherein said substituted aryl or substituted heteroaryl is aryl or heteroaryl substituted with one or more substituents selected from halo, nitro, cyano, carboxyl, ester, formyl, alkyl, halo-substituted alkyl, alkoxy, haloalkoxy, aryl, and heteroaryl. In some embodiments, R2and / or R3 is an aryl or heteroaryl moiety that is di-substituted with an alkylene group that forms a ring fused to the aryl or heteroaryl moiety. In some embodiments, the alkylene group is a substituted or unsubstituted alkylenedioxy group (e.g., -O-CH2-O- or -O-CF2-O-). In some embodiments, R2and / or R3is / are halo- substituted phenyl, optionally fluoro-substituted phenyl, further optionally mono- or di-fluoro- substituted phenyl. In some embodiments, the compound is a compound of Formula (III) and R2and R3 are the same.

[0232] In some embodiments, R4, Rs, Re, and R7 are each independently H or methyl, optionally wherein two, three, or four of R4, Rs, Re, and R7 are H. In some embodiments, each of R4, Rs, Re, and R7is H.

[0233] In some embodiments, the compound of Formula (I) has a structure of Formula (IV): wherein: X, Y, and Z are independently CH or N, subject to the proviso that at least one of X, Y, and Z is N; Ri is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; Li is -(CH2)n-, wherein n is an integer selected from 3, 4, and 5; R2and Rs are each selected from aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and R4, R5, Re, and R7 are each independently selected from the group consisting ofH and alkyl; or a pharmaceutically acceptable salt thereof. In some embodiments, two of X, Y, and Z are N. In some embodiments, Y and Z are each N and X is CH.

[0234] In some embodiments, Ri is substituted or substituted aryl or substituted or unsubstituted heteroaryl. In some embodiments, Ri is substituted aryl (e.g., substituted phenyl) or substituted heteroaryl (e.g., substituted pyridinyl). In some embodiments, Ri is pyridinyl substituted with alkoxy or halo-substituted alkoxy. In some embodiments, Ri is substituted phenyl substituted with alkoxy or alkylenedioxy (e.g., -O-CH2-O-).

[0235] In some embodiments, n is 4 or 5. In some embodiments, n is 4.

[0236] In some embodiments, R2and R3 are each fluoro-substituted phenyl (e.g., mono- or di-fluoro- substituted phenyl).

[0237] In some embodiments, R4, Rs, R«, and R7 are selected from H and methyl. In some embodiments, at least two of R4, Rs, R«, and R7 are H. In some embodiments, each of R4, Rs, R«, and R7is H.

[0238] In some embodiments, the compound of Formula (I) is selected from the group consisting of XJ-3-9, XJ-3-17, XJ-3-23; XJ-3-45, XJ-3-41, XJ-3-51, XJ-3-71, XJ-3-77, XJ-3-109, XJ-3-113, XJ-

[0239] 3-115, XJ-3-119, XJ-3-123, XJ-3-125, XJ-3-129, XJ-3-131, XJ-3-133, XJ-3-137, XJ-3-139, XJ-3- 149, XJ-3-65, XJ-4-7, XJ-4-5, XJ-4-9, XJ-4-17, XJ-4-27, XJ-4-31, XJ-4-39, XJ-4-65, XJ-4-85, XJ-

[0240] 4-89, XJ-4-97, and XJ-4-99 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is selected from the group consisting of XJ-3-41, XJ-4-27, XJ-3-129, XJ-3-71, XJ-4-65, XJ-4-85, XJ-4-99, XJ-4-89, XJ-4-97, XJ-3-9, XJ-3-65, XJ-4-5, XJ-4-7, and pharmaceutically acceptable salts thereof.

[0241] In some embodiments, the compound is selected from XJ-3-71, XJ-3-129, XJ-3-113, XJ-4-17, XJ-4-99, XJ-4-65, and XJ-4-85. In some embodiments, the compound is selected from XJ-3-129, XJ- 4-65, and XJ-4-85. In some embodiments, the compound is XJ-4-85.

[0242] As noted above, in some embodiments, the presently disclosed compounds can be provided as a pharmaceutically acceptable salt. As used herein, the term “physiologically acceptable salt” means a salt form of the recited compound which is compatible with any other ingredients of a pharmaceutical composition and / or which is not deleterious to a subject to which the composition is to be administered (e.g., a human or other mammalian subject).

[0243] Such salts include, but are not limited to, pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts, and combinations thereof.

[0244] Acid addition, salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethane sulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzene sulfonic, p- toluenesulfonic acids, sulphates, nitrates, phosphates, perchlorates, borates, acetates, benzoates, hydroxynaphthoates, glycerophosphates, ketoglutarates and the like.

[0245] Base addition salts include but are not limited to, ethylenediamine, N-methyl -glucamine, lysine, arginine, ornithine, choline, N, N’- dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris (hydroxymethyl)- aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e. g., lysine and arginine dicyclohexylamine and the like.

[0246] Examples of metal salts include lithium, sodium, potassium, magnesium salts, and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like.

[0247] In some embodiments, the presently disclosed compounds can further be provided as a solvate.

[0248] In some embodiments, the presently disclosed subject matter encompasses the preparation and use of pharmaceutical compositions comprising a ligand compound as described herein. The pharmaceutical compositions can be useful for treatment of diseases and disorders as would be apparent upon review of the instant disclosure as an active ingredient. Such a pharmaceutical composition can comprise, consist essentially of, or consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition can comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient can be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art. Thus, in some embodiments, the presently disclosed subject matter provides a pharmaceutical composition comprising (a) a compound of formula (I), (II), (III) or (IV), or a pharmaceutical salt and / or solvate thereof; and (b) a pharmaceutically acceptable carrier.

[0249] The compositions of the presently disclosed subject matter can comprise at least one active ingredient (e.g., at least one compound of formula (I), (II), (III), or (IV) or a pharmaceutically acceptable salt or solvate thereof), one or more acceptable carriers, and optionally other active ingredients or therapeutic agents.

[0250] Pharmaceutically acceptable carriers include physiologically tolerable or acceptable diluents, excipients, solvents, or adjuvants. The compositions are in some embodiments sterile and nonpyrogenic. Examples of suitable carriers include, but are not limited to, water, normal saline, dextrose, mannitol, lactose or other sugars, lecithin, albumin, sodium glutamate, cysteine hydrochloride, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, kaolin, agar-agar and tragacanth, or mixtures of these substances, and the like.

[0251] The pharmaceutical compositions can also contain minor amounts of nontoxic auxiliary pharmaceutical substances or excipients and / or additives, such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like). Suitable additives include, but are not limited to, physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions (e.g., 0.01 to 10 mole percent) of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA or CaNaDTPA-bisamide), or, optionally, additions (e.g., 1 to 50 mole percent) of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). If desired, absorption enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) can be used. The compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Pharmaceutical compositions according to the presently disclosed subject matter can be prepared in a manner fully within the skill of the art.

[0252] The compositions of the presently disclosed subject matter or pharmaceutical compositions comprising these compositions can be administered so that the compositions can have a physiological effect. Administration can occur enterally or parenterally; for example, orally, rectally, intracistemally, intravaginally, intraperitoneally, locally (e.g., with powders, ointments or drops), or as a buccal or nasal spray or aerosol. Parenteral administration is an approach. Particular parenteral administration methods include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intraarterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature), peri- and intra-target tissue injection, subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps), intramuscular injection, and direct application to the target area, e.g., intratumoral injection, for example by a catheter or other placement device.

[0253] Where the administration of the composition is by injection or direct application, the injection or direct application can be in a single dose or in multiple doses. Where the administration of the compound is by infusion, the infusion can be a single sustained dose over a prolonged period of time or multiple infusions.

[0254] The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi -dose unit.

[0255] It will be understood by the skilled artisan that such pharmaceutical compositions are generally suitable for administration to animals of all sorts. Subjects to which administration of the pharmaceutical compositions of the presently disclosed subject matter is contemplated include, but are not limited to, humans and other primates, mammals including commercially and / or socially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs, birds including commercially and / or socially relevant birds such as chickens, ducks, geese, parrots, and turkeys.

[0256] A pharmaceutical composition of the presently disclosed subject matter can be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0257] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the presently disclosed subject matter will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition can comprise between 0.1% and 100% (w / w) active ingredient.

[0258] In addition to the active ingredient, a pharmaceutical composition of the presently disclosed subject matter can further comprise one or more additional pharmaceutically active agents.

[0259] Controlled- or sustained-release formulations of a pharmaceutical composition of the presently disclosed subject matter can be made using conventional technology.

[0260] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fdlers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the presently disclosed subject matter are known in the art and described, for example in Gennaro (1990) Remington’s Pharmaceutical Sciences, 18th ed.. Mack Pub. Co., Easton, Pennsylvania, United States of America and / or Gennaro (ed.) (2003) Remington: The Science and Practice of Pharmacy, 20th edition Lippincott, Williams & Wilkins, Philadelphia, Pennsylvania, United States of America, each of which is incorporated herein by reference.

[0261] The compositions may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type of cancer being diagnosed, the type and severity of the condition or disease being treated, the type and age of the animal, etc.

[0262] Other approaches include but are not limited to nanosizing the composition comprising a ligand compound as described herein to be delivered as a nanoparticle intravenously, intraperitoneal injection, or implanted beads with time release of a ligand compound as described herein.

[0263] Suitable preparations include injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, suspension in, liquid prior to injection, may also be prepared. The preparation may also be emulsified, or the compositions encapsulated in liposomes. The active ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants.

[0264] The presently disclosed subject matter also includes a kit comprising the composition of the presently disclosed subject matter and an instructional material which describes administering the composition to a cell or a tissue of a subject. In some embodiments, this kit comprises a (in some embodiments sterile) solvent suitable for dissolving or suspending the composition of the presently disclosed subject matter prior to administering the compound to the subject and / or a device suitable for administering the composition such as a syringe, injector, or the like or other device as would be apparent to one of ordinary skill in the art upon a review of the instant disclosure.

[0265] As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition of the presently disclosed subject matter in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material may describe one or more methods of using the compositions for diagnostic or identification purposes or of alleviation the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the presently disclosed subject matter can, for example, be affixed to a container which contains a composition of the presently disclosed subject matter or be shipped together with a container which contains the composition. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the composition be used cooperatively by the recipient. IV. Synthesis

[0266] The compounds of the presently disclosed subject matter can be prepared using organic group transformations known in the art of organic synthesis, as further described in the Examples below, and via methods analogous to those described in PCT International Publication No. WO 2020 / 214336 to Hsu et al., published October 22, 2020, and PCT International Publication No. WO 2023 / 023664, published February 23, 2023, the disclosure of each of which is incorporated herein by reference in its entirety.

[0267] By way of example, SuTEx probes and ligands comprising a substituted 1,2,4-triazole group can be prepared by reacting sulfonyl chlorides with N-heteroaryl compounds. For example, Scheme 2, below shows an exemplary synthetic route to a sulfonyl-triazole compound starting from an amide reagent precursor of a substituted triazole. Thus, as shown in Scheme 2, an amide starting material (compound A in Scheme 2, where J represents the triazole substituent in the final SuTEx compound) can be coupled with DMF-DMA to produce an amidine intermediate (B). The amidine intermediate can undergo cyclization in acetic acid with hydrazine hydrate to form the corresponding 1,2,4-triazole, i.e., compound C in Scheme 2. The 1,2,4-triazole can then be reacted with a suitable sulfonyl chloride to provide the final SuTEx probe or ligand or a compound that can be further reacted to provide the SuTEx probe or ligand. J’ in Scheme 2 represents the adduct group (AG) of a SuTEx compound or a moiety that can be further reacted to provide the AG. Additional compounds for sulfur heterocycle exchange chemistry can be prepared by reacting the sulfonyl chlorides of with other N-heteroaryl compounds, e.g., imidazole, a substituted imidazole, pyrazole, a substituted pyrazole, tetrazole, or a substituted pyrazole.

[0268] Scheme 2, General Synthesis of 1.2.4-Triazole SuTEx Compounds

[0269] SuTEx probes comprising a 1,2,3-triazole group can be prepared as using a previously reported procedure (Mertins et al., 2018), involving a copper catalyzed azide-alkyne cycloaddition using copper(I) thiophene-2-carboxylate (CuTC) in toluene. See Scheme 3, below. This initial cycloaddition provides a 1,4-regioisomer of the 1,2, 3 -triazole (compound D in Scheme 3), which can be converted to the 2,4-regioisomer (compound E) using dimethylaminopyridine (DMAP) in acetonitrile.

[0270] Scheme 3, General Synthesis of 1,2,3 -Triazole SuTEx Compounds Alternatively, sulfonyl-triazole compounds can be prepared by synthetic routes involving a sulfide intermediate. Scheme 4, below, shows the synthesis of a sulfonyl-triazole compound by a route involving a benzyl sulfide intermediate. benzyl mercaptan (1.0 eq)

[0271] Pd2(dba)3(0.05 eq)

[0272] XantPhos (0.1 eq)

[0273] DIEA (2.0 eq.)

[0274] ,Ar' - BnxAr'

[0275] X 1,4-dioxane, 110 °C, 2 h S

[0276] F G

[0277] 1) 2,4-dichloro-5,5-dimethylhydantoin (2.0 eq)

[0278] BrK zAr' S -e-Ar'

[0279] G o' o

[0280] X = Cl or Br,

[0281] Ar' = aryl or heteroaryl,

[0282] Bn = benzyl

[0283] Scheme 4, General Synthesis of Sulfonyl-Triazole Compounds via Sulfide Intermediate

[0284] For example, as shown in Scheme 4, halo-substituted arene or heteroarene F can be reacted with benzyl mercaptan to provide benzyl sulfide intermediate G. Treatment of benzyl sulfide G with l,2-dichloro-5,5-dimethylhydantoin in acetonitrile / water / acetic acid, followed by reaction with a 1,2,4- triazole provides the sulfonyl-triazole product. Other sulfonyl-heteroaryl compounds can be prepared by analogous routes using other nitrogen-containing heteroaryl compounds (e.g., imidazole) in place of the 1,2,4-triazole.

[0285] V, Methods for Activating PFKL

[0286] In some embodiments, the presently disclosed subject matter provides a method of activating PFKL. In some embodiments, the method comprises contacting a sample comprising PFKL with a compound of Formula (I), (II), (III), or (IV), or a pharmaceutical composition thereof. In some embodiments, the method comprises selectively activating PFKL (e.g., compared to other proteins in the sample, including other isoforms of phosphofructokinase). In some embodiments, the selectivity comprises a 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or more difference in level of activation. In some embodiments, the compound selectively modifies (i.e., selectively covalently modifies) PFKL compared to other proteins in the sample, e.g., PFKM or PFKP.

[0287] The sample can be any suitable sample. In some embodiments, the sample comprising PFKL is a cell extract, a biological fluid, a cell, a tissue, a tissue extract, an organ, or an organism (e.g., a mammal, such as a human).

[0288] In some embodiments, activating PFKL in the sample provides increased glycolysis and / or T cell activation (e.g., compared to the same sample prior to or without contact with the compound). In some embodiments, activating PFKL reactivates TILs. In some embodiments, activating PFKL provides neuroprotection.

[0289] In some embodiments, the PFKL is human PFKL (UniProt ID P17858.6; Accession No. NP_002617.3 of the GENBANK biosequence database) In some embodiments, activating the PFKL comprises covalently modifying PFKL (e.g., human PFKL) at the lysine of residue 677 of human PFKL. VI. Therapeutic Uses and Pharmaceutical Compositions

[0290] In some embodiments, the presently disclosed compounds can be used to treat a disease or disorder associated with glycolytic flux imbalance, e.g., an inflammatory' related disease, a neurodegen erative disease (e.g., Parkinson’s Disease) or cancer.

[0291] In some embodiments, the presently disclosed subject matter provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of Formula (I), (II), (III), or (IV), or a pharmaceutical composition thereof. In some embodiments, the subject is a human. In some embodiments, the subject is a human subject diagnosed with or suspected of having a cancer, such as a breast cancer, a liver cancer or a colorectal cancer. In some embodiments, the cancer is a breast cancer, a liver cancer, or a colorectal cancer.

[0292] In some embodiments, the compound is a compound of Formula (III) or (IV). In some embodiments, the compound is a compound of Formula (IV). In some embodiments, the compound is an activator of PFKL (e.g., a selective activator of PFKL). In some embodiments, the compound improves glycolytic flux. In some embodiments, the compound activates T-cells. In some embodiments, the compound has an EC50 for PFKL of about 5 pM or less.

[0293] In some embodiments, the compound is selected from the group consisting of XJ-3-41, XJ-4- 27, XJ-3-129, XJ-3-71, XJ-4-65, XJ-4-85, XJ-4-89, XJ-4-97, XJ-3-9, XJ-3-65, and pharmaceutically acceptable salts thereof.

[0294] In some embodiments, the compound is XJ-4-85.

[0295] VIE Cells, Analytical Techniques, and Instrumentation

[0296] In some embodiments, one or more of the methods disclosed herein comprise a sample (e.g., a cell sample, or a cell lysate sample). In some embodiments, the sample for use with the methods described herein is obtained from cells of an animal. In some instances, the animal cell includes a cell from a marine invertebrate, fish, insects, amphibian, reptile, or mammal. In some instances, the mammalian cell is a primate, ape, equine, bovine, porcine, canine, feline, or rodent. In some instances, the mammal is a primate, ape, dog, cat, rabbit, ferret, or the like. In some cases, the rodent is a mouse, rat, hamster, gerbil, hamster, chinchilla, or guinea pig. In some embodiments, the bird cell is from a canary, parakeet or parrots. In some embodiments, the reptile cell is from a turtles, lizard or snake. In some cases, the fish cell is from a tropical fish. In some cases, the fish cell is from a zebrafish (e.g. Danino rerio). In some cases, the worm cell is from a nematode (e.g. C. elegans). In some cases, the amphibian cell is from a frog. In some embodiments, the arthropod cell is from a tarantula or hermit crab.

[0297] In some embodiments, the sample for use with the methods described herein is obtained from a mammalian cell. In some instances, the mammalian cell is an epithelial cell, connective tissue cell, hormone secreting cell, a nerve cell, a skeletal muscle cell, a blood cell, or an immune system cell. Exemplary mammalian cell lines include, but are not limited to, 293A cells, 293FT cells, 293F cells, 293H cells, HEK 293 cells, CHO DG44 cells, CHO-S cells, CH0-K1 cells, and PC 12 cells.

[0298] In some embodiments, the sample for use with the methods described herein is obtained from cells of a tumor cell line. In some instances, the sample is obtained from cells of a solid tumor cell line. In some instances, the solid tumor cell line is a sarcoma cell line. In some instances, the solid tumor cell line is a carcinoma cell line. In some embodiments, the sarcoma cell line is obtained from a cell line of alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, clear cell sarcoma of soft tissue, dedifferentiated liposarcoma, desmoid, desmoplastic small round cell tumor, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma, esthesioneuroblastoma, Ewing sarcoma, extrarenal rhabdoid tumor, extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma, infantile fibrosarcoma, inflammatory myofibroblastic tumor, Kaposi sarcoma, leiomyosarcoma of bone, liposarcoma, liposarcoma of bone, malignant fibrous histiocytoma (MFH), malignant fibrous histiocytoma (MFH) of bone, malignant mesenchymoma, malignant peripheral nerve sheath tumor, mesenchymal chondrosarcoma, myxofibrosarcoma, myxoid liposarcoma, myxoinflammatory fibroblastic sarcoma, neoplasms with perivascular epitheioid cell differentiation, osteosarcoma, parosteal osteosarcoma, neoplasm with perivascular epitheioid cell differentiation, periosteal osteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, PNET / extraskeletal Ewing tumor, rhabdomyosarcoma, round cell liposarcoma, small cell osteosarcoma, solitary fibrous tumor, synovial sarcoma, and telangiectatic osteosarcoma.

[0299] In some embodiments, the carcinoma cell line is obtained from a cell line of adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0300] In some instances, the sample is obtained from cells of a hematologic malignant cell line. In some instances, the hematologic malignant cell line is a T-cell cell line. In some instances, B-cell cell line. In some instances, the hematologic malignant cell line is obtained from a T-cell cell line of: peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma, angioimmunoblastic lymphoma, cutaneous T-cell lymphoma, adult T-cell leukemia / lymphoma (ATLL), blastic NK-cell lymphoma, enteropathy-type T-cell lymphoma, hematosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma, nasal NK / T-cell lymphomas, or treatment-related T-cell lymphomas.

[0301] In some instances, the hematologic malignant cell line is obtained from a B-cell cell line of: acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), high-risk chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk small lymphocytic lymphoma (SLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Waldenstrom’s macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt’s lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B- lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[0302] In some embodiments, the sample for use with the methods described herein is obtained from a tumor cell line. Exemplary tumor cell lines include, but are not limited to, 600MPE, AU565, BT-20, BT-474, BT-483, BT-549, Evsa-T, Hs578T, MCF-7, MDA-MB-231, SkBr3, T-47D, HeLa, DU145, PC3, LNCaP, A549, H1299, NCI-H460, A2780, SKOV-3 / Luc, Neuro2a, RKO, RKO-AS45-1, HT-29, SW1417, SW948, DLD-1, SW480, Capan-1, MC / 9, B72.3, B25.2, B6.2, B38.1, DMS 153, SU.86.86, SNU-182, SNU-423, SNU-449, SNU-475, SNU-387, Hs 817.T, LMH, LMH / 2A, SNU-398, PLHC-1, HepG2 / SF, OCI-Lyl, OCI-Ly2, OCI-Ly3, OCI-Ly4, OCI-Ly6, OCI-Ly7, OCI-LylO, OCI-Lyl8, OCI- Lyl9, U2932, DB, HBL-1, RIVA, SUDHL2, TMD8, MEC1, MEC2, 8E5, CCRF-CEM, MOLT-3, TALL-104, AML-193, THP-1, BDCM, HL-60, Jurkat, RPMI 8226, MOLT-4, RS4, K-562, KASUMI- 1, Daudi, GA- 10, Raji, JeKo-1, NK-92, and Mino.

[0303] In some embodiments, the sample for use in the methods is from any tissue or fluid from an individual. Samples include, but are not limited to, tissue (e.g. connective tissue, muscle tissue, nervous tissue, or epithelial tissue), whole blood, dissociated bone marrow, bone marrow aspirate, pleural fluid, peritoneal fluid, central spinal fluid, abdominal fluid, pancreatic fluid, cerebrospinal fluid, brain fluid, ascites, pericardial fluid, urine, saliva, bronchial lavage, sweat, tears, ear flow, sputum, hydrocele fluid, semen, vaginal flow, milk, amniotic fluid, and secretions of respiratory, intestinal or genitourinary tract. In some embodiments, the sample is a tissue sample, such as a sample obtained from a biopsy or a tumor tissue sample. In some embodiments, the sample is a blood serum sample. In some embodiments, the sample is a blood cell sample containing one or more peripheral blood mononuclear cells (PBMCs). In some embodiments, the sample contains one or more circulating tumor cells (CTCs). In some embodiments, the sample contains one or more disseminated tumor cells (DTC, e.g., in a bone marrow aspirate sample).

[0304] In some embodiments, the samples are obtained from the individual by any suitable approach of obtaining the sample using well-known and routine clinical methods. Procedures for obtaining tissue samples from an individual are well known. For example, procedures for drawing and processing tissue sample such as from a needle aspiration biopsy is well-known and is employed to obtain a sample for use in the methods provided. Typically, for collection of such a tissue sample, a thin hollow needle is inserted into a mass such as a tumor mass for sampling of cells that, after being stained, will be examined under a microscope.

[0305] VIII. Sample Preparation and Analysis

[0306] In some embodiments, the sample (e.g., cell sample, cell lysate sample, or comprising isolated proteins) is a sample solution. In some instances, the sample solution comprises a solution such as a buffer (e.g. phosphate buffered saline) or a media. In some embodiments, the media is an isotopically labeled media. In some instances, the sample solution is a cell solution.

[0307] In some embodiments, the sample (e.g., cell sample, cell lysate sample, or comprising isolated proteins) is incubated with one or more compound probes for analysis of protein-probe interactions. In some instances, the sample (e.g., cell sample, cell lysate sample, or comprising isolated proteins) is further incubated in the presence of an additional compound probe prior to addition of the one or more probes. In other instances, the sample (e.g., cell sample, cell lysate sample, or comprising isolated proteins) is further incubated with a non-probe small molecule ligand, in which the non-probe small molecule ligand does not contain a photoreactive moiety and / or an alkyne group. In such instances, the sample is incubated with a probe and non-probe small molecule ligand for competitive protein profiling analysis.

[0308] In some cases, the sample is compared with a control. In some cases, a difference is observed between a set of probe protein interactions between the sample and the control. In some instances, the difference correlates to the interaction between the small molecule fragment and the proteins.

[0309] In some embodiments, one or more methods are utilized for labeling a sample (e.g. cell sample, cell lysate sample, or comprising isolated proteins) for analysis of probe protein interactions. In some instances, a method comprises labeling the sample (e.g. cell sample, cell lysate sample, or comprising isolated proteins) with an enriched media. In some cases, the sample (e.g. cell sample, cell lysate sample, or comprising isolated proteins) is labeled with isotope-labeled amino acids, such as13C or15N- labeled amino acids. In some cases, the labeled sample is further compared with a non-labeled sample to detect differences in probe protein interactions between the two samples. In some instances, this difference is a difference of a target protein and its interaction with a small molecule ligand in the labeled sample versus the non-labeled sample. In some instances, the difference is an increase, decrease or a lack of protein-probe interaction in the two samples. In some instances, the isotope-labeled method is termed SILAC, stable isotope labeling using amino acids in cell culture.

[0310] In some embodiments, a method comprises incubating a sample (e.g. cell sample, cell lysate sample, or comprising isolated proteins) with a labeling group (e.g., an isotopically labeled labeling group) to tag one or more proteins of interest for further analysis. In such cases, the detectable labeling group comprises a biotin, a streptavidin, bead, resin, a solid support, or a combination thereof, and further comprises a linker that is optionally isotopically labeled. As described above, the linker can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more residues in length and might further comprise a cleavage site, such as a protease cleavage site (e.g., TEV cleavage site). In some cases, the labeling group is a biotin-linker moiety, which is optionally isotopically labeled with13C and15N atoms at one or more amino acid residue positions within the linker. In some cases, the biotin-linker moiety is a isotopically- labeled TEV-tag.

[0311] In some embodiments, an isotopic reductive dimethylation (ReDi) method is utilized for processing a sample. In some cases, the ReDi labeling method involves reacting peptides with formaldehyde to form a Schiff base, which is then reduced by cyanoborohydride. This reaction dimethylates free amino groups on N-termini and lysine side chains and monomethylates N-terminal prolines. In some cases, the ReDi labeling method comprises methylating peptides from a first processed sample with a “light” label using reagents with hydrogen atoms in their natural isotopic distribution and peptides from a second processed sample with a “heavy” label using deuterated formaldehyde and cyanoborohydride. Subsequent proteomic analysis (e.g., mass spectrometry analysis) based on a relative peptide abundance between the heavy and light peptide version might be used for analysis of probe-protein interactions.

[0312] In some embodiments, isobaric tags for relative and absolute quantitation (iTRAQ) method is utilized for processing a sample. In some cases, the iTRAQ method is based on the covalent labeling of the N-terminus and side chain amines of peptides from a processed sample. In some cases, reagent such as 4-plex or 8-plex is used for labeling the peptides.

[0313] In some embodiments, the probe-protein complex is further conjugated to a chromophore, such as a fluorophore. In some instances, the probe-protein complex is separated and visualized utilizing an electrophoresis system, such as through a gel electrophoresis, or a capillary electrophoresis. Exemplary gel electrophoresis includes agarose based gels, polyacrylamide based gels, or starch based gels. In some instances, the probe-protein is subjected to a native electrophoresis condition. In some instances, the probe-protein is subjected to a denaturing electrophoresis condition.

[0314] In some instances, the probe-protein after harvesting is further fragmentized to generate protein fragments. In some instances, fragmentation is generated through mechanical stress, pressure, or chemical approach. In some instances, the protein from the probe-protein complexes is fragmented by a chemical approach. In some embodiments, the chemical approach is a protease. Exemplary proteases include, but are not limited to, serine proteases such as chymotrypsin A, penicillin G acylase precursor, dipeptidase E, DmpA aminopeptidase, subtilisin, prolyl oligopeptidase, D-Ala-D-Ala peptidase C, signal peptidase I, cytomegalovirus assemblin, Lon-A peptidase, peptidase Clp, Escherichia coli phage KIF endosialidase CIMCD self-cleaving protein, nucleoporin 145, lactoferrin, murein tetrapeptidase LD-carboxypeptidase, or rhomboid-1; threonine proteases such as ornithine acetyltransferase; cysteine proteases such as TEV protease, amidophosphoribosyltransferase precursor, gamma-glutamyl hydrolase (Rattus norvegicus), hedgehog protein, DmpA aminopeptidase, papain, bromelain, cathepsin K, calpain, caspase- 1, separase, adenain, pyroglutamyl -peptidase I, sortase A, hepatitis C virus peptidase 2, sindbis virus-type nsP2 peptidase, dipeptidyl -peptidase VI, or DeSI-1 peptidase; aspartate proteases such as beta-secretase 1 (BACE1), beta-secretase 2 (BACE2), cathepsin D, cathepsin E, chymosin, napsin-A, nepenthesin, pepsin, plasmepsin, presenilin, or renin; glutamic acid proteases such as AfuGprA; and metalloproteases such as peptidase_M48.

[0315] In some instances, the fragmentation is a random fragmentation. In some instances, the fragmentation generates specific lengths of protein fragments, or the shearing occurs at particular sequence of amino acid regions.

[0316] In some instances, the protein fragments are further analyzed by a proteomic method such as by liquid chromatography (LC) (e.g. high performance liquid chromatography), liquid chromatographymass spectrometry (LC-MS), matrix-assisted laser desorption / ionization (MALDI-TOF), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or nuclear magnetic resonance imaging (NMR).

[0317] In some embodiments, the LC method is any suitable LC methods well known in the art, for separation of a sample into its individual parts. This separation occurs based on the interaction of the sample with the mobile and stationary phases. Since there are many stationary / mobile phase combinations that are employed when separating a mixture, there are several different types of chromatography that are classified based on the physical states of those phases. In some embodiments, the LC is further classified as normal-phase chromatography, reverse-phase chromatography, sizeexclusion chromatography, ion-exchange chromatography, affinity chromatography, displacement chromatography, partition chromatography, flash chromatography, chiral chromatography, and aqueous normal-phase chromatography.

[0318] In some embodiments, the LC method is a high performance liquid chromatography (HPLC) method. In some embodiments, the HPLC method is further categorized as normal-phase chromatography, reverse-phase chromatography, size-exclusion chromatography, ion-exchange chromatography, affinity chromatography, displacement chromatography, partition chromatography, chiral chromatography, and aqueous normal-phase chromatography. In some embodiments, the HPLC method of the present disclosure is performed by any standard techniques well known in the art. Exemplary HPLC methods include hydrophilic interaction liquid chromatography (HILIC), electrostatic repulsion-hydrophilic interaction liquid chromatography (ERLIC) and reverse phase liquid chromatography (RPLC).

[0319] In some embodiments, the LC is coupled to a mass spectroscopy as a LC-MS method. In some embodiments, the LC-MS method includes ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS), ultra-performance liquid chromatography-electro spray ionization tandem mass spectrometry (UPLC-ESI-MS / MS), reverse phase liquid chromatography-mass spectrometry (RPLC-MS), hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS), hydrophilic interaction liquid chromatography-triple quadrupole tandem mass spectrometry (HILIC-QQQ), electrostatic repulsion-hydrophilic interaction liquid chromatography-mass spectrometry (ERLIC-MS), liquid chromatography time-of-flight mass spectrometry (LC-QTOF-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), multidimensional liquid chromatography coupled with tandem mass spectrometry (LC / LC-MS / MS). In some instances, the LC-MS method is LC / LC-MS / MS. In some embodiments, the LC-MS methods of the present disclosure are performed by standard techniques well known in the art.

[0320] In some embodiments, the GC is coupled to amass spectroscopy as a GC-MS method. In some embodiments, the GC-MS method includes two-dimensional gas chromatography time-of-flight mass spectrometry (GC*GC-TOFMS), gas chromatography time-of-flight mass spectrometry (GC-QTOF- MS) and gas chromatography-tandem mass spectrometry (GC-MS / MS).

[0321] In some embodiments, CE is coupled to a mass spectroscopy as a CE-MS method. In some embodiments, the CE-MS method includes capillary electrophoresis-negative electrospray ionizationmass spectrometry (CE-ESI-MS), capillary electrophoresis-negative electrospray ionizationquadrupole time of flight-mass spectrometry (CE-ESI-QTOF-MS) and capillary electrophoresisquadrupole time of flight-mass spectrometry (CE-QTOF-MS).

[0322] In some embodiments, the nuclear magnetic resonance (NMR) method is any suitable method well known in the art for the detection of one or more cysteine binding proteins or protein fragments disclosed herein. In some embodiments, the NMR method includes one dimensional (ID) NMR methods, two dimensional (2D) NMR methods, solid state NMR methods and NMR chromatography. Exemplary ID NMR methods include 'Hydrogen,13Carbon,15Nitrogen,17Oxygen,19Fluorine,31Phosphorus, "Potassium,23Sodium,33Sulfur,87Strontium,27Aluminium,43Calcium,35Chlorine,37Chlorine,63Copper, “Copper,57Iron, “Magnesium, '"Mercury or67Zinc NMR method, distortionless enhancement by polarization transfer (DEPT) method, attached proton test (APT) method and ID- incredible natural abundance double quantum transition experiment (INADEQUATE) method. Exemplary 2D NMR methods include correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), 2D-INADEQUATE, 2D-adequate double quantum transfer experiment (ADEQUATE), nuclear overhauser effect spectroscopy (NOSEY), rotating-frame NOE spectroscopy (ROESY), heteronuclear multiple -quantum correlation spectroscopy (HMQC), heteronuclear single quantum coherence spectroscopy (HSQC), short range coupling and long range coupling methods. Exemplary solid state NMR method include solid state13Carbon NMR, high resolution magic angle spinning (HR- MAS) and cross polarization magic angle spinning (CP-MAS) NMR methods. Exemplary NMR techniques include diffusion ordered spectroscopy (DOSY), DOSY-TOCSY and DOSY-HSQC.

[0323] In some embodiments, the protein fragments are analyzed by a method as previously described. See PCT International Publication No. WO 2020 / 214336 to Hsu et al., published October 22, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0324] In some embodiments, the results from the mass spectroscopy method are analyzed by an algorithm for protein identification. In some embodiments, the algorithm combines the results from the mass spectroscopy method with a protein sequence database for protein identification. In some embodiments, the algorithm comprises ProLuCID algorithm, Probity, Scaffold, SEQUEST, or Mascot.

[0325] In accordance with the presently disclosed subject matter, as described above or as discussed in the EXAMPLES below, there can be employed conventional chemical, cellular, histochemical, biochemical, molecular biology, microbiology, recombinant DNA, and clinical techniques which are known to those of skill in the art. Such techniques are explained fully in the literature. See for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Publications, Cold Spring Harbor, New York, United States of America; Glover (1985) DNA Cloning: A Practical Approach. Oxford Press, Oxford; Gait (1984) Oligonucleotide Synthesis: A Practical Approach. IRL Press, Oxford, England; Harlow & Lane (1988) Antibodies. A Laboratory Manual. Cold Spring Harbor Publications, New York; Roe et al. (1996) DNA Isolation and Sequencing: Essential Techniques. John Wiley, New York, New York, United States of America; and Ausubel et al. (2003) Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York, New York, United States of America.

[0326] EXAMPLES

[0327] The following EXAMPLES provide illustrative embodiments. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following EXAMPLES are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

[0328] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative EXAMPLES, make and utilize the compounds of the presently disclosed subject matter and practice the methods of the presently disclosed subject matter. The following EXAMPLES therefore particularly point out embodiments of the presently disclosed subject matter and are not to be construed as limiting in any way the remainder of the disclosure. Materials and Methods for the EXAMPLES

[0329] Mice. All studies were conducted in 6-12-week-old C57BL / 6J from the Jackson Laboratory. Mice were allowed free access to a standard chow diet and water under specific pathogen-free conditions in the animal facility of the Animal Resources Center (ARC) at the University of Texas at Austin. Animal studies were performed in compliance with National Institutes of Health (NIH) guidelines. Studies were approved by the Animal Care and Use Committee (ACUC) of the University of Texas at Austin.

[0330] Cell culture. All cell lines were cultured at 37 °C in a humidified atmosphere with 5% CO2 and procedures were conducted under aseptic conditions in a biosafety cabinet according to standard operating procedures. HEK293T, HepG2, A549, MDA-MB-231 and B16-F10-Luc2 cells were obtained from the American Type Culture Collection (ATCC) and cultured in complete DMEM media (10% FBS (U.S. Source, Omega Scientific) and 1% L-glutamine (Thermo Fisher Scientific)), penicillin (100 U / mL), streptomycin (100 pg / mL) in 10 cm2plates. Molml4 cells were cultured in RPMI-1640 medium (Sigma) containing 2 mM L-glutamine, supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific), and 25 mM HEPES. Human neuroblastoma cells SH-SY5Y were maintained in Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F12, Sigma) supplemented with 4 mM L-glutamine, 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific), and 25 mM HEPES. For starvation experiments, glucose was omitted from the medium to induce a state of nutrient deprivation. Prior to drug treatment, cells were re-supplied with 5 mM glucose (Sigma) to assess cellular responses under re-nourished conditions. The human monocyte cell line THP-1 and Jurkat cells were cultured in RPMI-1640 medium supplemented with 2.05 mM glutamine, 1% antibiotic-antimitotic (penicillin-streptomycin, Gibco), 10% heat-inactivated FBS. To differentiate THP-1 cells into macrophage -like cells, cells were treated with 100 ng / mL PMA in 10% FBS culture medium for 24 h and treated with RPMI-1640 medium containing 5% FBS for another 2 d. To activate M0 macrophages, macrophages were incubated with 5% FBS RPMI-1640 medium containing 100 ng / mL LPS for 4 h. All the cell lines were routinely tested negative for mycoplasma contamination.

[0331] In situ gel-based competitive activity-based protein profiling. HEK293T cells were grown to 90% confluence. Cells were carefully washed with Dulbecco’s phosphate -buffered saline (DPBS) and replenished with 10 mL of serum -free medium containing the DMSO vehicle or SuTEx ligands at the indicated concentrations, with the final concentration of DMSO at 0.1%. After incubation at 37 °C for 1 h in CO2 incubator, the medium was aspirated and 10 mL of probe TH211 (25 pM) in serum -free medium was added. After incubation at 37 °C for another 2 h, the cells were then harvested in cold DPBS by scraping. After centrifugation at 400 x g for 5 min, the cell pellets were washed with cold DPBS (2 times). Pellets were either directly processed or kept frozen at -80 °C until further use. The cell pellets were resuspended and lysed by sonication (1 sec pulse, 20% amplitude, 3 times) in DPBS in the presence of EDTA-free protease inhibitor cocktail tablet (Pierce). The cell lysates were subject to ultracentrifugation (100,000 x g, 45 min at 4 °C) to yield the cytosolic fraction in the supernatant and the insoluble fraction as a pellet. Protein concentrations were determined by the Bio-Rad DC protein assay. Proteome aliquots (2 mg / mL, 50 pL) were conjugated with fluorophore which was accomplished by copper-catalyzed azide-alkyne cycloaddition (CuAAC) with rhodamine-azide (TAMRA-azide, 1.25 mM, 1 pL, final concentration of 25 pM) in the presence oftris(2- carboxyethyljphosphine (TCEP, 50 mM fresh in water, 1 pL, final concentration of 1 mM), tris[(l- benzyl-lH-l,2,3-triazol-4- yl)methyl] amine (TBTA, 1.7 mM in 4: 1 / -butanol / DMSO. 3 pL, final concentration of 100 pM) and CuSO4(50 mM, 1 pL, final concentration of 1 mM). After incubation at room temperature (RT) for 1 h, reactions were quenched by adding 4X SDS-PAGE loading buffer with beta-mercaptoethanol (17 pL) and samples were resolved by SDS-PAGE and imaged by in-gel fluorescence scanning using a BioRad image system.

[0332] ADP-GLO™ Kinase Assay. A 250x stock solution was prepared in 100% DMSO. 2 pL ofthese dilutions was added to 198 pl assay buffer and mixed well by vortex. A visual inspection of these dilutions was carried out to check for solubility at 1% DMSO. The PFK reaction in the presence of inhibitor was carried out using 10 pL of 1 pg / mL PFKL (final concentration 0.4 pg / ml) which was added to 10 pL of the 1% DMSO compound in a white, non-binding 96-well plate. The protein / compound mixture was incubated for 30 min at RT. The kinase reaction was started by adding a 5 pL of 5x ATP / F6P stock in the assay buffer (final concentrations 0.5 mM F6P and 0. 1 mM ATP). Plates were then sealed, centrifuged at 600 x g for 1 min and incubated at RT for 30 min. 5 pL of ADP- GLO™ Reagent was then added to each well, mixed using a multichannel pipette and incubated at RT for 40 min. 10 pL of Kinase Detection Reagent was added and incubated for 50 min at RT. The luminescence was recorded using a CLARIOSTAR® multi-mode Plate Reader in single-point mode, with an integration time of 750 milliseconds.

[0333] Enzyme-linked immunosorbent assay. Supernatants from THP-1 macrophages were treated with different reagents for 2 h before stimulation with or without LPS for 4 h (n = 3 biologically independent samples). The supernatants were collected and stored at -80 °C for further measurements. The concentration of cytokines in the extracellular medium was measured using ELISA Duoset kits (R&D Systems) for human TNF-a and IL- 1 P according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader. Concentrations were calculated using the corresponding standard curve after accounting for the fourfold dilution of sample in the assay.

[0334] Cell viability assay. The cell viability assay was performed using the CELLTITER-GLO® Luminescent Cell Viability Assay (Promega, Catalog No.: G7572) according to the manufacturer’s protocol. Cells were seeded in white-opaque 96-well plates in 100 pL of full growth media at a density of 2, 000 ~ 5,000 cells per well and were incubated for 12 h at 37 °C in a humidified 5% CC>2 atmosphere. The cells were subsequently treated with the indicated compounds or DMSO (0.1% DMSO final for all wells) in four replicates and incubated at 37 °C in a humidified 5% CO2 atmosphere for 48 h. DMSO without drugs was used as a negative control to normalize the cell viability of drug treated wells. 100 pL of Cell Titer-Gio (Promega) reagent was added to each well. Microplates were incubated with Cell Titer Gio at RT for an additional 10 min to stabilize the luminescence signal. Cell viability was determined by the luminescence measured using the BioTek Synergy Hl microplate reader.

[0335] Recombinant protein expression and purification. Recombinant His-tagged human PFKL (Accession No. NP 002617 of the GENBANK® biosequence library) was expressed and purified as previously described (59). Briefly, PFKL cDNA was cloned into the pFastBac HTa vector and baculovirus generated using the Bac-to-Bac Expression system (ThermoFisher Scientific). PFKL was expressed in 2 x 106sf9 cells at an MOI of two for 48 h. Cells were pelleted and stored at -80 °C until puri fication. Cell pellets were lysed with 20 passes of a Dounce homogenizer after resuspension in lysis buffer (20 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.5; 80 mM potassium phosphate; 1 mM 2-mercapentoethanol; 10% glycerol; 10 mM imidazole) supplemented with EDTA- free Protease Inhibitor Cocktail (Abeam). Cell debris was pelleted by centrifugation and the lysate incubated with HisPur cobalt resin (ThermoFisher Scientific). The resin was washed with 5 bed volumes lysis buffer, 10 bed volumes lysis buffer containing 2 M NaCl, and 5 further bed volumes lysis buffer. PFKL was eluted in lysis buffer containing 100 mM imidazole. Fractions containing protein were pooled, passed over a desalting column equilibrated in freezing buffer (20 mM HEPES pH 7.5, 1 mM DTT, 500 pM ammonium sulfate, 5% glycerol, 1 mM ATP, and 100 pM EDTA) and concentrated using an Amicon Ultracel-SOK Centrifugal Filter Unit (MiiliporeSigma). Protein was quantified using a Bradford Protein Assay kit (ThermoFisher Scientific) and the integrity and purity of the protein determined by analysis of Coomassie-stamed SDS-PAGE gels. Aliquots of protein were frozen in liquid nitrogen and stored at -80 °C. PFKL point mutants N702T and R315K were generated as previously described (60,61).

[0336] Cytoplasmic FBP measurement using the FBP biosensor. SH-SY5Y and MOLM-14 cells were transduced with a lentiviral vector expressing a biosensor for fructose- 1,6-bisphosphate (FBP). After incubated with 5% CO2 at 37 °C for 48 h, the cells underwent selection with 1 pg / ml puromycin (ThermoFisher Scientific) to enrich populations stably expressing both the sensor and the resistance gene. Ultimately, cells expressing GFP were sorted using a flow cytometer, and populations demonstrating successful sensor expression were considered stable when GFP expression reached 90%.

[0337] For SH-SY5Y-sensor cells, seeding occurred at a density of I / 10scells per well in a 12-well plate pre-coated with 25 pg / ml poly-L-lysine (Sigma). After 24 h, the medium was switched to glucose- free DMEM to initiate a starvation environment. These cells were then maintained under starvation conditions for an additional 48 h. Subsequently, to evaluate the cellular response to XJ-4-85, these cells were exposed for 2 h to a medium containing 10 pM XJ-4-85, prepared by dissolving in DMSO and further diluting in DMEM supplemented with 5 mM glucose. In the control group, DMSO concentration was kept at 0.5%o to maintain solvent exposure consistency.

[0338] MOLM-14 cells stably expressing the sensor were cultured at a density of 3x l05cells / mL in T25 flasks under glucose-free RPMI 1640 medium for a 2-d starvation treatment. Post-starvation, 3 x 105cells were transferred to each well of a 12-well plate with a volume of 500 pl per well. An additional 500 pL of medium, containing either XJ-4-85 or XJ-4-97, was added to achieve a final concentration of 10 pM of the compound.

[0339] After a 2 h treatment with these compounds, the cells were harvested and analyzed using flow cytometry within 5min on the Novocyte 3000 VYB. Detection was performed using lasers 488-1 (excitation at 488 nm and emission at 530 / 30 nm) and 405-2 (excitation at 405 nm and emission at 525 / 50 nm), and gated a stop condition of 10,000 cells with FITC positive. Analysis was done on flowjo generating a derived value (488 / 405 ratio).

[0340] Confocal imaging of SH-SY5Y cells expressing Hylight sensor. SH-SY 5Y cells, which stably express the FBP sensor, were seeded at a density of Ix lO4cells per well in a 96-well plate and subsequently subjected to 2 d of starvation treatment. Prior to imaging, the cells underwent a 2 h treatment with either 10 pM XJ-4-85 or 0.5% DMSO. For each group, images were collected from eight distinct wells, and each condition was replicated three different times to ensure biological consistency. Images were captured on an Olympus IXplore SpinSR with a Yokogawa W2 spinning disk and used 100 mW lasers at 405 nm and 488 nm, a 10X Olympus objective, and a Photonics Prime 95B sCMOS camera. Ratiometric measurements were captured between 519 nm excited at 405 nm and 485 nm Method- 1 with an excitation wavelength of 495 nm and an emission wavelength of 519 nm, and Method-2 with an excitation wavelength of 405 nm and an emission wavelength of 519 nm. All images were analyzed using ImageJ software, with the average fluorescence intensity of the cells calculated for each frame.

[0341] Cryo-electron microscopy: Protein expression and purification. PFKL was codon optimized for expression in mammalian cells and synthesized commercially (Twist Bioscience). It was subcloned into the pcDNA3.1(+) vector with an N-terminal Twin-Strep tag and TEV cleavage site using Gibson assembly. PFKL was expressed in Expi293F cells (ThermoFisher). Expi293F cells were thawed, maintained, and passaged as described by the manufacturer. To transfect PFKL into cells, Expi293F cells in log phase were seeded in suspension culture at a density of 3 x 106live cells / mL. Plasmid DNA (1 mg DNA / mL culture) was diluted in Opti-MEM (ThermoFisher, 35 mL / mL culture). PEI-MAX (Polysciences), at a ratio of 1:3 (w / w) DNA: PEI MAX, was diluted in Opti-MEM (35 mL / mL culture) and mixed with the diluted DNA. The DNA-PEI MAX mixture was incubated for 10 min at RT before it was added dropwise to the cell culture. The cells were maintained in a humid incubator at 37 °C with 8% CO2 and 120 rpm. After 120 h, the cells were harvested, washed with cold PBS, and stored at -20 For purification, the cell pellet was thawed and resuspended in 50 mM HEPES pH 8, 50 mM potassium phosphate, 150 mM KC1, 2 mM DTT, 10% glycerol, 1% triton X and cOmplete EDTA-free protease inhibitor cocktail (Roche). The resuspended cell pellet was incubated on ice for 30 min. The suspension was then clarified by centrifugation at 32500 x g at 4 °C for 45 min. The clarified supernatant was collected and passed over a pre-equilibrated 1 mb Strep-Tactin XT 4Flow (IBA Life Sciences) gravity column. Once bound, the protein was washed with 5 column volumes (CV) of 50 mM HEPES pH 7.5, 50 mM potassium phosphate, 150 mM KC1, 2 mM DTT, 10% glycerol, 1% triton X. The resin was further washed with 5 CV of 50 mM HEPES pH 8, 50 mM potassium phosphate, 750 mM KC1, 2 mM DTT, 5% glycerol, then 5 CV of 50 mM HEPES pH 8.0, 50 mM potassium phosphate, 150 mM KC1, 2 mM DTT, 5% glycerol. The protein was eluted in 1 mb fractions with 50 mM HEPES pH 8, 50 mM potassium phosphate, 150 mM KC1, 50 mM biotin, 0.5 mM fructose-6-phosphate (F6P), 2 mM DTT, 5% glycerol. The elution fractions were assessed by SDS-PAGE and selected based on purity. Pure fractions were buffer exchanged into 20 mM HEPES pH 8.0, 150 mM KC1, 3% glycerol, 0.5 mM F6P, 2 mM DTT in a 50k MWCO Amicon centrifugal filter (Millipore Sigma). The buffer-exchanged protein was concentrated, filtered, and flash frozen in liquid N2. Protein concentration was determined using a Bradford assay with BSA as the standard. Sample quality was assessed by negative stain electron microscopy and mass photometry.

[0342] Cryo-electron microscopy: Sample preparation, data collection, and image processing. To prepare samples for cryoEM, 4.2 pM PFKL in 20 mM HEPES pH 8.0, 150 mM KC1, 3% glycerol, 500 pM F6P, and 2 mM DTT was combined with 25 pM XJ-4-85 and incubated for 30 min at RT prior to addition of 2 mM F6P and 1 mM ATP. Sample was applied to glow -discharged ANTcryo M04-Au300- 2.0 / 1.0 grids (SingleParticle) and blotted away four times sequentially, then plunged into liquid ethane using a Vitrobot (Thermofisher). Movies were acquired using a Glacios microscope (Thermofisher) operating at 200kV, equipped with a K3 Direct Detect camera operating in counting mode with a pixel size of 0.885 A / pixel, 99 frames, and a total dose of 60 electrons / A2. Data collection was automated with SerielEM. Data processing was performed using cryoSPARC, where movies were aligned, dose- weighted, and summed by patch motion correction, and CTF parameters were estimated using patch CTF. Particles were picked using the blob picker and subjected to 5 rounds of 2D classification. Particles from selected 2D classes were then refined by homogenous 3D refinement (Cl symmetry) followed by 3D classification with a focus mask encompassing a tetramer. Particles selected from high quality 3D classes were then subjected to non-uniform refinement (tetramer mask, D2 symmetry), symmetry expanded (D2), then subjected to 3D classification with a mask encompassing a monomer. Each monomer class was then refined by local refinement (monomer mask). A single monomer class with the best XJ-4-85 ligand density was selected for local refinement with a tetramer mask, to provide a view of the overall PFKL conformation in the XJ-4-85-bound state. Density modification was performed using ResolveCryoEM in Phenix. Atomic models were refined using ISOLDE, with ligands refined in both ISOLDE and Coot. A final round of real space refinement was performed in Phenix, with grid searches, Ramachandran restraints, and rotamer restraints disabled, and with starting model restraints enabled. Custom geometry restraints were used to restrain the sulfone bond of the K677- covalent moiety to approximately tetrahedral geometry. Ligand restraints for the XJ-4-85 leaving group were generated using eLBOW in Phenix. The cryoEM data processing workflow is summarized in Figure 38.

[0343] Seahorse assays. Seahorse XFp glycolysis stress test kit (Agilent Technologies, 103017-100) was used to measure glycolytic flux (extracellular acidification rate; ECAR) according to the manufacture’s protocol. Briefly, cells (2 x 104cells per well) were seeded in a Cell-Tak (Coming, 354240) coated XFp miniplates. At 1 h prior to analysis, the medium was replaced with Seahorse XF media (Agilent Technologies, 103681-100) and plates were incubated in a CCE-free incubator at 37 °C. Basal extracellular acidification rate (ECAR) was then analyzed, followed by ECAR measurements after sequential injections of 10 mM glucose, 1 mM oligomycin, and 50 mM 2-deoxyglucose.

[0344] Chemical proteomics. Cells were grown to 90% confluence for HEK293T, SH-SY 5Y and B 16- F10-Luc2 or until the cell density reached 2 x 107cells / mL for Jurkat, THP-1 macrophages and MOLM- 14. Cells were treated with SuTEx ligands at the indicated concentrations or DMSO in serum-free media for 1 h, followed by the treatment of 25 pM of TH211 for 2 h (with the final concentration of DMSO maintained at 0.1%). The cells were washed and harvested in cold Dulbecco’s phosphate-buffered saline by centrifugation at 400 x g for 5 min. The pellets after DPBS wash for a second time were either directly processed or snap-frozen using liquid nitrogen and stored at -80 °C for further use.

[0345] Cell pellets were resuspended and lysed in DPBS with protease inhibitor (10 mb per one tablet) by sonication at 4 °C and the protein concentrations were determined using the Bio-Rad DC protein assay. Protein concentration was adjusted to 2.3 mg / mL with lysis buffer and probe-modified proteomes (437 pL, 1 mg of proteome) were subjected to CuAAC conjugation to desthiobiotin-PEG3 -azide (10 pL of 10 mM stock in DMSO; final concentration of 200 pM) using TCEP (10 pL of fresh 50 mM stock in water, 1 mM final concentration), TBTA ligand (33 pL of a 1.7 mM 4: 1 / -butanol / DMSO stock, 100 pM final concentration) and CuSO4(10 pL of 50 mM stock, 1 mM final concentration). Samples were mixed by vortexing and then incubated for 1 h at RT. Excess click reagents were removed by chloroform-methanol extraction as previously described (63). Protein pellets were resuspended in 500 pL of 6 M urea: 25 mM ammonium bicarbonate and reduced using 10 mM dithiothreitol at 65 °C for 15 min followed by alkylation with 40 mM iodoacetamide at RT for 30 min in the dark. Next, proteins were precipitated with chloroform-methanol, and the protein pellet was resuspended in 500 pL of 25 mM ammonium bicarbonate and then digested overnight with trypsin / Lys-C (7.5 pg in 15 pL of25 mM ammonium bicarbonate) at 37 °C on a rotator. The resulting peptides were desalted by using Pierce Peptide Desalting Spin Columns. Peptide concentrations were normalized to 5 pg / pL in EPPS (pH 8.5) using the Pierce Quantitative Colorimetric Peptide Assay (Thermo #23275) according to Manufacturer’s instruction. 100 pg peptides from each sample were labelled by TMT6plex reagent (Thermo Fisher Scientific) following the SL-TMT protocol (5). 1 pL of each sample were pooled, desalted, and analyzed to check labeling efficiency. After labeling efficiency was verified, the reaction was quenched and acidified with 5% formic acid (pH reduced to ~2 - 3). TMT-labeled peptides were combined, and dried using a SpeedVac. Samples were reconstituted in 550 pL of DPBS and washed streptavidin beads were then added. The SuTEx-modified peptides were enriched for 1 h at RT with rotation and eluted using 150 pL of 50% ACN + 0.1% formic acid (3x). Enriched peptides were dried, desalted using in-house Cl 8 StageTips following standard procedure, and vacuum centrifuged to dryness. The resulting samples were resuspended in 25 pL of 0.1% formic acid and stored at -80 °C until analysis.

[0346] Mass spectrometry samples were analyzed on an Exploris 480 or Orbitrap Eclipse Tribrid Mass Spectrometer coupled with a Vanquish Neo UHPLC System (Thermo Fisher Scientific). Peptide separation were achieved on a 75-pm capillary column packed with 20 cm of C18 resin (3 pm, 120 A; YMC Co., Ltd.) using an 85 min or 120 min gradient of 4 - 50 % mobile phase B with a flow rate of 300 nL / min, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% acetonitrile. Eluted peptides were acquired by data-dependent acquisition (DDA) mode. In brief, MSI spectra were acquired in the scan range of 400 -1600 m / z at an orbitrap resolution of 120,000 with an AGC target set to 1 x 106and a maximum injection time of 20 ms. The top thirty precursors were then selected for MS / MS analysis. Determined charge states between 2 and 5 were required for sequencing, and an 80 s dynamic exclusion window was used with isotopes excluded. ForMS2 scans, HCD collision energy set to 36; normalized AGC target set to 50%; maximum injection time of 75 ms; isolation window of 0.7 Da; resolution set to 15,000 (Turbo); fixed first mass of 110 m / z.

[0347] Peptide search was performed in Proteome Discover 3.0 (Thermo Fisher Scientific) against UniProt human protein database or Uniprot mouse protein database with the following parameters: up to two missed cleavages, 10 ppm precursor mass tolerance, 20 ppm fragment mass tolerance and fully tryptic peptides were allowed with a minimum of 6 peptide length. Cysteine residues were searched with a static modification for carboxyamidomethylation (+57.02146 Da). SuTEx probe on tyrosine and lysine (+635.2737), TMT6plex on lysine and peptide N termini (+304.2071 Da) and oxidation of methionine (+15.9949 Da) as variable modifications were included. For the TMT reporter ion quantification, all the identified peptide spectral matches from the MS2 scans were extracted by an inhouse program and the reporter ion intensities were adjusted for impurity correction according to the manufacturer’s specifications. Peptides used for quantification met the following quality control criteria: PMI-Byonic score > 300, delta ppm err. 5, co-isolation threshold <50%, reporter ion S / N threshold > 10. Results were filtered to a peptide false discovery rate of 1%. Volcano plots were generated by grouping PSMs using the peptide isoform node with cutoffs of Log2 fold change > 1.0, and / -’-value < 0.05.

[0348] Phosphoproteomics . B16-F10-Luc2 cells were seeded in 15 cm2plates and treated with 5 pM of XJ-4-85 for 2 h upon the confluency reached around 90%. Cells were washed with ice-cold PBS and scraped into 8 M urea lysis buffer (8 M urea, 200 mM EPPS, pH 8.5) with EDTA-free protease / phosphatase inhibitors (Thermo #A32961). Cells were further probe-sonicated on ice. The protein concentrations were determined using the Bio-Rad DC protein assay and adjusted to 2 mg / mL. To 1.0 milligram of proteomes was added 0.5 pL of universal nuclease (Thermo#88700) and incubated at RT for 15 min. The proteomes were then subjected to disulfide bond reduction with 10 mM tris(2- carboxyethyljphosphine hydrochloride (TCEP) at RT for 30 min followed by alkylation with 20 mM iodoacetamide for 30 min at RT in the dark. Excess iodoacetamide was quenched with dithiothreitol (DTT, 10 mM) at RT for 15 min. Proteins were precipitated by chloroform -methanol precipitation. The resulting protein pellet was resuspended in 500 pL of 25 mM ammonium bicarbonate and then digested with trypsin / Lys-C (7.5 pg in 15 pL of 25 mM ammonium bicarbonate) at 37 °C overnight on a rotator. The resulting peptides were desalted by using Pierce Peptide Desalting Spin Columns. Peptide concentrations were normalized to 5 pg / pL in EPPS (pH 8.5) using the Pierce Quantitative Colorimetric Peptide Assay (Thermo #23275) according to Manufacturer’s instruction. 100 pg peptides from each sample were labelled by TMT6plex reagent (Thermo Fisher Scientific) following the SL-TMT protocol (63). The labeled peptides were acidified with 10% formic acid (pH reduced to ~2 - 3), pulled together, and dried down in a SpeedVac. TMT-labeled samples were subsequently desalted by using Pierce Peptide Desalting Spin Columns. Ten percent of the resulting peptides were used for protein abundance analysis. The rest were subjected to phosphopeptide enrichment sequentially using the High-Select TiCF (Thermo Scientific; #A32993) and Fe-NTA (Thermo Scientific; #A32992) Phosphopeptide Enrichment Kits in accordance with the manufacturer’s instructions. The resulting phosphopeptides were resuspended with 25 pL 0. 1% formic acid before LC-MS / MS analysis.

[0349] For the LC-MS / MS analysis, 5 pL of resuspended peptides were separated on an in-house packed 75-pm capillary column with 20 cm of C18 resin (3 pm, 120 A; YMC Co., Ltd.) using an 140 min gradient of 4 - 45% mobile phase B with a flow rate of 300 nL / min, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% ACN. Peptide separations were achieved on an THERMO SCIENTIFIC™ ORBITRAP ECLIPSE™ TRIBRID™ Mass Spectrometer operating in data-dependent acquisition (DDA) mode. For MSI spectra, resolution for the precursor scan (m / z 400 - 1600) was set to 120,000 with a normalized AGC target set to 250% and a maximum injection time of 50 ms. The 30 most intense ions were selected for MS / MS analysis. Determined charge states between 2 and 5 were required for sequencing, and an 80 s dynamic exclusion window was used with isotopes excluded. For MS2 scans, HCD collision energy set to 36; normalized AGC target set to 50%; maximum injection time of 86 ms; isolation window of 0.7 Da; resolution set to 50,000; fixed first mass of 110 m / z. For data analysis, THERMO SCIENTIFIC™ PROTEOME DISCOVERER™ 3.1 software was used with a precursor mass tolerance of 10 ppm and MS / MS m / z tolerance of 0.02 Da. Carbamidomethylation (+57.021464 Da) on cysteine was used as a fixed modification with methionine oxidation (+15.994915 Da), TMT tags on peptide N terminus / lysine residues (+229.162932 Da) and phosphorylation (+79.966331 Da, T, Y, S) as variable modifications with phosphoRS for site localization. Data was searched against a Uniprot mouse protein database with 1% FDR criteria using Percolator.

[0350] Untargeted Metabolomic analysis. Metabolites were extracted and measured according to a previously reported protocol (64,65). Jurkat cells (1 million) were treated with DMSO vehicle, XJ-4- 85 (5 pM) or XJ-4-97 (5 pM) for 2 h. B10-F10-Luc2 cells in 10 cm plates at ~80% confluence were treated with DMSO, XJ-4-85 (5 pM) or XJ-4-97 (5 pM) for 2 h. At the time of collection, cells washed with ice-cold saline solution (Saline: 2F7123 Baxter 0.9% Sodium Chloride Irrigation, USP), lysed with an 80:20 ratio of methanol to water and quickly scraped into an Eppendorf tube, followed by three freeze-thaw cycles between liquid nitrogen and 37 °C. The insoluble material was pelleted in a cooled centrifuge (4 °C), and the metabolite-containing supernatant was transferred to a new tube. The protein concentration of the supernatant was determined, and 10 pg of protein was transferred to a new Eppendorf tube and evaporated until dry using a SpeedVac concentrator (Eppendorf). Metabolites were reconstituted in 100 pL acetonitrile / water 80:20 (vol / vol) for a final concentration of [0.1 mg / mL] of protein, mixed by vortexing rigorously for 1 min and centrifuged at 20,000 x g for 15 min in a refrigerated centrifuge to remove debris. The metabolite -containing supernatant was transferred to an LC-MS vial (with insert) and capped for LC-MS / MS analysis. Chromatographic separation was achieved on a Thermo Scientific (Bremen, Germany) Vanquish Flex liquid chromatography system equipped with a Millipore-Sigma (St. Louis, MO) ZIC-pHILIC column. Metabolite mass spectra were acquired on a Thermo Scientific Orbitrap Exploris 480 mass spectrometer collecting both precursor and product ion spectra. Metabolites were identified with CompoundDiscoverer 3.3 (Thermo Scientific) equipped with an in-house spectral library generated from purified chemical standards, or previously characterized metabolites from biological extracts. Spectra were also searched against the publicly available mzCloud database (Thermo Scientific). Our method compares spectra collect from experimental samples to precursor and product ion spectra from our library or mzCloud. All metabolites were identified with a 5 ppm mass tolerance for precursor ions and a 10 ppm tolerance for product ions. All data were reviewed by an analytical scientist to confirm IDs generated by the software. The normalized areas were used as variables for the multivariate and univariate statistical data analysis. All multivariate analyses and modelling on the normalized data were carried out using MetaboAnalyst v.5.0 (http: / / www.metaboanalyst.ca). Univariate statistical differences in the metabolites between two groups were analyzed using a two-tailed Student’s / -test. Animal studies. C57BL / 6 mice (male, 18 ± 20 g, 6-8 weeks old) were purchased from Jackson Laboratory. Mice were housed in an animal facility of the University of Texas at Austin under constant environmental conditions (RT, 21 ± 1 °C; relative humidity, 40-70%; a 12 h light-dark cycle). To establish the B16-F10-Luc2 tumor-bearing model, mice were shaved at the injection site, and 200 pL of 3.0 x 105B16-F10-Luc2 melanoma cells in serum -free DMEM medium were subcutaneously injected into the flanks of male C57BL / 6 mice (8-10 weeks old, average mice weight: 28 g) with a 29 G needle. In all mouse experiments, age-matched and weight-matched mice were randomized to each group to eliminate age and weight differences. All animal experiments were approved by the Animal Resources Center of the University of Texas at Austin. When the tumor volume of B16-F10-Luc2 tumor-bearing mice reached approximately 50 mm3in size (around one week), the mice were administered intraperitoneally with 100 pL of vehicle (sterile saline : PEG40 Castor oil : 100% ethanol solution = 18 : 1 : 1), XJ-4-85, XJ-4-97, XJ-4-119 at indicated dose every day. Tumor dimensions were measured daily with a digital caliper, and tumor volume was calculated using the formula (length x width2x 0.5), where length represents the largest tumor diameter and width represents the perpendicular tumor diameter. For in vivo bioluminescent assays, a fresh stock solution of luciferin was prepared at 15 mg / mL in DPBS and sterilized through a 0.2 pm fdter. Each mouse was intraperitoneally (i.p.) injected with 100 pL of luciferin solution (15 mg / mL) 10 minutes before imaging. The imaging was then collected on an Xenogen IVIS Spectrum system. During imaging, mice were under isoflurane anesthesia on a bed maintained at physiological temperature. The imaging results were analyzed using Living Image 4.1. Mice weight was recorded daily to monitor potential drug toxicity. Mice were sacrificed when tumors reached 1500 mm3or upon ulceration / bleeding.

[0351] Chemical Synthesis. All chemical reagents and solvents were obtained from commercial suppliers such as Sigma Aldrich, Ambeeds, Alfa Aesar, Combi-Blocks, and used as supplied without further purification unless stated otherwise. Analytical thin layer chromatography (TLC) was performed on Merck Silica gel 60 F254 plates (0.25 mm). Flash column chromatography was accomplished with Silica Gel 60 (230- 400 mesh) purchased from Fisher Scientific. Detection was accomplished by using UV-light (254 nm). Nuclear magnetic resonance (NMR) spectra (’H and13C) were recorded on a Varian spectrometer at 600 MHz at RT. Chemical shifts were provided in parts per million (ppm) with coupling constants in Hz. ’H and13C spectra were calibrated in relation to deuterated solvents, namely CDCL (7.26 ppm for 'H and 77. 16 ppm for13C) and CD3COCD3(2.05 ppm for 'H, 29.84 and 206.26 ppm for13C). Splitting patterns for apparent multiplets were indicated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broadened) as well as combinations of them. High resolution mass spectrometry was obtained with an Agilent 6545B LC / Q-TOF (Agilent Technologies, Santa Clara, CA, USA). Purity of all final products was greater than 95% as determined by analytical high performance liquid chromatography (HPLC) with a Shimadzu 1100 Series spectrometer detector. General synthetic procedures for preparation of terminal alkynes.

[0352] Method A: The synthetic procedure was performed following previously described methods (Turner et al., 2003). To a solution of secondary amines (1.0 equiv., 10.0 mmol) in DMF (50 mL) was added K2CO3 (3.0 equiv., 4.15 g, 30.0 mmol) and corresponding p-toluene sulfonates (1.2 equiv., 12.0 mmol) sequentially. The reaction mixture was heated to 60 °C and stirred for 6 h. Upon completion, which was indicated by TLC, the reaction solution was concentrated under reduced pressure. And the residue was dissolved with ethyl acetate (200 mL) and washed with brine (50 mL x 3). The combined organic layers were dried over Na2SC>4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to yield alkynes as a yellowish oil.

[0353] Method B: To a solution of secondary amines (1.0 equiv., 10.0 mmol) in DMF (50 mL) was added K2CO3 (3.0 equiv., 4.15 g, 30.0 mmol), potassium iodide (2.0 equiv., 3.32 g, 20.0 mmol) and 6- chloro-1 -hexyne (1.1 equiv., 1.83 g, 11.0 mmol) sequentially. The reaction mixture was heated to 60 °C and stirred for 6 h. Upon completion, which was indicated by TLC, the reaction solution was concentrated under reduced pressure. And the residue was dissolved with ethyl acetate (200 mL) and washed with brine (50 mL x 3). The combined organic layers were dried over Na2SC>4, fdtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to yield alkynes as a yellowish oil.

[0354] Method C: A solution of 4-pentynoic acid (1.0 equiv., 2.0 g, 20.4 mmol) and 200 pL of DMF (cat.) in CH2C12 (20 mL) was cooled (0° C) and oxalyl chloride (1.2 equiv., 2.1 mL, 24.5 mmol) was added dropwise. The reaction mixture was warmed to RT and stirred for 2 h. Excess solvent and oxalyl chloride were removed in vacuo, and the dark red residue was redissolved in 20 mL of CH2C12 . This solution was added to a pre-cooled (0° C) solution of RF001 (1.1 equiv., 6.4 g, 22.4 mmol) and DIPEA (2.0 equiv., 7.0 mL, 40.8 mmol). The reaction mixture was warmed to RT and stirred for 2 h. The reaction was poured into a saturated sodium bicarbonate aqueous solution, and the aqueous layer was extracted with CH2C12. The combined organic layers were washed with brine, dried over Na2SO4and concentrated under reduced pressure. The crude product was purified by column chromatography to afford the alkynes.

[0355] EXAMPLE 1

[0356] Synthesis of SuTEx Compounds

[0357] SuTEx compounds with bis((4-fluorophenyl)methylene)piperidine-based or bis((4- fluorophenyl)methyl)piperazine-based leaving groups were prepared via routes analogous to those described previously for compounds such as TH211. See PCT International Patent Application Publication No. 2023 / 023664, the disclosure of which is incorporated herein by reference in its entirety. For example, the compounds can be prepared using via reactions between alkynes and azides to form sulfonyl -triazoles. See Scheme 3, above. The alkyne 4-((Bis(4-fluorophenyl)methylene)-l-but-3-yn-l-yl)piperidine was prepared as previously described. See PCT International Patent Application Publication No. WO 2020 / 214336 and U.S. Patent Application Publication No. 2022 / 0214355, the disclosures ofwhich are incorporated herein by reference in their entireties. Additional substituted alkynes were prepared as follows:

[0358] Representative Procedure for the Preparation of substituted alkynes. To a solution of piperidine or piperazine (1.0 mmol) in anhydrous DMF was added potassium carbonate, potassium iodide (2.0 mmol) and the corresponding halogenated alkynes (1.2 mmol). The resulting mixture was heated to 60 °C for 6 h. Once the starting material was fully consumed, the reaction solution was diluted with ethyl acetate (100 mb) and the organic layer was washed with brine (3 x 50 mb) and then dried over anhydrous DMSO. The crude was purified by column chromatography (20 to 60% EtOAc in hexane) to provide the desired substituted alkynes as a yellowish oil.

[0359] 4-(Bis(4-fluorophenyl)methylene)-l-(pent-4-yn-l-yl)piperidine (XJ-3-7)

[0360] Method A, light yellowish oil, 72% yield, 2.5 g, Rf = 0.20 (hexane / EtOAc=3: 1, UV detection on TLC plate). ' H NMR (800 MHz, CDC1A 5 7.09 - 7.02 (m, 4H), 6.99 - 6.94 (m, 4H), 2.56 - 2.43 (m, 6H), 2.37 (t, J= 5.7 Hz, 4H), 2.24 (td, J= 7.1, 2.6 Hz, 2H), 1.94 (t, J= 2.6 Hz, 1H), 1.74 (q, J= 7.4 Hz, 2H).13CNMR (201 MHZ, CDC1A 5 161.66. 160.44, 137.72, 137.70, 135.89, 133.31, 130.86, 130.82, 114.54, 114.44, 83.67, 68.02, 56.78, 54.76, 31.15, 25.46, 16.05.19F NMR (564 MHz, CDC1A 5 -119.29. HRMS (ESI): Calcd for C23H23F2N [M+H]+: 352.1871, found: 352.1868.

[0361] 4-(Bis(4-fluorophenyl)methylene)-l-(hept-6-yn-l-yl)piperidine (XJ-3-61)

[0362] Method A, yellowish oil, 65% yield, 2.5 g, Rf = 0.15 (hexane / EtOAc=4: 1, UV detection on TLC plate).1H NMR (800 MHz, Acctonc< / „) 5 7.19 - 7.13 (m, 4H), 7.10 - 7.05 (m, 4H), 2.45 (t, J= 5.6 Hz, 4H), 2.34 - 2.29 (m, 7H), 2.17 (td, J = 7.1, 2.7 Hz, 2H), 1.55 - 1.50 (m, 2H), 1.51 - 1.46 (m, 2H), 1.47 - 1.40 (m, 2H).13C NMR (200 MHz, Acctonc< / „) 5 161.59, 160.38, 138.20, 138.18, 136.63, 132.88, 131.00, 130.96, 114.35, 114.24, 83.59, 68.46, 68.42, 57.46, 54.54, 31.18, 27.90, 26.02, 25.97, 17.36.19F NMR (564 MHz, Acetone- J6) 5 -117.97. HRMS (ESI): Calcd for C25H27F2N [M+H]+: 380.2184, found: 380.2180.

[0363] 4-(Bis(4-fluorophenyl)methylene)-l-(hex-5-yn-l-yl)piperidine (XJ-3-69)

[0364] Method B, yellowish oil, 52% yield, 1.9 g, Rf = 0.30 (hexane / EtOAc=3: l, UV detection on TLC plate).1H NMR (800 MHz, Acctonc- 5 7.18 - 7.13 (m, 4H), 7.09 - 7.04 (m, 4H), 2.45 (t, J= 5.6 Hz, 4H), 2.35 - 2.28 (m, 7H), 2.20 - 2.17 (m, 2H), 1.60 - 1.57 (m, 2H), 1.56 - 1.52 (m, 2H).13C NMR (201 MHz, Acetone-J6) 5 161.59, 160.38, 138.19, 138.17, 136.58, 132.91, 131.00, 130.96, 114.35, 114.25, 83.61, 68.52, 56.90, 54.51, 31.16, 25.87, 25.47, 17.27.19F NMR (564 MHz, Acetone- J6) 5 -117.97. HRMS (ESI): Calcd for C24H25F2N [M+H]+: 366.2028, found: 366.2045. l-(Hex-5-yn-l-yl)piperidine (XJ-3-155)

[0365] XJ-3-155

[0366] ’H NMR (800 MHz, Acctonc-t / ,.) 52.37 - 2.26 (m, 4H), 2.25 - 2.22 (m, 2H), 2.20 - 2.15 (m, 2H), 2.08 - 2.02 (m, 1H), 1.62 - 1.46 (m, 8H), 1.43 - 1.33 (m, 2H).13C NMR (201 MHz, Acctonc-t / ,.) 5 83.65, 68.44, 57.84, 53.97, 25.90, 25.53, 25.38, 24.02, 17.27. HRMS (ESI): Calcd for C11H19N [M+H]+: 166.1596, found: 166.1598. l-(4-(bis(4-fluorophenyl)methylene)piperidin-l-yl)pent-4-yn-l-one (XJ-3-19)

[0367] A solution of the 3-butynoic acid or 4-pentynoic acid (1 equiv.) and 200 uL of DMF (cat.) in CH2C12 (20 mL) was cooled (0° C) and oxalyl chloride (1.2 equiv., 2.2 mL) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 2 h. Excess solvent and oxalyl chloride were removed in vacuo, and the dark red residue was redissolved in 20 mL of CH2C12. This solution was added to a pre-cooled (0° C) solution ofRFOOl (1.1 equiv., 6.4 g) and DIPEA (1.1 equiv., 3.9 mL). The reaction mixture was warmed to room temperature and stirred for 2 h. The reaction was poured into a saturated NaHCO3(aq) solution and the aqueous layer was extracted with CH2C12. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography with elution system (hexane s / EtOAc=3: 1) to afford the amide.

[0368] Method C, white solid, 72% yield, 5.9 g, Rf = 0.25 (hexane / EtOAc=3: 1, UV detection on TLC plate). ' H NMR (800 MHz, CPC13) 5 7.16 - 7.12 (m, 4H), 7.11 - 7.06 (m, 4H), 3.74 (t, J= 5.9 Hz, 2H), 3.59 - 3.56 (m, 2H), 2.2.71 - 2.66 (m, 2H), 2.65 - 2.63 (m, 2H), 2.48 - 2.46 (m, 2H), 2.43 (t, J= 5.9 Hz, 2H), 2.06 (t, J= 2.6 Hz, 1H).13C NMR (201 MHz, CPC13) 5 168.84, 161.84, 160.61, 137.24, 137.22, 137.05, 137.03, 135.48, 133.61, 130.70, 130.66, 114.79, 114.78, 114.69, 114.67, 83.10, 68.25, 45.95, 42.62, 31.81, 31.53, 30.70, 14.10.19F NMR (564 MHz, CPC13) 5 -118.49. HRMS (ESI): Calcd for C23H2IF2NO [M+H]+: 388.1483, found: 388.1489.

[0369] 4-(Bis(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)methylene)-l-(hex-5-yn-l-yl)piperidine (XJ-4-29)

[0370] ’H NMR (800 MHz, Acctonc-d.) 5 7.33 (d, J = 8.2 Hz, 2H), 7.18 (d, J = 1.5 Hz, 2H), 7.10 (ddd, J = 8.2, 1.7, 0.7 Hz, 2H), 2.55 (t, J= 5.6 Hz, 5H), 2.43 - 2.40 (m, 4H), 2.40 - 2.38 (m, 1H), 2.31 - 2.25(m, 2H), 2.14 (p, J= 2.2 Hz, 1H), 1.70 - 1.65 (m, 2H), 1.65 - 1.59 (m, 2H). 13C NMR (201 MHz, Acetonede) 8 204.75, 142.88, 141.65, 138.27, 138.20, 132.44, 132.26, 131.18, 129.93, 125.09, 110.62, 108.95, 83.59, 68.52, 56.82, 54.30, 31.19, 25.85, 25.45, 17.25. HRMS (ESI): Calcd for C26H23F4NO4[M+H]+: 490.1641, found: 490.1621.

[0371] (4-Fluorophenyl)(l-(hex-5-yn-l-yl)piperidin-4-yl)methanone (XJ-4-79) ’H NMR (800 MHz, Acctonc-t / ,.) 5 8.20 - 8.15 (m, 2H), 7.38 - 7.32 (m, 2H), 3.55 - 3.37 (m, 1H), 3.08 - 3.00 (m, 2H), 2.45 - 2.40 (m, 2H), 2.39 - 2.37 (m, 1H), 2.33 - 2.25 (m, 2H), 2.20 - 2.09 (m, 2H), 1.94 - 1.87 (m, 2H), 1.83 - 1.74 (m, 2H), 1.70 - 1.65 (m, 2H), 1.64 - 1.60 (m, 2H).13C NMR (201 MHz, Acetone-Je) 5 204.77, 200.17, 165.61, 164.36, 130.62, 130.57, 115.12, 115.01, 83.63, 68.51, 57.34, 52.58, 42.98, 25.86, 25.39, 17.27. 19F NMR (564 MHz, Acctonc-t / ,.) 5 -108.23. HRMS (ESI): Calcd for CI8HI2FNO [M+H]+: 288.1764, found: 288.18761.

[0372] Representative Procedure for the Preparation of Sulfonyl 1,2.3-triazoles. To a stirred mixture of alkynes (6.0 mmol), sulfonyl azide (5.0 mmol), and Cui (0.25 mmol) in chloroform (20 mL) was slowly added 2,6-lutidine (6.0 mmol) at 0 °C. After stirring the reaction mixture for 12 h at 0 °C, it was diluted by adding CH2CI2 (20 mL) and aqueous NH4CI solution (50 mL). The mixture was stirred for an additional 30 min and two layers were separated. The aqueous layer was extracted with CH2Q2 (50 mL x 3). The combined organic layers were dried over anhydrous sulfate, and filtered. Then the organic layers were concentrated in vacuo and the crude residue was purified by column chromatography with elution system to provide the desired compounds.

[0373] 4-(Bis(4-fluorophenyl)methylene)-l-(3-(l-((4-methoxyphenyl)sulfonyl)-lH-l,2,3-triazol-4- yl)propyl)piperidine (XJ-3-9)

[0374] ’H NMR (800 MHz, CDCh) 5 8.10 - 7.94 (m, 2H), 7.85 (s, 1H), 7.05 - 7.02 (m, 4H), 7.02 - 7.00 (m, 2H), 6.98 - 6.94 (m, 4H), 3.87 (s, 3H), 2.74 (t, J= 7.6 Hz, 2H), 2.44 (t, J= 5.7 Hz, 4H), 2.40 - 2.36 (m, 2H), 2.33 (t, J= 5.7 Hz, 4H), 1.86 (p, J= 7.6 Hz, 2H).13C NMR (201 MHz, CDCh) 5 164.78, 161.65, 160.43, 147.21, 137.72, 137.70, 135.85, 133.29, 130.86, 130.82, 130.65, 126.75, 119.82, 114.54, 114.43, 56.99, 55.46, 54.69, 31.20, 25.85, 22.89.19F NMR (564 MHz, CDCh) 5 -119.21. HRMS (ESI): Calcd for C30H30F2N4O3S [M+H]+: 565.2085, found: 565.2082.

[0375] 4-(Bis(4-fluorophenyl)methylene)-l-(3-(l-tosyl-LH-l,2,3-triazol-4-yl)propyl)piperidine (XJ-3-17)

[0376] 1H NMR (800 MHz, CDC13) 5 7.99 - 7.95 (m, 2H), 7.86 (s, 1H), 7.37 (d, J= 8. 1 Hz, 2H), 7.06 - 7.02 (m, 4H), 6.98 - 6.94 (m, 4H), 2.75 (t, J= 7.6 Hz, 2H), 2.48 - 2.42 (m, 7H), 2.41 - 2.36 (m, 2H), 2.36 - 2.32 (m, 4H), 1.93 - 1.81 (m, 2H).13C NMR (200 MHz, CDC13) 5 161.66, 160.44, 147.28, 146.67, 137.68, 132.80, 130.85, 130.81, 129.92, 128.15, 119.97, 114.55, 114.44, 56.96, 54.68, 31.16, 25.79, 22.87, 21.37.19F NMR (564 MHz, CDCh) 5 -119.20. HRMS (ESI): Calcd for C30H30F2N4O2S [M+H]+: 549.2136, found: 549.2130.

[0377] 4-(Bis(4-fluorophenyl)methylene)-l-(3-(l-((2,3-dihydrobenzo[Z>] [l,4]dioxin-6-yl)sulfon-yl)-EH- l,2,3-triazol-4-yl)propyl)piperidine (XJ-3-51)

[0378] ’H NMR (800 MHz, CDCh) 5 7.84 (s, 1H), 7.60 - 7.56 (m, 2H), 7.06 - 7.02 (m, 4H), 7.00 - 6.93 (m, 5H), 4.33 - 4.31 (m, 2H), 4.28 - 4.26 (m, 2H), 2.75 (t, J= 7.6 Hz, 2H), 2.53 - 2.24 (m, 10H), 1.88 (p, J = 7.7 Hz, 2H).13C NMR (200 MHz, CDCh) 5 161.66, 160.44, 149.55, 147.17, 143.54, 137.68, 137.66, 130.85, 130.81, 127.42, 122.13, 119.95, 117.99, 117.79, 114.55, 114.45, 64.25, 63.57, 56.94, 54.65, 31.10, 25.73, 22.86.19F NMR (564 MHz, CDCh) 5 -119.18. HRMS (ESI): Calcd for C31H30F2N4O4S [M+H]+: 593.2033, found: 593.2030.

[0379] 4-(Bis(4-fluorophenyl)methylene)-l-(4-(l-((4-methoxyphenyl)sulfonyl)-lH-l,2,3-triazol-4- yl)butyl)piperidine (XJ-3-41)

[0380] 'H NMR (800 MHz, Acctonc-t / ,.) 5 8.29 (s, 1H), 8.10 - 8.04 (m, 2H), 7.22 - 7.18 (m, 2H), 7.18 - 7.13 (m, 4H), 7.10 - 7.04 (m, 4H), 3.93 (s, 3H), 2.75 - 2.64 (m, 2H), 2.42 (t, J= 5.5 Hz, 4H), 2.33 (t, J= 7.2 Hz, 2H), 2.30 (t, J= 5.6 Hz, 4H), 1.75 - 1.67 (m, 2H), 1.51 (p, J= 7.3 Hz, 2H).

[0381] 13C NMR (200 MHz, Acetone- d6) 5 204.84, 164.98, 161.59, 160.38, 147.64, 138.18, 138.16, 136.52, 132.93, 131.02, 130.98, 130.42, 126.96, 120.58, 114.79, 114.39, 114.29, 57.11, 55.24, 54.50, 31.13, 26.19, 25.87, 24.41.19F NMR (564 MHz, CDCh) 5 -119.17. HRMS (ESI): Calcd for C31H32F2N4O3 [M+H]+: 579.2241, found: 579.2246. 4-(Bis(4-fluorophenyl)methylene)-l-(4-(l-tosyl-l / / -l,2,3-triazol-4-yl)butyl)piperidine (XJ-3-45)

[0382] ’H NMR (800 MHz, CDC13) 5 7.99 - 7.97 (m, 2H), 7.86 (s, 1H), 7.39 - 7.35 (m, 2H), 7.07 - 7.03 (m, 4H), 7.00 - 6.94 (m, 4H), 2.79 - 2.71 (m, 2H), 2.46 (t, J= 5.3 Hz, 4H), 2.44 (s, 3H), 2.37 (q, J= 5.3 Hz, 6H), 1.69 (p, J = 7.7 Hz, 2H), 1.60 - 1.52 (m, 2H).13C NMR (200 MHz, CDCh) 5 161.66, 160.44,

[0383] 147.43, 146.65, 137.69, 137.68, 135.76, 133.34, 132.81, 130.86, 130.82, 129.92, 128.15, 119.87, 114.54,

[0384] 114.44, 57.61, 54.76, 31.09, 26.48, 25.99, 24.82, 21.35.19F NMR (564 MHz, CDCh) 5 -119.19. HRMS (ESI): Calcd for C31H32F2N4O2S [M+H]+: 563.2292, found: 563.2288.

[0385] 4-(Bis(4-fluorophenyl)methylene)-l-(5-(l-((4-methoxyphenyl)sulfonyl)-EH-l,2,3-triazol-4- yl)pentyl)piperidine (

[0386] 'H NMR (800 MHz, Acctonc-t / ,.) 5 8.29 (s, 1H), 8.08 - 8.04 (m, 2H), 7.22 - 7.19 (m, 2H), 7.18 - 7.13 (m, 4H), 7.10 - 7.04 (m, 4H), 3.93 (s, 3H), 2.72 - 2.66 (m, 2H), 2.46 - 2.39 (m, 4H), 2.32 - 2.25 (m, 6H), 1.71 - 1.63 (m, 2H), 1.53 - 1.44 (m, 2H), 1.40 - 1.32 (m, 2H).13C NMR (200 MHz, Acctonc-t / ,.) 5 204.91, 164.98, 161.60, 160.38, 147.64, 138.17, 138.16, 136.52, 132.95, 131.04, 131.00, 130.44, 126.96, 120.58, 114.81, 114.41, 114.31, 57.37, 55.27, 54.52, 31.13, 28.17, 26.24, 26.07, 24.52.19F NMR (564 MHz, CDCh) 5 - 119.21. HRMS (ESI) : Calcd for C32H34F2N4O3S [M+H]+: 593.2398, found: 593.2393. l-(4-(l-(Benzo[t / ] [l,3]dioxol-5-ylsulfonyl)-EH-l,2,3-triazol-4-yl)butyl)-4-(bis(4-fluo- rophenyl)methylene)piperidine (XJ-3-71) ’H NMR (800 MHz, Acctonc-t / ,.) 5 8.30 (s, 1H), 7.75 - 7.72 (m, 1H), 7.51 - 7.45 (m, 1H), 7.18 - 7.43 (m, 4H), 7.12 - 7.09 (m, 1H), 7.09 - 7.05 (m, 4H), 6.23 (s, 2H), 2.84 - 2.59 (m, 2H), 2.59 - 2.38 (m, 4H), 2.34 - 2.31 (m, 2H), 2.32 - 2.28 (m, 4H), 1.73 - 1.67 (m, 2H), 1.54 - 1.48 (m, 2H).13C NMR (200 MHz, Acetone-Je) 5 161.60, 160.38, 153.78, 148.57, 147.70, 138.18, 138.16, 136.51, 132.93, 131.02, 131.00, 130.98, 130.96, 128.49, 124.87, 120.72, 114.36, 114.25, 108.33, 106.95, 103.15, 57.08, 54.49, 31.11, 26.15, 25.84, 24.37.19F NMR (564 MHz, Acctonc-t / ,.) 5 -117.92. HRMS (ESI): Calcd for C31H30F2N4O4S [M+H]+: 593.2034, found: 593.2031.

[0387] 4-(Bis(4-fluorophenyl)methylene)-l-(4-(l-((2,3-dihydrobenzofuran-5-yl)sulfonyl)-EH-l,2,3- triazol-4-yl)butyl)piperidine

[0388] 'H NMR (800 MHz, Acctonc-t / ,.) 5 8.27 (s, 1H), 7.95 - 7.93 (m, 1H), 7.93 - 7.89 (m, 1H), 7.19 - 7.12 (m, 4H), 7.10 - 7.03 (m, 4H), 6.98 - 6.92 (m, 1H), 4.76 - 4.70 (m, 2H), 3.37 - 3.29 (m, 2H), 2.76 - 2.67 (m, 2H), 2.44 - 2.37 (m, 4H), 2.32 (t, J= 6.9 Hz, 2H), 2.31 - 2.26 (m, 4H), 1.73 - 1.66 (m, 2H), 1.54 - 1.47 (m, 2H).13C NMR (200 MHz, Acctonc-t / ,.) 5 166.03, 161.59, 160.38, 147.54, 138.18, 138.17, 136.56, 132.90, 131.00, 130.96, 130.16, 130.00, 126.68, 125.34, 120.49, 114.36, 114.25, 109.47, 72.64, 57.10, 54.50, 31.14, 27.89, 26.19, 25.87, 24.38.

[0389] 19F NMR (564 MHz, Acetone-^) 5 -117.95. HRMS (ESI): Calcd for C32H32F2N4O3S [M+H]+: 591.2241, found: 591.2240.

[0390] 4-(Bis(4-fluorophenyl)methylene)-l-(4-(l-((2,3-dihydrobenzo[Z>] [l,4]dioxin-6-yl)sulfo-nyl)-EH- l,2,3-triazol-4-yl)butyl)piperidine (XJ-3-109)

[0391] ’HNMR (800 MHz, Acetone- d6) 5 8.40 (s, 1H), 7.69 (dd, J= 8.7, 2.4 Hz, 1H), 7.61 (d, J = 2.4 Hz, 1H), 7.28 - 7.22 (m, 4H), 7.20 - 7.13 (m, 5H), 4.51 - 4.48 (m, 2H), 4.46 - 4.43 (m, 2H), 2.82 (t, J= 7.6 Hz, 2H), 2.51 (t, J = 5.6 Hz, 4H), 2.41 (t, J= 7.2 Hz, 2H), 2.40 - 2.35 (m, 4H), 1.80 (p, J = 7.6 Hz, 2H), 1.63 - 1.55 (m, J= 7.9, 7.3 Hz, 2H).13CNMR(200 MHz, Acctonc-t / ,.) 5 161.59, 160.38, 149.88, 147.68, 143.85, 138.17, 136.56, 132.90, 131.01, 130.97, 130.94, 127.58, 121.75, 120.71, 117.96, 116.86, 114.36, 114.26, 64.42, 63.77, 57.10, 54.51, 31.14, 31.12, 26.16, 25.87, 24.38.19F NMR (564 MHz, Acctonc-t / ,.) 5 -117.93. HRMS (ESI): Calcd for C32H32F2N4O4S [M+H]+: 607.2191, found: 607.2191. 4-(Bis(4-fluorophenyl)methylene)-l-(4-(l-((4-(difluoromethoxy)phenyl)sulfonyl)-l / / -l ,2,3- triazol-4-yl)butyl)piperidine (XJ-3-113)

[0392] ’H NMR (800 MHz, Acctonc-t / ,.) 5 8.35 (s, 1H), 8.22 (dt, J = 9.2, 2.7 Hz, 2H), 7.52 - 7.43 (m, 2H), 7.27 - 7.22 (m, 1H), 7.19 - 7.13 (m, 4H), 7.09 - 7.04 (m, 4H), 2.73 (t, J = 7.6, 2H), 2.46 - 2.38 (m, 4H), 2.35 - 2.31 (m, 2H), 2.30 - 2.26 (m, 4H), 1.75 - 1.65 (m, 2H), 1.55 - 1.45 (m, 2H).13C NMR (200 MHz, Acetone-Je) 5 161.59, 160.38, 156.42, 156.41, 156.39, 147.90, 138.18, 138.16, 136.52, 132.92, 131.97, 130.99, 130.95, 130.58, 128.31, 120.85, 118.81, 118.20, 116.63, 115.34, 114.35, 114.25, 114.05, 57.07, 54.50, 31.12, 26.12, 25.86, 24.34.19F NMR (564 MHz, Acctonc-t / ,.) 5 -85.28 (d, J= 76.8 Hz), - 117.95. HRMS (ESI): Calcd for C31H30F4N4O3 [M+H]+: 615.2053, found: 615.2047.

[0393] 6-((4-(4-(4-(Bis(4-fluorophenyl)methylene)piperidin-l-yl)butyl)-EH-l,2,3-triazol-l-yl)sulfonyl)- 2 / 7-chromen-2-one (XJ-3-115)

[0394] ’H NMR (800 MHz, Acctonc-t / ,.) 5 8.54 (d, J= 2.4 Hz, 1H), 8.37 (s, 1H), 8.28 (dd, J= 8.9, 2.4 Hz, 1H), 8.19 (d, J= 9.8 Hz, 1H), 7.60 (d, J= 8.8 Hz, 1H), 7.17 - 7.13 (m, 4H), 7.11 - 7.03 (m, 4H), 6.62 (d, J = 9.7 Hz, 1H), 2.73 (t, J= 7.6 Hz, 2H), 2.42 (t, J= 5.7 Hz, 4H), 2.32 (t, J= 7.1 Hz, 2H), 2.29 (t, J= 5.6 Hz, 4H), 1.70 (p, J = 7.6 Hz, 2H), 1.51 (p, J = 7.2 Hz, 2H).13C NMR (200 MHz, Acctonc-t / ,.) 5 161.59, 160.38, 157.95, 157.83, 147.94, 142.03, 138.17, 138.16, 136.50, 132.93, 131.54, 130.99, 130.95, 130.45, 129.17, 120.92, 119.57, 118.15, 118.10, 114.36, 114.25, 57.06, 54.49, 31.12, 26.11, 25.85, 24.34.19F NMR (564 MHz, Acctonc-t / ,.) 5 -117.94. HRMS (ESI): Calcd for C33H30F2N4O4S [M+H]+: 617.2034, found: 617.2032. 5-((4-(4-(4-(Bis(4-fluorophenyl)methylene)piperidin-l-yl)butyl)-LH-l,2,3-triazol-l-yl)su-lfonyl)-

[0395] JV^V-dimethylnaphthalen-l-amine (XJ-3-119)

[0396] ’H NMR (800 MHz, Acctonc-t / ,.) 5 8.83 (dt, J= 8.6, 1.1 Hz, 1H), 8.71 (dd, J= 7.4, 1.3 Hz, 1H), 8.58 (s, 1H), 8.45 (dt, J= 8.7, 1.0 Hz, 1H), 7.86 (t, J= 8.0 Hz, 1H), 7.72 (t, J= 8.6 Hz, 1H), 7.36 (d, J= 7.6 Hz, 1H), 7.26 - 7.21 (m, 4H), 7.19 - 7.14 (m, 4H), 2.94 (s, 6H), 2.79 (t, J= 7.5 Hz, 2H), 2.47 - 2.42 (m, 4H), 2.38 - 2.32 (m, 4H), 2.14 (p, J= 2.2 Hz, 2H), 1.76 (p, J = 7.6 Hz, 2H), 1.53 (p, J = 7.3 Hz, 2H).13C NMR (200 MHz, Acctonc-t / ,.) 5 161.60, 160.39, 152.00, 147.51, 138.19, 138.18, 136.54, 133.31, 132.89, 131.65, 131.12, 131.01, 130.97, 129.27, 129.16, 128.99, 123.12, 121.13, 117.18, 115.53, 114.36, 114.26, 57.04, 54.45, 44.21, 31.12, 26.03, 25.68, 24.28.19F NMR (564 MHz, Acctonc-t / ,.) 5 - 117.90. HRMS (ESI): Calcd for C36H37F2N5O2S [M+H]+: 642.2706, found: 642.2706.

[0397] 5-((4-(4-(4-(bis(4-fluorophenyl)methylene)piperidin-l-yl)butyl)-lH-l,2,3-triazol-l-yl)su-lfonyl)- 2-(difluoromethoxy)pyridine (XJ-3-129)

[0398] XJ-3-129

[0399] ’H NMR (800 MHz, Acctonc-t / ,.) 5 8.80 (s, 1H), 8.44 (dd, J = 8.6, 2.5 Hz, 1H), 8.21 - 8.11 (m, 1H), 7.87 - 7.64 (m, 1H), 7.33 - 7.25 (m, 5H), 7.23 - 7.13 (m, 5H), 3.34 (t, J= 5.8 Hz, 2H), 3.16 - 3.09 (m, 2H), 2.81 - 2.66 (m, 2H), 2.33 (t, J= 7.0 Hz, 2H), 1.88 - 1.83 (m, 2H), 1.70 - 1.63 (m, 2H), 1.48 - 1.42 (m, 2H).13C NMR (201 MHZ, Acctonc-t / ,.) 5 161.88, 161.85, 161.82, 160.67, 160.63, 160.61, 146.11, 146.09, 139.32, 139.31, 137.40, 135.50, 130.89, 130.85, 114.05 (t, J = 254.9 Hz), 114.50, 114.05, 110.29, 56.06, 53.24, 37.34, 28.45, 25.35, 23.42.19F NMR (564 MHz, Acctonc-t / ,.) 5 -89.56, -116.91. HRMS (ESI): Calcd for C30H29F4N5O3S [M+H]+: 616.2005, found: 616.2004. 7-((4-(4-(4-(Bis(4-fluorophenyl)methylene)piperidin-l-yl)butyl)-l / / -l ,2,3-triazol-l-yl)sulfonyl)-

[0400] 2,3-dihydro-[l,4]dioxino[2,3-Z>]pyridine (XJ-3-131)

[0401] 'H NMR (800 MHz, Acctonc-d.) 5 8.46 - 8.44 (m, 1H), 8.37 (s, 1H), 7.78 - 7.75 (m, 1H), 7.19 - 7.13 (m, 4H), 7.11 - 7.02 (m, 4H), 4.77 - 4.44 (m, 2H), 4.44 - 4.36 (m, 5H), 2.76 - 2.71 (m, 2H), 2.45 (t, J = 5.6 Hz, 4H), 2.35 (t, J= 7.2 Hz, 2H), 2.31 (t, J= 5.6 Hz, 4H), 2.06 - 2.02 (m, 2H), 1.76 - 1.65 (m, 2H), 1.53 (p, J = 7.3 Hz, 2H).13C NMR (201 MHz, Acctonc-d.) 5 161.60, 160.39, 156.19, 147.89, 139.75, 139.43, 138.16, 138.14, 137.33, 136.38, 132.99, 131.00, 130.96, 126.37, 122.39, 120.89, 114.37, 114.26, 65.30, 63.51, 57.06, 54.46, 31.05, 26.09, 25.79, 24.36.19F NMR (564 MHz, Acetone-d6) 5 - 117.93. HRMS (ESI): Calcd for C31H31F2N5O4 [M+H]+: 608.2135, found: 608.2131.

[0402] 5-((4-(4-(4-(Bis(4-fluorophenyl)methylene)piperidin-l-yl)butyl)-EH-l,2,3-triazol-l-yl)sulfonyl)-

[0403] 2,4-dimethylthiazole (XJ-3-133)

[0404] ’H NMR (800 MHz, Acetone-d6) 5 8.38 (s, 1H), 7.18 - 7.13 (m, 4H), 7.10 - 7.04 (m, 4H), 2.76 (t, J = 7.6 Hz, 2H), 2.72 (s, 3H), 2.66 (s, 3H), 2.43 (t, J= 5.5 Hz, 4H), 2.34 (t, J= 7.2 Hz, 2H), 2.30 (t, J= 5.6 Hz, 4H), 1.73 (p, J= 7.3 Hz, 2H), 1.53 (p, J= 7.3 Hz, 2H).13C NMR (200 MHz, Acetone-d6) 5 173.70, 161.60, 161.03, 160.38, 147.88, 138.18, 138.16, 136.52, 132.92, 131.00, 130.96, 124.41, 120.78, 120.66, 114.35, 114.25, 57.06, 54.50, 31.13, 26.10, 25.81, 24.33, 18.35, 15.61.19F NMR (564 MHz, Acetonede) 5 -117.95. HRMS (ESI): Calcd for C29H31F2N5O2S2 [M+H]+: 584.1965, found: 584.1963.

[0405] 5-((4-(4-(4-(Bis(4-fluorophenyl)methylene)piperidin-l-yl)butyl)-EH-l,2,3-triazol-l- yl)sulfonyl)benzo[d|thiazole (XJ-3-135) ’H NMR (800 MHz, Acctonc-t / ,.) 5 9.63 (s, 1H), 9.08 (d, J= 2.0 Hz, 1H), 8.39 (s, 1H), 8.36 - 8.31 (m, 1H), 8.25 - 8.19 (m, 1H), 7.18 - 7.12 (m, 4H), 7.11 - 7.02 (m, 4H), 2.72 (t, J= 7.6 Hz, 2H), 2.41 - 2.37 (m, 4H), 2.31 (t, J= 7.1 Hz, 2H), 2.28 (t, J= 5.6 Hz, 4H), 1.69 (p, J= 7.7 Hz, 2H), 1.50 (p, J= 7.3 Hz, 2H).13C NMR (200 MHz, Acctonc-t / ,.) 5 161.60, 161.41, 160.38, 157.04, 147.90, 138.16, 138.15, 136.42, 134.73, 132.95, 132.60, 131.00, 130.96, 124.81, 124.24, 124.14, 120.96, 114.37, 114.26, 57.04, 54.45, 31.06, 26.07, 25.77, 24.33.19F NMR (564 MHz, Acctonc-t / ,.) 5 -117.91. HRMS (ESI): Calcd for C31H29F2N3O2S2 [M+H]+: 606.1800, found: 606.1803.

[0406] 8-((4-(4-(4-(Bis(4-fluorophenyl)methylene)piperidin-l-yl)butyl)-LH-l,2,3-triazol-l- yl)sulfonyl)quinoline (XJ-3-137)

[0407] ’H NMR (800 MHz, Acctonc-t / ,.) 5 9.00 (dd, J= 4.2, 1.7 Hz, 1H), 8.78 (dd, J= 7.4, 1.4 Hz, 1H), 8.68 (s, 1H), 8.52 (dd, J= 8.3, 1.7 Hz, 1H), 8.47 (dd, J= 8.2, 1.4 Hz, 1H), 7.96 - 7.89 (m, 1H), 7.67 (dd, J = 8.3, 4.2 Hz, 1H), 7.19 - 7.14 (m, 4H), 7.12 - 7.06 (m, 4H), 2.76 (t, J= 7.3 Hz, 2H), 2.40 (t, J= 5.6 Hz, 4H), 2.33 (t, J = 7.2 Hz, 2H), 2.31 - 2.26 (m, 4H), 1.77 - 1.69 (m, 2H), 1.52 - 1.44 (m, 2H).13C NMR(200 MHz, Acetone-Je) 5 161.60, 160.39, 151.56, 145.88, 142.75, 138.19, 138.18, 136.56, 133.17, 132.90, 132.78, 131.02, 130.98, 128.61, 125.32, 123.84, 122.60, 114.37, 114.27, 57.11, 54.51, 31.15, 26.18, 25.62, 24.22. HRMS (ESI): Calcd for C33H31F2N5O2S [M+H]+: 600.2236, found: 600.2236.

[0408] 4-Butyl-l-((4-methoxyphenyl)sulfonyl)-LH-l,2,3-triazole (XJ-4-5)

[0409] 'HNMR (800 MHz, Acctonc-t / ,.) 5 8.37 (s, 1H), 8.18 - 8.10 (m, 2H), 7.32 - 7.23 (m, 2H), 4.03 (s, 3H), 2.80 - 2.73 (m, 2H), 1.75 - 1.66 (m, 2H), 1.48 - 1.29 (m, 2H), 1.01 - 0.93 (m, 2H).13C NMR (201 MHz, Acetone-Je) 5 164.97, 147.73, 130.45, 126.91, 120.58, 114.81, 55.32, 30.47, 24.29, 21.52, 12.81. HRMS (ESI): Calcd for C13H17N3O3S [M+H]+: 296.1069, found: 296.1063. l-(4-(l-((4-Methoxyphenyl)sulfonyl)-l / f-l,2,3-triazol-4-yl)butyl)piperidine (XJ-4-7)

[0410] 'HNMR (800 MHz, Acctonc-t / ,.) 5 8.58 (s, 1H), 8.24 - 8.16 (m, 2H), 7.35 - 7.26 (m, 2H), 4.03 (s, 3H), 3.63 - 3.34 (m, 2H), 3.07 (brs, 2H), 2.97 - 2.64 (m, 4H), 2.33 - 2.18 (m, 2H), 2.05 - 1.96 (m, 2H), 1.91 - 1.71 (m, 4H), 1.55 (brs, 2H).13C NMR (201 MHz, Acctonc-t / ,.) 5 164.97, 146.86, 130.57, 126.98, 121.26, 114.83, 55.52, 55.22, 51.73, 25.40, 23.79, 21.89, 21.52. HRMS (ESI): Calcd for C18H26N4O3S [M+H]+: 379.1804, found: 379.1797.

[0411] 5-((4-(4-(4-(Bis(4-fluorophenyl)methylene)piperidin-l-yl)butyl)-lH-l,2,3-triazol-l-yl)sul-fonyl)-

[0412] 2-methoxypyridine (

[0413] 'HNMR (800 MHz, Acctonc-t / ,.) 5 8.99 (s, 1H), 8.44 (s, 1H), 8.39 - 8.35 (m, 1H), 7.27 - 7.20 (m, 4H), 7.18 - 7.12 (m, 4H), 7.12 - 7.06 (m, 1H), 4.08 (dt, J= 5.5, 2.9 Hz, 2H), 2.83 (q, J= 6.4 Hz, 2H), 2.50 (q, J= 5.1 Hz, 4H), 2.39 (dq, J= 10.5, 5.5 Hz, 4H), 1.80 (h, J= 7.6 Hz, 2H), 1.60 (h, J= 7.1 Hz, 2H).13C NMR (201 MHZ, Acctonc-t / ,.) 5 167.71, 161.59, 160.38, 148.87, 147.89, 138.17, 138.16, 137.96, 136.52, 132.93, 131.01, 130.97, 125.62, 120.73, 114.38, 114.27, 111.81, 57.09, 54.51, 53.87, 31.13, 26.13, 25.85, 24.39.19F NMR (564 MHz, Acctonc-t / ,.) 5 -117.52. HRMS (ESI): Calcd for C30H31F2N5O3S [M+H]+: 580.2194, found: 580.2192.

[0414] 4-(Bis(4-fluorophenyl)methylene)-l-(4-(l-(propylsulfonyl)-lH-l,2,3-triazol-4-yl)butyl)- piperidine (XJ-4-27) ' H NMR (800 MHz, Acctonc-d.) 5 8.30 (s, 1H), 7.27 - 7.23 (m, 4H), 7.18 - 7.13 (m, 4H), 3.90 - 3.82 (m, 2H), 2.88 (t, J= 7.6 Hz, 2H), 2.53 (t, J= 5.7 Hz, 4H), 2.44 (t, J= 7.2 Hz, 2H), 2.42 - 2.32 (m, 4H), 1.89 - 1.74 (m, 4H), 1.64 (p, J = 7.4 Hz, 2H), 1.10 (t, J= 7.5 Hz, 3H). 13C NMR (201 MHz, Acetonede) 5 161.59, 160.38, 147.37, 138.18, 138.17, 136.57, 132.91, 131.00, 130.96, 121.31, 114.36, 114.25, 57.12, 55.69, 54.52, 31.16, 26.24, 25.84, 24.35, 16.29, 11.14.19F NMR (564 MHz, Acctonc-d.) 5 - 117.65. HRMS (ESI): Calcd for C27H32F2N4O2 [M+H]+: 515.2292, found: 515.2291.

[0415] 5-((4-(4-(4-(Bis(2,2-difluorobenzo[d] [l,3]dioxol-5-yl)methylene)piperidin-l-yl)butyl)-EH-l,2,3- triazol-l-yl)sulfonyl)-2-(difluoromethoxy)pyridine (XJ-4-31)

[0416] ’H NMR (800 MHz, Acetone-d6) 5 8.74 (s, 1H), 8.38 (dd, J = 8.6, 2.5 Hz, 1H), 7.95 - 7.56 (m, 2H), 7.24 (d, J= 8.2 Hz, 2H), 7.22 (dd, J= 8.6, 0.7 Hz, 1H), 7.09 (d, J= 1.7 Hz, 2H), 7.01 (dd, J= 8.3, 1.7 Hz, 2H), 2.92 - 2.87 (m, 2H), 2.85 - 2.73 (m, 2H), 2.48 (t, J= 5.6 Hz, 2H), 2.39 - 2.30 (m, 2H), 1.80 - 1.73 (m, 2H), 1.53 (p, J= 7.5 Hz, 2H), 1.47 - 1.40 (m, 2H), 1.28 (t, J= 7.1 Hz, 2H).13C NMR (201 MHz, Acetone-de) 5 176.35, 145.43, 142.88, 141.65, 138.66, 138.28, 138.20, 135.53, 132.44, 132.26, 131.18, 129.93, 125.09, 115.25, 113.99, 112.71, 111.21, 110.62, 108.95, 64.38, 57.17, 54.34, 33.41, 31.20, 26.37, 25.97, 25.32, 12.51.19F NMR (564 MHz, Acetone-d6) 5 -51.05, -89.91. HRMS (ESI): Calcd for C32H27F6N5O7S [M+H]+: 740.1614, found: 740.1614.

[0417] 5-((4-(4-(4-(Bis(benzo[d] [l,3]dioxol-5-yl)methylene)piperidin-l-yl)butyl)-EH-l,2,3-triaz-ol-l- yl)sulfonyl)-2-(difluoromethoxy)pyridine (XJ-4-39)

[0418] ' H NMR (800 MHz, Acetone-d6) 5 8.75 (s, 1H), 8.38 (dd, J = 8.7, 2.5 Hz, 1H), 7.84 - 7.57 (m, 2H), 7.22 (dd, J= 8.6, 0.8 Hz, 2H), 6.79 (d, J= 7.9 Hz, 2H), 6.62 (dd, J= 7.9, 1.6 Hz, 2H), 6.59 (d, J= 1.7 Hz, 2H), 5.96 (s, 4H), 4.22 (d, J= 7.1 Hz, 2H), 3.02 - 2.83 (m, 2H), 2.45 (t, J= 5.6 Hz, 2H), 2.33 (dd, J = 6.3, 5.0 Hz, 2H), 1.86 - 1.70 (m, 2H), 1.53 (p, J= 7.4 Hz, 2H), 1.46 - 1.39 (m, 2H), 1.30 - 1.25 (m, 2H).13C NMR (201 MHz, Acctonc-d.) 5 176.36, 146.97, 145.68, 145.44, 138.66, 136.11, 135.54,

[0419] 135.36, 134.20, 122.34, 115.26, 113.99 (t, J= 255.7 Hz), 109.37, 107.21, 100.54, 64.37, 64.36, 57.32, 54.65, 33.41, 31.27, 26.40, 26.00, 25.33, 12.51.19F NMR (564 MHz, Acetone-d6) 5 -89.90. HRMS (ESI): Calcd for C32H3iF2N5O7S [M+H]+: 668.1991, found: 668.1990. l-(4-(l-(Benzo[d] [l,3]dioxol-5-ylsulfonyl)-EH-l,2,3-triazol-4-yl)butyl)-4-(bis(3,4-difluor- ophenyl)methylene)piperidine (XJ-4-85)

[0420] 'HNMR (800 MHz, Acetone-d6) 5 8.39 (d, J= 0.9 Hz, 1H), 7.83 (dd, J= 8.4, 2.0 Hz, 1H), 7.56 (d, J= 2.0 Hz, 1H), 7.36 (dt, J= 10.7, 8.4 Hz, 2H), 7.21 - 7.16 (m, 3H), 7.10 - 7.07 (m, 2H), 6.32 (d, J= 0.7 Hz, 2H), 2.82 (t, J= 7.6 Hz, 3H), 2.52 (t, J= 5.6 Hz, 6H), 2.42 (t, J= 7.2 Hz, 3H), 2.41 - 2.37 (m, 6H),

[0421] I.83 - 1.74 (m, 3H), 1.64 - 1.56 (m, 3H).13C NMR (201 MHz, Acetone-d6) 5 153.78, 149.89 (d, J = 13.0 Hz), 149.12 (d, J = 12.6 Hz), 148.66 (d, J = 12.7 Hz), 147.90 (d, J = 12.5 Hz), 138.76, 138.74, 138.72, 138.50, 131.11, 128.48,13C 149.89 (d, J = 13.0 Hz), 149.12 (d, J = 12.6 Hz), 148.66 (d, J = 12.7 Hz), 147.90 (d, J= 12.5 Hz), 125.85 - 125.92 (m), 124.88 (d, J= 6.5 Hz), 120.71 (d, J= 13.9 Hz), 117.99 (dd, J= 16.7, 12.8 Hz), 116.60 (dd, J= 17.2, 13.1 Hz), 108.33 (d, J= 12.6 Hz), 106.96 (d, J =

[0422] I I.8 Hz), 103.49 - 102.46 (m), 56.99, 54.25, 31.10, 26.15, 25.85, 24.37.19F NMR (564 MHz, Acetonede) 5 -140.00, -142.52. HRMS (ESI): Calcd for C31H28F4N4O4 [M+H]+: 629.1846, found: 629.1846.

[0423] (l-(4-(l-(Benzo[d] [l,3]dioxol-5-ylsulfonyl)-l / 7-l,2,3-triazol-4-yl)butyl)piperidin-4-yl)(4- fluorophenyl)methanone (

[0424] ’H NMR (800 MHz, Acetone-d6) 5 8.39 (s, 1H), 7.84 - 7.80 (m, 1H), 7.61 - 7.48 (m, 2H), 7.27 - 7.20 (m, 2H), 7.19 - 7. 11 (m, 3H), 6.32 (s, 2H), 2.85 - 2.76 (m, 2H), 2.51 (m, 2H), 2.44 - 2.40 (m, 2H), 2.40

[0425] - 2.36 (m, 5H), 1.83 - 1.76 (m, 2H), 1.64 - 1.56 (m, 2H).13C NMR (201 MHz, Acctonc-d.) 5 161.59, 160.38, 153.78, 148.56, 147.70, 138.18, 138.16, 136.50, 132.93, 131.59 - 130.45 (m), 128.48, 131.59

[0426] - 130.45 (m), 124.87 (d, J= 6.7 Hz), 120.72 (d, J= 14.6 Hz), 114.87 - 113.22 (m), 108.34 (d, J= 14.0 Hz), 106.96 (d, J = 13.2 Hz), 103.15, 57.08, 54.49, 31.11, 26.15, 25.84, 24.38.19F NMR (564 MHz, Acctonc-7.) 5 -117.61. HRMS (ESI): Calcd for CAIEYFN^S [M+H]+: 515.1764, found: 515.1761. (4-Fluorophenyl)((2S,5R)-4-(hex-5-yn-l-yl)-2,5-dimethylpiperazin-l-yl)methanone (XJ-4-95)

[0427] Synthetic procedures for the intermediates XJ-4-91 and XJ-4-92: To a solution of tert-butyl (2R,5S)- 2,5-dimethylpiperazine-l-carboxylate (1.0 equiv., 9.0 g, 42.0 mmol) in dichloromethane (100 mL) was added 4-fluorobenzoyl chloride (1.2 equiv., 8.0 g, 50.4 mmol) and triethylamine (2.0 equiv., 11.7 mL, 84.0 mmol), respectively. The resulting solution was stirred at RT for 1 h. Once completion, the reaction solution was concentrated under reduced pressure. The residue was diluted with ethyl acetate and washed with brine. The organic layers were dried over anhydrous sodium sulfate, fdtered and concentrated under reduced pressure. The crude was purified by column chromatography to give XJ- 4-91 (85% yield). To a solution of XJ-4-91 (12.0 g) in dichloromethane (100 mL) was added 50 mL of trifluoroacetic acid (TFA). The resulting mixture was stirred at RT overnight. Upon completion, the reaction solution was concentrated under reduced pressure. The residue was dissolved with ethyl acetate (200 mL) and washed with sodium hydroxide solution (4 mol / L) and brine, respectively. The organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was resuspended in dichloromethane followed by addition of hexane. The white precipitate was filtered and dried to provide XJ-4-92. tert-butyl (2R,5S)-4-(4-fluorobenzoyl)-2,5-dimethylpiperazine-l-carboxylate (XJ-4-91)

[0428] White powder, 85% yield, 12.0 g, R / = 0.25 (hexane / EtOAc=5: 1, UV detection on TLC plate). 'HNMR (500 MHz, Acetone- J6) 5 7.74 - 7.40 (m, 2H), 7.23 (t, J = 8.6 Hz, 2H), 4.91 - 4.40 (m, 1H), 4.27 (s, 1H), 4.02 - 3.45 (m, 2H), 3.39 - 3.09 (m, 2H), 1.46 (s, 9H), 1.34 - 1.02 (m, 6H).13C NMR (126 MHz, Acctonc-7.) 5 169.50. 163.06 (d, 7= 247.0 Hz), 154.64, 154.42, 133.19, 129.47, 129.06, 115.38, 115.21, 79.01, 50.10, 47.29, 46.29, 45.78, 44.53, 43.13, 42.61, 41.80, 41.35, 39.85, 27.70, 14.31.19F NMR (471 MHz, Acctonc-7,) 5 -113.30. HRMS (ESI) calculated for CI8H25FN2O3 [M+H]+: 359.1741, found: 359.1748. ((2iS,57?)-2,5-dimethylpiperazin-l-yl)(4-fluorophenyl)methanone (XJ-4-92)

[0429] White powder, 50% yield, 4.2 g, R / = 0.40 (dichloromethane / methanol = 20 : 1, UV detection on TLC plate).1H NMR (500 MHz, AcctoncA) 5 7.67 - 7.47 (m, 2H), 7.30 - 7.15 (m, 2H), 4.76 - 4.45 (m, 1H), 3.98 (d, J= 14.6 Hz, 1H), 3.92 - 3.81 (m, 1H), 3.75 (dd, J= 14.5, 3.6 Hz, 1H), 3.59 (dd, J= 13.5, 5.0 Hz, 1H), 3.21 (dd, J = 13.4, 2.0 Hz, 1H), 1.50 (d, J = 7.1 Hz, 3H), 1.47 (d, J = 6.9 Hz, 3H).13C NMR (126 MHz, Acetone-Je) 5 169.85, 163.30 (d, J = 247.4 Hz), 132.21, 129.48, 129.41, 115.52, 115.34, 47.56, 45.33, 41.30, 41.04, 15.03, 12.62.19F NMR (471 MHz, Acetone-Je) 5 -112.69. HRMS (ESI) calculated for CI3HI7FN2O [M+H]+: 237.1398, found: 237.1402.

[0430] (4-Fluorophenyl)((2iS,57?)-4-(hex-5-yn-l-yl)-2,5-dimethylpiperazin-l-yl)methanone (XJ-4-95)

[0431] Method B, light yellow oil, 60% yield, 1.9 g, R^ = 0.25 (hexane / EtOAc=3: 1, UV detection on TLC plate).1H NMR (500 MHz, Acctonc-A) 7.49 - 7.42 (m, 2H), 7.26 - 7.16 (m, 2H), 4.33 (s, 1H), 3.73 (s, 1H), 3.43 (d, J= 12.9 Hz, 1H), 3.03 (s, 1H), 2.80 (t, J= 1.1 Hz, 1H), 2.72 (dd, J= 11.8, 4.2 Hz, 1H), 2.47 (dt, J= 12.4, 6.9 Hz, 1H), 2.41 - 2.37 (m, 1H), 2.34 (t, J= 2.7 Hz, 1H), 2.25 - 2.19 (m, 1H), 1.68 - 1.50 (m, 4H), 1.34 (d, J= 6.7 Hz, 3H), 0.93 (d, J= 6.6 Hz, 3H).13C NMR (200 MHz, AcctoncA) 5 169.22, 162.89 (d, J= 246.5 Hz), 133.74, 133.71, 129.12, 129.05, 115.24, 115.07, 84.14, 68.99, 53.21, 52.36, 48.83, 26.12, 26.05, 17.70, 15.98. 6.32.19FNMR(471 MHz, Acetone-Js) 5 -113.67. HRMS (ESI) calculated for C19H25FN2O [M+Na]+: 339.1843, found: 339.1830.

[0432] ((2iS,57?)-4-(4-(l-(Benzo[tZ| [l,3] dioxol-5-ylsulfonyl)-LH-l,2,3-triazol-4-yl)butyl)-2,5-dime- thylpiperazin-l-yl)(4-fluorophenyl)methanone (XJ-4-97)

[0433] F XJ-4-97 ' H NMR (800 MHz, Acctonc-t / ,.) 5 8.31 (s, 1H), 7.75 (dt, J= 8.3, 1.9 Hz, 1H), 7.47 (t, J= 1.8 Hz, 1H), 7.45 > 7.40 (m, 2H), 7.22 - 7.17 (m, 2H), 7.12 (dt, J= 8.3, 1.4 Hz, 1H), 6.26 (s, 2H), 3.38 (s, 1H), 2.95 (s, 1H), 2.74 (t, J= 7.6 Hz, 2H), 2.68 (dd, J= 12.0, 4.1 Hz, 2H), 2.44 (dt, J= 13.4, 7.2 Hz, 2H), 2.38 - 2.30 (m, 2H), 1.81 - 1.67 (m, 2H), 1.48 (p, J= 7.3 Hz, 2H), 1.28 (d, J= 6.8, 3H), 0.89 (d, J= 6.6 Hz, 3H).13C NMR (201 MHz, Acctonc-t / ,.) 5 168.75, 163.02, 161.80, 153.81, 148.59, 147.73, 133.22, 128.61 (d, J= 8.4 Hz), 124.88, 120.72, 114.70 (d, J= 21.5 Hz), 108.35, 106.94, 103.19, 52.85, 51.87, 48.30, 25.94, 25.84, 24.26, 15.50, 5.82.19F NMR (564 MHz, Acctonc-t / ,.) 5 -113.40. HRMS (ESI): Calcd for C26H30FN5O5S [M+H]+: 544.2030, found: 544.2028.

[0434] Synthetic procedures for the intermediates XJ-4-43 and XJ-4-45: The synthetic procedure for Grignard reagent was performed following previously described methods (Baron & Knochel, 2005). A dry flask (500 mL) was equipped with magnesium turnings (3.5 equiv., 2.6 g, 108.8 mmol) and a magnetic stirring bar. The flask was evacuated and heated with a heat gun for 10 min. When the flask was cooled down to RT, it was refdled with argon. THF (200 mL) was then added followed by 1,2- dibromoethane (1.25 equiv., 38.9 mmol, 3.4 mL) and the mixture was heated with a heat gun until ebullition started. After 2 min, the flask was placed into an ice bath and 3,4-difuoro-l -bromobenzene (2.5 equiv., 15.0 g, 77.7 mmol) was added dropwise over 1 h by syringe. 1-Boc-isonipecotic acid ester (1.0 equiv., 8.0 g, 31.1 mmol) was added and the reaction solution was stirred overnight at RT. The reaction was quenched with saturated ammonia chloride solution and extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was resuspended in dichloromethane followed by addition of hexane. The re suspension was cooled down in ice bath and the white precipitate was filtered and dried to provide XJ-4-43.

[0435] To a solution of XJ-4-43 in dichloromethane (100 mL) was added 50 mL of trifluoroacetic acid (TFA). The resulting mixture was stirred overnight at RT. Upon completion, the reaction solution was concentrated under reduced pressure. The residue was dissolved with ethyl acetate (200 mL) and washed with sodium hydroxide solution (4 mol / L) and brine, respectively. The organic layers were dried over anhydrous sodium sulfate, fdtered and concentrated under reduced pressure. The residue was resuspended in dichloromethane followed by addition of hexane. The white precipitate was filtered and dried to provide XJ-4-45. tert- Butyl 4-(bis(3,4-difluorophenyl)(hydroxy)methyl)piperidine-l-carboxylate (XJ-4-43)

[0436] White powder, 42% yield, 5.7 g, R^= 0.30 (hexane / EtOAc=4: l, UV detection on TLC plate). 'HNMR (500 MHz, CDCE) 5 7.35 - 7.22 (m, 2H), 7.04 - 7.17 (m, 4H), 4.27 - 4.00 (m, 2H), 2.69 (t, J= 12.9 Hz, 2H), 2.31 - 2.48 (m, 1H), 2.24 (s, 1H), 1.43 (s, 9H), 1.16 - 1.35 (m, 2H).13C NMR (126 MHz, CPC13) 5 154.61. 151.17 (d, J= 12.5 Hz), 150.11 (d, J= 12.7 Hz), 149.19 (d, J= 12.7 Hz), 148.13 (d, J= 12.6 Hz), 142.39 (t,J= 4.2 Hz), 121.61 (dd, J= 6.1, 3.5 Hz), 117.15, 117.02, 115.32, 115.17, 79.69, 78.51, 44.34, 28.36, 26.24.19F NMR (471 MHz, CDCh) 5 -139.96, -143.28. HRMS (ESI) calculated for C23H25F4NO3 [M+Na]+: 426.1663, found: 462.1676.

[0437] 4-(Bis(3,4-difluorophenyl)methylene)piperidine (XJ-4-45)

[0438] 1H NMR (500 MHz, Acctonc-A) 5 7.38 - 7.28 (m, 2H), 7.27 - 7.20 (m, 2H), 7.14 - 7.06 (m, 2H), 3.35 (t, J= 6.0 Hz, 4H), 2.69 (t, J= 6.0 Hz, 4H).13C NMR (126 MHz, Methanol- J4) 5 151.00 (d, J= 12.8 Hz), 150.48 (d, J= 12.7 Hz), 149.02 (d, J= 13.0 Hz), 148.51 (d, J= 12.7 Hz), 137.69, 135.71, 131.44, 125.75, 125.72, 125.69, 125.67, 118.01, 117.87, 117.23, 117.09, 44.53, 27.75.19F NMR (471 MHz, Mcthanol- 5 - 139.51 (d, J= 20.8 Hz), - 141.57 (d, J= 20.8 Hz) . HRMS (ESI) calculated for Ci8HI5F4N [M+Na]+: 322.1213, found: 322.1219. 4-(Bis(3,4-difluorophenyl)methylene)-l-(hex-5-yn-l-yl)piperidine (XJ-4-49)

[0439] Method B, colorless oil, 65% yield, 2.0 g, R / = 0.30 (hexane / acetone=4: 1, UV detection on TLC plate) .1H NMR (500 MHz, Acctonc-A) 5 7.30 (dt, J = 10.8, 8.5 Hz, 2H), 7.13 (ddd, J= 11.5, 7.8, 2.1 Hz, 2H), 7.06 - 6.99 (m, 2H), 2.82 (d, J= 16.9 Hz, 1H), 2.48 (t, J= 5.6 Hz, 4H), 2.39 - 2.30 (m, 6H), 2.22 (td, J= 6.8, 2.7 Hz, 2H), 1.66 - 1.48 (m, 4H).13C NMR (126 MHz, Acctonc- 5 150.74 (d, J= 12.7 Hz), 149.97 (d, J = 12.6 Hz), 148.78 (d, J = 12.8 Hz), 148.01 (d, J= 12.6 Hz), 139.39 - 139.04 (m), 139.01, 131.60, 126.33 (dd, J= 6.2, 3.3 Hz), 118.51, 118.37, 117.12, 116.99, 84.06, 68.94, 57.26, 54.72, 31.60, 26.33, 25.93, 17.73.19F NMR (471 MHz, Acctonc-A) 5 -140.33, -140.38, -142.89, -142.93. HRMS (ESI) calculated for C24H23F4N [M+H]+: 402.1839, found: 402.1849.

[0440] Representative preparation of sulfonyl 1,2,3-triazoles by click chemistry. The synthetic procedure was performed following previously described methods (68). To a stirred mixture of alkynes (1.2 equiv., 6 mmol), sulfonyl azides (1.0 equiv., 5 mmol), and copper(I) iodide (Cui, 0.05 mmol) in CHCh (30 mL) was slowly added 2,6-lutidine (0.070 mb, 6 mmol) at 0 °C. After stirring the reaction mixture for 12 h at 0 °C, it was diluted with CH2Q2 (30 mL) and then quenched with aqueous ammonia chloride solution (50 mL). The mixture was stirred for an additional 30 min and two layers were separated. The aqueous layer was extracted with CH2Q2 (50 mL x 3) and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by column chromatography to give the desired products.

[0441] 4-(Bis(4-fluorophenyl)methylene)-l-(2-(l-tosyl-lH-l,2,3-triazol-4-yl)ethyl)piperidine (TH207)

[0442] Light brown oil, 63% yield, 1.6 g, R / = 0.20 (hexane / EtOAc =2: 1, UV detection on TLC plate).

[0443] 1H NMR (800 MHz, CDCL) 5 8.00 7.95 (m, 3H), 7.37 (d, J= 8.2 Hz, 2H), 7.10 - 7.02 (m, 4H), 7.00 - 6.95 (m, 4H), 2.93 (t, J= 7.5 Hz, 2H), 2.68 (t, J= 7.5 Hz, 2H), 2.52 (t, J = 5.6 Hz, 4H), 2.44 (s, 3H), 2.37 (t, J= 5.7 Hz, 4H).13C NMR (201 MHz, CDCE) 5 162.14. 160.92, 147.15, 146.20, 138.13, 138.11, 135.97, 134.04, 133.27, 131.41, 131.40, 131.38, 131.36, 131.33, 131.27, 131.25, 131.23, 131.21, 131.19, 130.47, 130.37, 130.32, 128.65, 128.62, 128.59, 121.06, 121.03, 121.00, 115.13, 115.11, 115.08, 115.02, 115.01, 114.98, 114.96, 114.95, 114.89, 114.86, 114.84, 56.89, 54.96, 31.73, 23.53, 21.88.19F NMR (564 MHz, CDCL) 5 -119.10. HRMS (ESI) calculated for C29H28F2N4O2S[M+H]+: 535.1974, found: 535.1979. l-(Bis(4-fluorophenyl)methyl)-4-(2-(l-tosyl-LH-l,2,3-triazol-4-yl)ethyl)piperazine (TH208)

[0444] White powder, 56% yield, 1.5 g, R / = 0.25 (hexane / EtOAc=2: 1, UV detection on TLC plate).

[0445] 1H NMR (800 MHz, CPC13) 5 8.00 - 7.97 (m, 2H), 7.95 (s, 1H), 7.43 - 7.32 (m, 6H), 7.02 - 6.93 (m, 4H), 4.22 (s, 1H), 2.96 - 2.83 (m, 2H), 2.71 - 2.62 (m, 2H), 2.60 - 2.21 (m, 11H).13C NMR (200 MHz, CDCI Q 5 162.44, 161.22, 147.14, 146.09, 138.22, 133.26, 130.46, 130.38, 130.31, 129.32, 129.29, 129.18, 129.14, 128.66, 128.63, 128.60, 121.04, 120.97, 115.56, 115.45, 115.41, 115.30, 56.90, 53.17, 51.74, 23.21, 21.89, 21.86, 21.82.19F NMR (564 MHz, CDCE) 5 -118.83. HRMS (ESI) calculated for C28H29F2N5O2S [M+H]+: 538.2083, found: 538.2075.

[0446] 4-(Bis(4-fluorophenyl)methylene)-l-(2-(l-tosyl-LH-l,2,3-triazol-4-yl)ethyl)piperidine (TH220)

[0447] White powder, 69% yield, 1.9 g, R / = 0.35 (hexane / EtOAc=l: 1, UV detection on TLC plate).

[0448] 1H NMR (800 MHz, CDC1A 5 8.08 - 8.01 (m, 2H), 7.97 (s, 1H), 7.09 - 7.04 (m, 4H), 7.04 - 7.01 (m, 2H), 7.00 - 6.96 (m, 4H), 3.88 (s, 3H), 2.98 - 2.85 (m, 2H), 2.74 - 2.63 (m, 2H), 2.57 - 2.48 (m, 4H), 2.42 - 2.10 (m, 4H).13C NMR (200 MHz, CDCL) 5 165.27, 162.15, 160.93, 146.07, 138.11, 135.98, 134.04, 132.89, 131.5 - 130.89 (m, 4C), 131.40, 131.36, 131.33, 131.27, 131.23, 131.20, 131.17, 131.14, 131.12, 129.40, 120.91, 120.87, 120.84, 115.30 - 114.74 (m, 4C), 57.11 - 56.70 (m, 1C), 55.94 (q, J = 49.0 Hz, 1C), 54.94, 31.64, 23.43.19F NMR (564 MHz, CDCL) 5 -119.12. HRMS (ESI) calculated for C29H28F2N4O3S [M+H]+: 551.1923, found: 551.1919.

[0449] 4-(Bis(4-fluorophenyl)methylene)-l-(2-(l-((4-fluorophenyl)sulfonyl)-l / 7-l,2,3-triazol-4-yl)e- thyl)piperidine (TH221)

[0450] Light yellow oil, 45% yield, 1.2 g, R^= 0.30 (hexane / EtOAc=2: 1, UV detection on TLC plate). *HNMR (800 MHz, CDCL) 5 8.17 - 8.13 (m, 2H), 7.99 (s, 1H), 7.30 - 7.24 (m, 2H), 7.09 - 7.03 (m, 4H), 7.01 - 6.94 (m, 4H), 2.95 (t, J= 7.5 Hz, 2H), 2.69 (t, J= 7.5 Hz, 2H), 2.53 (t, J= 5.6 Hz, 4H), 2.37 (t, J = 5.6 Hz, 4H).13C NMR (200 MHz, CDCL) 5 167.47. 166.18, 162.16, 160.93, 146.34, 138.85, 138.08 (d, J = 3.5 Hz), 135.83, 134.13, 132.23 (d, J = 3.2 Hz), 131.75 (d, J = 10.2 Hz), 131.29 (d, J = 7.5 Hz), 121.06, 117.31 (d, J= 23.1 Hz), 115.00 (d, J= 21.2 Hz), 77.21, 77.05, 76.89, 56.81, 54.94, 31.61, 23.38.19F NMR (564 MHz, CPC13) 5 -102.34. HRMS (ESI) calculated for C28H25F3N4O2S [M+H]+: 539.1723, found: 539.1716.

[0451] 4-(bis(4-fluorophenyl)methylene)-l-(2-(l-(cyclopropylsulfonyl)-LH-l,2,3-triazol-4-yl)ethyl) piperidine (TH223)

[0452] Light brown oil, 70% yield, 1.7 g, R / = 0.25 (hexane / EtOAc =1: 1, UV detection on TLC plate). ]H NMR (800 MHz, CDCL) 5 7.92 (s, 1H), 7.12 - 7.04 (m, 4H), 7.00 - 6.92 (m, 4H), 2.98 (t, J= 7.5 Hz, 2H), 2.86 (tt, J= 7.9, 4.7 Hz, 1H), 2.71 (t, J = 7.5 Hz, 2H), 2.54 (t, J = 5.6 Hz, 4H), 2.44 - 2.30 (m, 4H), 1.67 - 1.47 (m, 2H), 1.36 - 1.16 (m, 2H).13C NMR (200 MHz, CDCL) 5 162.12, 160.90, 145.98, 138.13, 138.12, 135.96, 134.04, 131.33, 131.29, 121.42, 115.02, 114.92, 56.90, 54.94, 32.16, 23.38, 7.80.19F NMR (564 MHz, CDCL) 5 -119.07. HRMS (ESI) calculated for C25H26F2N4O2S [M+H]+: 485.1717, found: 485.1796. 4-((4-(2-(4-(Bis(4-fluorophenyl)methylene)piperidin-l-yl)ethyl)-l / / -l,2,3-triazol-l-yl)sul- fonyl)-

[0453] JV-propylbenzamide

[0454] White powder, 80% yield, 2.4 g, R / = 0.20 (hexane / EtOAc=l: 1, UV detection on TLC plate).

[0455] ' H NMR (800 MHz, CDCL) 5 8.14 - 8.10 (m, 2H), 8.00 (s, 1H), 7.96 - 7.92 (m, 2H), 7.09 - 7.01 (m, 4H), 7.00 - 6.94 (m, 4H), 6.43 (t, J= 5.9 Hz, 1H), 3.45 - 3.35 (m, 2H), 2.92 (t, J= 7.5 Hz, 2H), 2.67 (t, J= 7.5 Hz, 2H), 2.52 (t, J= 5.9 Hz, 4H), 2.36 (t, J= 5.6 Hz, 4H), 1.62 (h, J= 7.4 Hz, 2H), 0.96 (t, J = lA Hz, 3H).13C NMR (200 MHZ, CDCL) 5 165.28. 162.15, 160.93, 146.53, 141.43, 138.41, 138.08, 135.78, 134.16, 131.47-131.09 (m, 2C), 128.85-128.80 (m, 2C), 128.41-128.23 (m, 2C), 121.32, 121.25, 115.15- 114.87 (m, 2C) 56.75, 54.92, 42.11, 31.60, 23.35, 22.86, 22.76, 11.47, 11.34.19F NMR (564 MHz, CDCL) 5 -119.07. HRMS (ESI) calculated for C32H33F2N5O3S [M+H]+: 606.2345, found: 606.2329.

[0456] 4-(Bis(4-fluorophenyl)methylene)-l-(3-(l-((4-methoxyphenyl)sulfonyl)-lH-l,2,3-triazol-4- yl)propyl)piperidine (

[0457] Light brown oil, 74% yield, 2.1 g, R / = 0.40 (hexane / acetone=3: 1, UV detection on TLC plate)

[0458] 1H NMR (800 MHz, CPC13) 5 8.10 - 7.94 (m, 2H), 7.85 (s, 1H), 7.05 - 7.02 (m, 4H), 7.02 - 7.00 (m, 2H), 6.98 - 6.94 (m, 4H), 3.87 (s, 3H), 2.74 (t, J= 7.6 Hz, 2H), 2.44 (t, J= 5.7 Hz, 4H), 2.40 - 2.36 (m, 2H), 2.33 (t, J= 5.7 Hz, 4H), 1.86 (p, J= 7.6 Hz, 2H).13C NMR (201 MHz, CDCI3) 5 164.78, 161.65,

[0459] 160.43, 147.21, 137.72, 137.70, 135.85, 133.29, 130.86, 130.82, 130.65, 126.75, 119.82, 114.54,

[0460] 114.43, 56.99, 55.46, 54.69, 31.20, 25.85, 22.89.19F NMR (564 MHz, CDCI3) 5 -119.21. HRMS (ESI) calculated for C30H30F2N4O3S [M+H]+: 565.2079, found: 565.2069. l-(4-(Bis(4-fluorophenyl)methylene)piperidin-l-yl)-3-(l-((4-methoxyphenyl)sulf-onyl)-l / / -l ,2,3- triazol-4-yl)propan-l-one

[0461] Light yellow powder, 68% yield, 2.0 g, Rf = 0.10 (hexane / acetone=3: l, UV detection on TLC plate). ' H NMR (800 MHz, CDCI3) 5 8.06 - 7.99 (m, 2H), 7.95 (s, 1H), 7.06 - 7.01 (m, 5H), 7.01 - 6.96 (m, 5H), 3.87 (s, 3H), 3.61 (t, J= 5.9 Hz, 2H), 3.43 (t, J= 5.8 Hz, 2H), 3.10 - 3.03 (m, 2H), 2.78 - 2.67 (m, 2H), 2.27 - 2.34 (m, 4H).13C NMR (201 MHz, CDCL) 5 169.22, 161.22 (d, J = 245.9 Hz), 160.61,

[0462] 146.34, 137.20, 137.18, 137.09, 137.07, 135.47, 133.55, 130.70, 130.66, 126.74, 120.86, 114.78, 114.67, 114.54, 55.46, 45.88, 42.59, 31.60, 31.43, 30.68, 20.46.19F NMR (564 MHz, CDCL) 5 -118.52. HRMS (ESI) calculated for CsoIMNXhS [M+H]+: 579.1872, found: 579.1870.

[0463] 4-(Bis(4-fluorophenyl)methylene)-l-(4-(l-((4-methoxyphenyl)sulfonyl)-lH-l,2,3-triazol-4- yl)butyl)piperidine (

[0464] Light brown oil, 82% yield, 2.4 g, Rf = 0.20 (hexane / acetone=4: 1, UV detection on TLC plate). 'H NMR (800 MHz, Acetone-Je) 5 8.29 (s, 1H), 8.10 - 8.04 (m, 2H), 7.22 - 7.18 (m, 2H), 7.18 - 7.13 (m, 4H), 7.10 - 7.04 (m, 4H), 3.93 (s, 3H), 2.75 - 2.64 (m, 2H), 2.42 (t, J= 5.5 Hz, 4H), 2.33 (t, J= 7.2 Hz, 2H), 2.30 (t, J = 5.6 Hz, 4H), 1.75 - 1.67 (m, 2H), 1.51 (p, J = 7.3 Hz, 2H).13C NMR (200 MHz, Acetone-Je) 5204.84. 164.98, 161.59, 160.38, 147.64, 138.18, 138.16, 136.52, 132.93, 131.02, 130.98, 130.42, 126.96, 120.58, 114.79, 114.39, 114.29, 57.11, 55.24, 54.50, 31.13, 26.19, 25.87, 24.41.19F NMR (564 MHz, CDC1A 5 -119.17, -164.90. HRMS (ESI) calculated for C31H32F2N4O3S [M+H]+: 579.2236, found: 579.2248. 4-(Bis(4-fluorophenyl)methylene)-l-(5-(l-((4-methoxyphenyl)sulfonyl)-l / / -l,2,3-triazol-4-yl) pentyl)piperidine (XJ-3-65)

[0465] Light brown oil, 67% yield, 2.0 g, Rf = 0.20 (hexane / acetone=3: 1, UV detection on TLC plate). 'H NMR (800 MHz, Acctonc- 5 8.29 (s, 1H), 8.08 - 8.04 (m, 2H), 7.22 - 7.19 (m, 2H), 7.18 - 7.13 (m, 4H), 7.10 - 7.04 (m, 4H), 3.93 (s, 3H), 2.72 - 2.66 (m, 2H), 2.46 - 2.39 (m, 4H), 2.32 - 2.25 (m, 6H), 1.71 - 1.63 (m, 2H), 1.53 - 1.44 (m, 2H), 1.40 - 1.32 (m, 2H).13C NMR (200 MHz, Acctonc-A) 5 204.91, 164.98, 161.60, 160.38, 147.64, 138.17, 138.16, 136.52, 132.95, 131.04, 131.00, 130.44, 126.96, 120.58, 114.81, 114.41, 114.31, 57.37, 55.27, 54.52, 31.13, 28.17, 26.24, 26.07, 24.52.19F NMR (564 MHz, CPC13) 5 -119.21, -164.90. HRMS (ESI) calculated for C32H34F2N4O3S [M+H]+: 593.2392, found: 593.2384.

[0466] 4-Butyl-l-((4-methoxyphenyl)sulfonyl)-LH-l,2,3-triazole (XJ-4-5)

[0467] White powder, 85% yield, 1.3 g, R^= 0.50 (hexane / EtOAc =4: 1, UV detection on TLC plate). *HNMR (800 MHz, Acetone-^ / ,,) 5 8.37 (s, 1H), 8.18 - 8.10 (m, 2H), 7.32 - 7.23 (m, 2H), 4.03 (s, 3H), 2.80 - 2.73 (m, 2H), 1.75 - 1.66 (m, 2H), 1.48 - 1.29 (m, 2H), 1.01 - 0.93 (m, 2H).13C NMR (201 MHz, Acetone-Je) 5 164.97, 147.73, 130.45, 126.91, 120.58, 114.81, 55.32, 30.47, 24.29, 21.52, 12.81.19F NMR (564 MHz, CPC13) 5 -119.21, -164.90. HRMS (ESI) calculated for C13H17N3O3S [M+H]+: 296.1063, found: 296.1057.

[0468] 4-(Bis(4-fluorophenyl)methylene)-l-(4-(l-(propylsulfonyl)-lH-l,2,3-triazol-4-yl)butyl)pipe- ridine (XJ-4-27) Light brown oil, 70% yield, 1.8 g, Rf = 0.40 (hexane / acetone=3: 1, UV detection on TLC plate). 'H NMR (800 MHz, Acctonc- 5 8.21 (s, 1H), 7.24 - 7.12 (m, 4H), 7.12 - 6.90 (m, 4H), 3.82 - 3.67 (m, 2H), 2.79 (t, J = 7.6 Hz, 2H), 2.44 (t, J = 5.7 Hz, 4H), 2.36 (t, J = 7.2 Hz, 2H), 2.33 - 2.26 (m, 4H), 1.80 - 1.67 (m, 4H), 1.55 (p, J= 7.4 Hz, 2H), 1.01 (t, J = 7.5 Hz, 3H).13C NMR (201 MHz, Acetone- de) 8 161.59, 160.38, 147.37, 138.18, 138.17, 136.57, 132.91, 131.00, 130.96, 121.31, 114.36, 114.25, 57.12, 55.69, 54.52, 31.16, 26.24, 25.84, 24.35, 16.29, 11.14.19F NMR (564 MHz, Acetone- 8 - 117.96. HRMS (ESI) calculated for C27H32F2N4O2S [M+H]+: 515.2287, found: 515.2298. l-(4-(l-(Benzo[<Z] [l,3]dioxol-5-ylsulfonyl)-LH-l,2,3-triazol-4-yl)butyl)-4-(bis(4-fluorophenyl) methylene)piperidine (

[0469] *

[0470] Light yellow oil, 78% yield, 2.3 g, R / = 0.30 (hexane / acetone=3: 1, UV detection on TLC plate). 'H NMR (800 MHz, Acctonc- 8 8.30 (s, 1H), 7.75 - 7.72 (m, 1H), 7.51 - 7.45 (m, 1H), 7.18 - 7.43 (m, 4H), 7.12 - 7.09 (m, 1H), 7.09 - 7.05 (m, 4H), 6.23 (s, 2H), 2.84 - 2.59 (m, 2H), 2.59 - 2.38 (m, 4H), 2.34 - 2.31 (m, 2H), 2.32 - 2.28 (m, 4H), 1.73 - 1.67 (m, 2H), 1.54 - 1.48 (m, 2H).13C NMR (200 MHz, Acetone-Jfi) 8 161.60, 160.38, 153.78, 148.57, 147.70, 138.18, 138.16, 136.51, 132.93, 131.02, 131.00, 130.98, 130.96, 128.49, 124.87, 120.72, 114.36, 114.25, 108.33, 106.95, 103.15, 57.08, 54.49, 31.11, 26.15, 25.84, 24.37.19F NMR (564 MHz, Acetone-^) 8 -117.92. HRMS (ESI) calculated for C31H30F2N4O4S [M+H]+: 593.2029, found: 593.2015. l-(4-(l-(Benzo[<Z] [l,3]dioxol-5-ylsulfonyl)-LH-l,2,3-triazol-4-yl)butyl)-4-(bis(3,4-difluor-op- henyl)methylene)piperidine (XJ-4-85)

[0471] Light yellow oil, 76% yield, 2.4 g, R / = 0.25 (hexane / acetone=3: 1, UV detection on TLC plate). 'H NMR (800 MHz, Acctonc- 8 8.30 (s, 1H), 7.74 (dd, J= 8.4, 2.0 Hz, 1H), 7.47 (d, J= 2.0 Hz, 1H), 7.28 (dt, J= 10.7, 8.4 Hz, 2H), 7.12 - 7.07 (m, 3H), 7.02 - 6.96 (m, 2H), 6.24 (s, 2H), 2.73 (t, J= 7.6 Hz, 2H), 2.43 (t, J= 5.6 Hz, 4H), 2.33 (t, J= 12 Hz, 2H), 2.31 - 2.28 (m, 4H), 1.71 (p, J= 7.6 Hz, 2H), 1.51 (p, J = 7.3 Hz, 2H).13C NMR (126 MHz, Acetone- J6) 5 154.22, 150.75 (d, J= 12.7 Hz), 149.98 (d, J= 12.7 Hz), 149.01, 148.79 (d, J= 12.9 Hz), 148.17, 148.02 (d, J= 12.6 Hz), 139.21 (dd, J = 5.1, 4.0 Hz), 139.17, 139.01, 131.60, 129.00, 126.32 (dd, J = 6.2, 3.3 Hz), 125.34, 121.14, 118.52, 118.39, 117.12, 116.99, 108.79, 107.47, 103.60, 57.47, 54.74, 31.60, 26.64, 26.36, 24.88.19F NMR (471 MHz, Acetone-Js) 5 -140.32, -140.37, -142.88, -142.92. HRMS (ESI) calculated for C31H28F4N4O4S [M+H]+: 629.1840, found: 629.1817.

[0472] ((25,57?)-4-(4-(l-(Benzo[J| [l,3] dioxol-5-ylsulfonyl)-LH-l,2,3-triazol-4-yl)butyl)-2,5-dime-th- ylpiperazin-l-yl)(4-fluorophenyl)methanone (XJ-4-97)

[0473] White solid, 62% yield, 1.7 g, R^= 0.20 (hexane / acetone=3: 1, UV detection on TLC plate). 'H NMR (800 MHz, Acetone-^ / ,,) 5 8.31 (s, 1H), 7.75 (dt, J = 8.3, 1.9 Hz, 1H), 7.47 (t, J = 1.8 Hz, 1H), 7.45 - 7.38 (m, 2H), 7.22 - 7.16 (m, 2H), 7.14 - 7.10 (m, 1H), 6.25 (s, 2H), 3.38 (s, 1H), 2.95 (s, 1H), 2.74 (t, J= 7.6 Hz, 2H), 2.68 (dd, J = 12.0, 4.1 Hz, 2H), 2.47 - 2.40 (m, 2H), 2.38 - 2.28 (m, 2H), 1.82 - 1.63 (m, 2H), 1.48 (p, J = 7.3 Hz, 2H), 1.28 (d, J= 6.8, 3H), 0.89 (d, J = 6.6 Hz, 3H).13C NMR (201 MHz, Acetone-J6) 5 168.75. 162.41 (d,J= 246.5 Hz), 153.81, 148.59, 147.73, 133.22, 128.63, 128.59, 124.88, 120.72, 114.75, 114.65, 108.35, 106.94, 103.19, 52.85, 51.87, 48.30, 28.79, 28.69, 28.60, 28.50, 28.40, 28.31, 28.21, 25.94, 25.84, 24.26, 15.50, 5.82.19F NMR (564 MHz, acetone- J6) 5 -113.75. HRMS (ESI) calculated for C26H30FN5O5S [M+H]+: 544.2024, found: 544.2002. l-(4-(LH-l,2,3-Triazol-4-yl)butyl)-4-(bis(3,4-difluorophenyl)methylene)piperidine (XJ-4-119)

[0474] Colorless oil, 57% yield, 0.4 g, R / = 0.10 (hexane / acetone=3: 1, UV detection on TLC plate). *H NMR (500 MHz, Acetone-J6) 5 7.55 (s, 1H), 7.35 - 7.23 (m, 2H), 7.18 - 7.09 (m, 2H), 7.05 - 6.96 (m, 2H), 2.84 (brs, 1H), 2.74 (t, J= 7.5 Hz, 2H), 2.48 (t, J= 5.5 Hz, 4H), 2.38 (t, J= 12 Hz, 2H), 2.33 (t, J= 5.6 Hz, 4H), 1.77 - 1.66 (m, 2H), 1.62 - 1.49 (m, 2H).13C NMR (126 MHz, Acctonc-A) 5 150.73 (d, J = 12.9 Hz), 149.97 (d, J= 12.7 Hz), 148.77 (d, J= 12.8 Hz), 148.01 (d, J= 12.7 Hz), 139.20 (t, J= 5.0 Hz), 147.96, 139.24, 139.20, 139.16, 138.90, 131.84, 131.63, 130.16, 126.33 (dd, J = 6.2, 3.3 Hz), 118.51, 118.38, 117.14, 117.00, 57.56, 54.71, 31.52, 27.03, 26.40, 24.52.19F NMR (471 MHz, Acetone- d6) 5 -140.30, -140.35, -142.85, -142.90. HRMS (ESI) calculated for C26H30FN5O5S [M+H]+: 445.2010, found: 445.2004.

[0475] EXAMPLE 2

[0476] Exemplary Proteomic and PFKL Activity Assays

[0477] Gel-based chemical proteomic assay (In situ ABPP analysis): As shown in Figure 24, HEK293T cells were incubated in 10 cm petri dishes with DMEM medium supplemented with 10% fetal bovine serum and 1% L-glutamine until the confluency reached about 90%. Aspirate the medium and then add serum-free-medium (SFM) with vehicle only (DMSO) or SuTEx compounds in DMSO, which afforded final concentrations of 0.1% DMSO. After incubation at 37°C for 1 hours in CO2 incubator, the medium was aspirated and 25 pM of probe TH211 in SFM was added. After incubation at 37°C for 2 hrs, the medium was aspirated and the cells were washed off in cold PBS. Centrifuge at 400 x g for 5 min to pellet and resuspend pellet again in PBS for 2X total washes. The cell pellets were resuspended and lysed by sonication (1 sec pulse, 20% amplitude, 3 times) in PBS in the presence of EDTA-free protease inhibitor cocktail tablet (Pierce Biotechnology, Waltham, Massachusetts, United States of America). The cell lysates were subject to ultracentrifugation (100,000 x g, 45 min at 4°C) to separate the cytosolic fraction in the supernatant and the membrane fraction as a pellet. Protein concentrations in soluble fraction were measured by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, California, United States of America). Proteome aliquots (2 mg / mL, 50 pL) were conjugated with fluorophore which was accomplished by copper-catalyzed azide-alkyne cycloaddition (CuAAC) with rhodamine-azide (TAMRA-azide, 1.25 mM, 1 pL, final concentration of 25 pM) in the presence of tris(2- carboxyethyljphosphine (TCEP, 50 mM fresh in water, 1 pL, final concentration of 1 mM), tris[(l- benzyl-lH-l,2,3-triazol-4-yl)methyl]amine (TBTA, 1.7 mM in 4: 1 t- butanol / DMSO, 3 pL, final concentration of 100 pM) and CuSO4 (50 mM, 1 pL, final concentration of 1 mM). After 1 hr incubation at room temperature, reactions were quenched by adding 4X SDS-PAGE loading buffer and beta-mercaptoethanol (17 pL) and samples were resolved by SDS-PAGE followed by in-gel fluorescence scanning.

[0478] SILAC sample preparation for competitive chemical proteomic assay: As shown in Figure 25, the light and heavy proteomes (2.3 mg / mL, 432 pL) prepared from HEK293T cells that were pretreated in situ with vehicle (DMSO) or SuTEx ligand (2 pM), respectively, at 37°C for 1 hr followed by probe TH211 treatment (50 pM) for 2 h, were directly were subjected to click reaction with the desthiobiotin- PEG3-azide (10 mM in DMSO, 10 pL, final concentration 200 pM) in the presence of TCEP (50 mM, 10 pL), TBTA (1.7 mM, 33 pL) and CuSO4 (50 mM, 10 pL) at room temperature for 1 hr. The light and the heavy samples were mixed in the chloroform-methanol extraction step. The subsequent steps including reduction with dithiothreitol, alkylation with iodoacetamide, digestion with Trypsin / Lys-C, enrichment with avidin beads were conducted as describedin Ho et al., 2015.

[0479] Gel-based chemical proteomic assay (In vitro ABPP analysis): HEK293T cells at 30-50% confluency were transfected with 2.6 pg of Flag-PFKL, Flag-PFKM or Flag-PFKP plasmid DNA a serum-free media for 48 hrs. After 48 hrs, the media was aspirated and cells washed with cold PBS and harvested. Cells were spun at 400 x g for 5 min at 4°C and supernatant was removed. Pellets were resuspended in 1 mb of cold DPBS and spun at 400 x g for 5 min at 4°C and supernatant was removed. The cells were resuspended and lysed by sonication (1 sec pulse, 20% amplitude, 3 times) in PBS in the presence of EDTA-free protease inhibitor cocktail tablet (Pierce Biotechnology, Waltham, Massachusetts, United States of America) by sonication and fractionated (100,000 x g, 45 min, 4°C). The protein concentration of the lysates in soluble fractions was determined on the Clariostar plate reader (BMG Labtech, Ortenberg, Germany) using the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, California, United States of America). The soluble fraction was diluted to 1 mg / mL in PBS in the presence of EDTA-free protease inhibitor cocktail tablet (Pierce Biotechnology, Waltham, Massachusetts, United States of America) and 48 pL was used for analysis. DMSO or compound was added and the tube gently flicked tube to mix. The tube was incubated for 1 h at 37°C in an incubator. Then 1 pL of probe TH211 was added (1.25 mM of TH211 stock) to a final concentration of 25 pM and the tube again incubated for 1 h at 37°C in an incubator. The probe- modified proteomes were conjugated to Rhodamine -azide (1 pl of 1.25 mM stock in DMSO) using TCEP ( 1 pl of fresh 50 mM stock in water), TBTA ligand (3 pl of a 1.7 mM 4: 1 t-butanol / DMSO stock,) and G1SO4 (1 pl of 50 mM stock) and incubated for 1 hr at room temperature. The reaction was quenched with 17 pL of 4X SDS-PAGE loading buffer + PME and vortexed to mix. The samples were analyzed by SDS-PAGE (30 pL) and imaged by in-gel fluorescence scanning.

[0480] TMT-Based Quantitative Chemical Proteomics: Tandem mass tag (TMT) technology (Rauniyar & Yates, 2014; Mertins et al., 2018), which introduces isotopic barcodes at the peptide level will be used to increase quantitative power in our LC-MS / MS chemical proteomics. See Figure 26. TH211 and TMT quantitative chemical proteomics can be used to determine potency of XJ-4-85 at individual binding sites of native PFKL. Both concentration- (0.001-100 pM) and time- (30 min - 20 hours) dependence of XJ-4-85 activity can be evaluated. XJ-4-97, which is structurally and mechanistically analogous to XJ-4-85 will be utilized as a negative control to demonstrate XJ-4-85 specific effects. The selectivity of XJ-4-85 against the kinome will also be assessed using ATP acyl phosphate activity-based probe methodology (Franks et al., 2017; Campbell et al., 2018; McCloud et al., 2018).

[0481] PFKL Activation Assay. The ability of the SuTEx compounds to activate PFKL activity was studied using His-tagged PFKL and a commercially available assay (sold under the tradename ADP- GLO™ Kinase Assay (Promega Corporation, Madison, Wisconsin, United States of America) according to the manufacturer’s instructions. NA-11 was used as a positive control of activation assays.

[0482] EXAMPLE 3

[0483] Additional Biological Activity Studies

[0484] Primary CD8+ T Cell Activation. Primary T cells will be isolated using commercial immunopurification kits (Stemcell). TCR activation of T cells will be achieved using plates coated (4 °C overnight) with anti-CD3 and -CD28 antibodies. T cells will be treated with DMSO vehicle or compound (e.g., XJ-4-85, XJ-4-97, or NA-11) at varying concentrations for short (30 min) and long incubation times (2 hr). T cells will be activated on antibody coated for 30 min (for phospho-ERK measurements) or 4 hrs (for cytokine measurements). Cell proliferation will be assessed by commercial WST-1 assays. Phospho-ERK will be measured by western blot method and cytokine (IFN-y, IL-2) activation will be measured by ELISA and intracellular cytokine staining (Paia et al., 2000) to assign signaling responses to CD8+ T cell populations. Lower antibody concentrations (6.7 nM) will also be used to evaluate PFKL activators under sub-threshold CD3 / CD28 activation conditions.

[0485] Glycolytic measurements (mode of action): To characterize SuTEx compound (e.g., XJ-4-85) activation of glycolysis in primary mouse and human T-cells, an improved CRISPR / Cas9-RNP method will be used to generate single-copy sensor knock-in lines at the TRAC locus (Oh et al., 2022). This approach is highly efficient (20-50%) for generating primary T-cell knock-in lines and will provide stable, non-viral expression for uniform sensor expression. To evaluate the heterogeneity of glycolytic states and to determine how this impacts SuTEx compound (e.g., XJ-4-85) activation, FBP (Koberstein et al., 2022) and lactate sensors (cytosolic and extracellular; Nasu et al., 2023) co-expressed from a single cassette will be introduced; the distinct spectra of these sensors permit multiplexing within a single cell. Longitudinal changes in individual cells will be studied using time-lapse microscopy, complemented with single cell analyses using cytometry measurements to obtain larger cell representations. Baseline heterogeneity of glycolytic states will be quantified based on FBP and Lactate measurements and determine the uniformity of cellular responses following SuTEx compound (e.g., XJ-4-85) activation at different doses, including determining the dynamics of observed effects. Singlecell analyses will be extended to evaluate effects from SuTEx compound (e.g., XJ-4-85) treatment under high or low glucose conditions, using hexokinase blocker 2-deoxyglucose as a control. The results will be deconvoluted to determine whether there exist permissive or restrictive initial metabolic states for SuTEx compound activation. The data can be further compared with Seahorse ECAR analyses performed on treated primary T-cells. To assess specificity, either PFKL WT or K677R mutants can be expressed into PFKL KO cells to evaluate responsivity to SuTEx compound (e.g., XJ-4-85) activation. Notably, as ratiometric sensors their readout is independent of sensor expression levels. Thus, there exists the additional option to individually express and analyze each sensor transiently using nucleofection of plasmid DNA. To assess site selectivity in SuTEx compound (e.g., XJ-4-85) pharmacology, PFKL WT and K677R mutants can be expressed into PFKL KO primary T cells and sensitivity to SuTEx-mediated activation of glycolysis can be evaluated. PFKL expression in PFKL KO T cells can be achieved using CRISPR / Cas9-mediated gene knock-in methods (see e.g., Oh et al., 2022).

[0486] EXAMPLE 4

[0487] Discovery of a Site-specific, Proteome-wide Selective Covalent PFKL Activator

[0488] Sulfur-triazole exchange (SuTEx) chemistry has emerged as a versatile platform for developing covalent inhibitors that target functional tyrosine and lysine residues on proteins (Hahm et al., 2020; Ciancone et al., 2023; Kim et al., 2025). In a prior study, alkynyl probe TH211 was produced by embedding a kinase binding fragment RF001 into a reactive SuTEx electrophile, allowing broad chemoproteomic profiling of kinases in cells (Huang et al., 2021). Whether RFOOl-based scaffolds can be optimized into selective ligands for targeting kinases remains largely unexplored. To address this question, a library of TH211 analogues bearing various modifications was synthesized as starting points for optimizing targeted covalent ligands of kinases (Figure 32). To identify hits capable of engaging target protein(s) in dynamic cellular environments, gel-based competitive activity-based protein profiling (ABPP) was conducted in situ. HEK293T cells were treated with SuTEx ligand (1 pM, 1 hour) followed by TH211 probe labeling (25 pM, 2 hours) and reduced fluorescent protein labeling from probe competition indicated cellular target engagement.

[0489] Notably, SuTEx ligands featuring electron-donating groups, such as methyl or methoxy, on the para position of the phenyl sulfone moiety effectively blocked labeling of a ~85 kDa protein (Figure 33A). Next, a competitive tandem mass tag (TMT)-ABPP assay (Grams et al., 2024) was deployed to deduce the molecular identity of the ~85 kDa cellular target (Figure 34). Probe-enriched sites detected by LC-MS / MS and exhibiting significant competition with ligand pretreatment compared to DMSO were designated as liganded sites (competition ratio or CR < 0.5, P < 0.05; Figure 33B). By cross referencing these liganded sites with the molecular weights of corresponding proteins, the glycolytic enzyme PFKL was identified as a prominent liganded target exhibiting a competition profile congruent with the gel-based analysis (Figures 33A and 33B). Among the active hits, TH220 demonstrated superior potency (IC50 = 305 nM; Figures 33C and 33D) for blockade of PFKL probe labeling that was site specific (K677) and isoform selective as evidenced by negligible activity against the homologous lysine site on PFKP (K688; Figures 33C-33E and 35).

[0490] To investigate the impact of TH220 on PFKL biochemical activity, a reported substrate assay using purified His-tagged PFKL was employed and conversion of F6P to FBP was monitored (McNae et al., 2021; see Figure 36A). K677 resides in the nucleotide effector site of PFKL and covalent modification at this residue with electrophilic natural products was previously shown to inactivate PFKL (Bracken et al., 2024). Unexpectedly, the K677-directed ligand TH220 activated PFKL in a dosedependent manner with a moderate half maximal effective concentration (EC50) of 56 pM (Figure 36B). Structure-activity relationship (SAR) exploration revealed: (i) molecular flexibility and length of the linker; and (ii) introduction of additional fluorine atoms as hydrogen bond acceptors as key for activity (Figures 27 A, 36C, and 36D). These efforts produced the optimal ligand XJ-4-85, which exhibited dose- and time -dependent activation of PFKL (Figure 27B). Notably, the leaving group alone (XJ-4- 119) or analogs with a different recognition motif (XJ-4-97) did not activate PFKL, identifying the latter as a suitable negative control (Figures 27A and 27B).

[0491] Gel-based competitive ABPP was performed, and it was determined that XJ-4-85 potently engaged PFKL in HEK293T cells (IC50 = 141 nM; Figure 27C). Next, competitive TMT-ABPP was performed to assess PFKL selectivity of XJ-4-85 in treated cells. Across -3,000 quantified probe- modified peptides, PFKL K677 was the only significantly liganded site detected with XJ-4-85 treatment (CR < 0.5, P < 0.05; Figure 27D). This exquisite selectivity was not cell line- or species-dependent as evidenced by nearly identical findings in human (Jurkat, THP-1, MOLM-14, SH-SY5Y) and mouse (B16-F10-Luc2) cell lines (Figures 27E-27I). These competition events were not due to PFKL expression changes (Figures 37A-37F). Collectively, these data established XJ-4-85 as a site specific (K677), proteome-wide selective covalent activator that effectively engaged PFKL but no other detectable PFK1 isoforms across a panel of diverse cell lines.

[0492] EXAMPLE 5

[0493] Structural Insights into PFKL Activation by XJ-4-85

[0494] To decipher the activation mechanism by XJ-4-85, the cryo-electron microscopy (cryo-EM) structure of PFKL in complex with XJ-4-85 and substrates ATP and F6P (PDB ID: 9P0J) were determined. See Figures 28A, 38, 39A, and 39B. 3D classification was performed to identify the highest quality PFKL tetramers, and subsequently performed D2 symmetry expansion, 3D classification, and local refinement to identify the highest quality monomers in the dataset. The monomer classes that reached the highest resolution (3.2-3.3A) exhibited clear density for the XJ-4-85 leaving group in the allosteric activating site between the PFKL catalytic and regulatory domains, adjacent to residue K677 (Figure 28B). The covalent moiety was less well-resolved, with additional density next to K677 only apparent in a subset of the monomer classes, suggesting flexibility in the covalently modified residue (Figure 28B). The XJ-4-85 binding pose overlapped with those of native activators (AMP and ADP) and the reversible PFKL activator NA-11 (Amara et al., 2021; see Figure 28C). The linear alkyl chain was not resolved, presumably due to conformational flexibility, and therefore was not included in the atomic model (Figures 28A-28H).

[0495] To understand the effect of XJ-4-85 on the overall conformation of PFKL, a local refinement was performed using particles from the best monomer class, expanding the refinement mask to encompass a full tetramer (Figure 28A). The resulting structure showed XJ-4-85-bound PFKL was in the active R-state conformation, with F6P and ADP bound at active sites, aligning with the activating function of XJ-4-85 (Figures 28D, 39A, and 39B). As previously described for R-state PFKL, the allosteric sugar-binding sites were also occupied and we modelled these ligands as FBP, likely produced by PFKL activity during sample preparation (Figures 39A and 39B). The active R-state conformation contrasts with the inactive T-state conformation, where the catalytic domains rotate to disrupt the enzyme active sites (Figure 28E). The R- to T-state transition also involved a rotation between the catalytic and regulatory domains within monomers, which compresses the allosteric activating site in the T-state conformation (Figure 28F). Akin to AMP, ADP, and NA-11, XJ-4-85 appeared to activate PFKL by sterically preventing compression of the allosteric activating site, thereby resisting transition to the T-state conformation. Rotation to the T-state would result in steric clashes between the XJ-4-85 leaving group and incoming catalytic domain (Figure 28F).

[0496] A washout experiment was performed after preincubation with compounds and showed that PFKL activation was retained with XJ-4-85 but not the reversible ligand NA-11 (Figure 28G). These data combined with the lack of activity for the free leaving group, supported covalency as a requirement for XJ-4-85-mediated PFKL activation (Figures 27B and 28G). It was determined that XJ-4-85 failed to activate FPKP or PFKM despite these isoforms containing a homologous lysine residue (K688 and K678, respectively) for covalent binding (Figures 28H, 40A, and 40B). To further probe PFK1 isoform- selective ligand recognition, the non-conserved K315 located in proximity to the bound leaving group was mutated, which abolished XJ-4-85-mediated PFKL activation (Figures 40A and 40C).

[0497] Collectively, it was determined that XJ-4-85 functions as an allosteric activator of PFKL that achieves isoform selectivity through covalent (K677) and non-covalent (K315) binding distinct from the reported reversible PFKL activator (Amara et al., 2021).

[0498] EXAMPLE 6

[0499] XJ-4-85 Reprograms Glycolysis Across Multiple Cell Types

[0500] Detection of intracellular FBP is widely utilized as an indicator of glycolytic pathway activity, as its intracellular concentration closely reflects the rate of glycolysis as the product of PFKL Whether XJ-4-85 could activate endogenous PFKL in cells was tested using the genetically encoded single- fluorescent protein HYlight biosensor (Koberstein et al., 2022) to monitor levels of FBP, the direct product of PFKL (Figure 29A).

[0501] An acute treatment (2 hours) with XJ-4-85 resulted in markedly elevated intracellular FBP levels in THP-1, Jurkat, MOLM-14, and SH-SY5Y cells relative to vehicle control. No significant changes were observed with the PFKL-inactive XJ-4-97 control or with a T152E control sensor that cannot bind FBP (Figures 29B-29D). In agreement with flow cytometry measurements, live cell imaging and the sensor was used to observe increased levels of intracellular FBP in XJ-4-85-treated SH-SY5Y cells (Figure 29E).

[0502] To determine whether the elevated FBP levels resulted in changes to glycolytic activity, the Seahorse extracellular acidification assay was employed, and increased extracellular acidification rates (ECAR) and a higher glycolytic capacity in XJ-4-85- but not XJ-4-97-treated cells were observed (Figures 29E and 41). Untargeted metabolomics was performed in cells to corroborate that acute XJ-4- 85 treatment resulted in a significant accumulation of glycolytic intermediates. Specifically, a prominent increase in FBP and dihydroxyacetone phosphate (DHAP) was detected compared to vehicle control (Figure 29G).

[0503] The effects of XJ-4-85 treatment in lipopolysaccharide (LPS) stimulated THP-1 macrophage cytokine release, a glycolysis-dependent process (Wang et al., 2024), was also investigated. Following LPS stimulation, XJ-4-85 significantly potentiated expression of the pro-inflammatory cytokines TNF- a and IL- 1 P compared to DMSO vehicle XJ-4-97 or NA-11 treatments (Figure 29H). This increase in inflammatory cytokine secretion supports that XJ-4-85-mediated glycolytic activation can functionally impact signaling responses.

[0504] EXAMPLE 7

[0505] The Covalent PFKL Activator XJ-4-85 is Cytotoxic to Cancer Cells

[0506] Next, a panel of cancer cell lines was treated with XJ-4-85 to assess the impact of covalent PFKL activation on tumor cell proliferation. The panel included THP-1 (monocytic leukemia), Jurkat (T-cell leukemia), HepG2 (hepatocellular carcinoma), A549 (lung adenocarcinoma), B16-F10-Luc2 (melanoma), MDA-MB-231 (triple -negative breast cancer), MOLM-14 (acute myeloid leukemia), and SH-SY5Y (neuroblastoma) cells. Unexpectedly, it was found that treatment with the PFKL activator was broadly cytotoxic across the cancer cell lines tested. The observed cytotoxicity appeared somewhat selective for cancer cells since non-cancerous HEK293T cells appeared less sensitive to XJ-4-85 treatment (Figure 30A).

[0507] Basal FBP levels was compared across the cell lines and observed that those with relatively higher basal FBP levels, including B16-F10-Luc2 and MOLM-14, were among the most sensitive to prolonged XJ-4-85 treatment (IC50 values of ~2.0 pM; Figures 30A and 30B). Washout experiments supported that the effect of extended XJ-4-85 treatment was mediated through irreversible PFKL binding, consistent with its covalent mode of action. The effects with XJ-4-119, which lacks the covalent warhead, were reversible after washout (Figure 30C).

[0508] EXAMPLE 8

[0509] XJ-4-85 Limited Melanoma Tumor Outgrowth In Vivo

[0510] The unexpected cytotoxic activity of XJ-4-85 in cancer cell lines prompted further investigations into the long-term effects of metabolic imbalance on tumor outgrowth in vivo. The observed selectivity of XJ-4-85 for PFKL and the cytotoxic activity of its leaving group present an opportunity for its delivery to FBP-high tumor cells. This electrophile-drug conjugate (EDC) concept was tested by directly comparing activity of XJ-4-85 and XJ-4-119 in a tumor model in vivo (Figure 30D).

[0511] B16-F10-Luc2 melanoma cells were selected for proof-of-concept studies in vivo because of enhanced basal FBP levels and sensitivity to XJ-4-85 (Figures 30A and 30b). A dose-escalation pilot study was performed to identify 10-50 mg / kg as the tolerated range for XJ-4-85 treatments in mice (Figure 42A). B16-F10-Luc2 melanoma cells (3 x 105cells) were subcutaneously injected into the right flanks of 6- to 8-week-old C57BL / 6 mice. Once tumors reached approximately 100 mm3, mice received daily intraperitoneal injections of XJ-4-85 (25 and 50 mg / kg), XJ-4-119 (leaving group, 25 mg / kg), XJ-4-97 (negative control, 100 mg / kg) or vehicle control (Figure 30E).

[0512] After two weeks, tumor expansion was evident in the vehicle -treated mice and dramatically and significantly reduced in XJ-4-85 treated mice (Figure 30F). Even at lower doses (25 mg / kg), XJ-4-85 treatment significantly controlled B16-F10-Luc2 tumor outgrowth. Mice treated with a high dose of XJ-4-97 (100 mg / kg) presented with tumor sizes comparable to vehicle -treated cohorts. Notably, administration of an equivalent amount of XJ-4-119, representing a maximal exposure of the payload in vivo, resulted in reduced efficacy compared to XJ-4-85 in support of an EDC mechanism. XJ-4-119 showed tumor-suppressive effects but was less effective at reducing tumor burden than XJ-4-85 (Figure 30F). Importantly, none of the treatments at these doses resulted in overt toxicity or weight loss in mice during the 2-week treatment paradigm (Figure 42B). In vivo luminescence imaging was employed to further confirm that XJ-4-85 was efficacious at reducing tumor burden compared to the vehicle group (Figure 30G, 43, and 44).

[0513] Collectively, the covalent PFKL activator XJ-4-85 blocked tumor growth in animal models with no apparent toxicity between 10 and 50 mg / kg and with evidence for an EDC mechanism.

[0514] EXAMPLE 9

[0515] XJ-4-85 Reprograms Cancer Cell Glycolysis and Signaling

[0516] To gain insights into XJ-4-85 mode of action, untargeted LC-MS / MS metabolomic profiling of XJ-4-85-treated B16-F10-Luc2 cells (5 pM, 2 hours) was performed. These studies revealed significant alterations across multiple metabolic pathways. KEGG pathway enrichment analysis of significantly altered metabolites indicated disruptions in starch and sucrose metabolism, pyrimidine metabolism, the pentose phosphate pathway, glutathione metabolism, and glycolysis / gluconeogenesis (Figure 31 A).

[0517] Deeper analysis uncovered a distinct glycolytic metabolic profile. XJ-4-85 treatment led to accumulation of key glycolytic metabolites, including glucose-6-phosphate, fructose-6-phosphate, FBP, and glyceraldehyde-3-phosphate (G3P), while pyruvate levels remained relatively unchanged (Figure 3 IB). This metabolic profile suggested that PFKL activation by XJ-4-85 may induce bottlenecks at specific steps of glycolysis and impair glucose utilization.

[0518] Next, TMT-based proteomics and phosphoproteomics were deployed to quantify changes in the proteome and phosphoproteome of B16-F10-Luc2 in response to XJ-4-85 treatments (5 pM). Cells were treated acutely (2 hours) to capture earlier changes in cell biology. Comparative analysis revealed that XJ-4-85 increased expression of ORMDL1, a protein associated with tumor suppression (Wang et al., 2021). XJ-4-85 significantly reduced the abundance of several proteins including SRGAP2, STMN2, S100A11, RhoA, and THUMPD3 (Figures 31C and 3 ID), previously reported to suppress cancer cell proliferation when down-regulated (Rodrigues et al., 2014; Kalpana et al., 2021; Li et al., 2021; Shao et al., 2023; Han et al., 2024; Klimontova et al., 2024).

[0519] Additionally, XJ-4-85 treatments led to the loss of site-specific phosphorylation for multiple oncogenic signaling proteins including HSP90AB1 (S255), eEF2 (T57), ERK2 (T183 / Y185), EIF4B (S406), and UCK2 (S254) (Mollapour et al., 2010; Wang et al., 2016; Wang et al., 2024). See Figure 31D. For example, inhibition of ERK2 phosphorylation at TI 83 / Y I 85 demonstrated anticancer effects against melanoma (Wong et al., 2014). XJ-4-85 treatment increased phosphorylation of HMGN1 at S87, which has been shown to disrupt DNA repair and chromatin regulation in cancer cells (Prymakowska-Bosak et al., 2002). Matching proteomics confirmed phosphorylation changes were not due to alterations in protein expression (Figure 31C). Collectively, these data suggested that XJ-4-85 exerts broader cellular effects beyond metabolic reprogramming, impacting key signaling pathways that regulate cell survival, growth, and stress responses.

[0520] Discussion of the EXAMPLES

[0521] TH211 (see Figure 2B) is an alkyne -modified SuTEx probe that contains a kinase fragment binding element (Franks et al., 2017) for chemical proteomic profiling of the kinome in live cells (Huang et al., 2021) SuTEx ligand analog of TH211, referred to as TH220 (see Figure 2C, has previously been reported and can block TH211 labeling of PFKL). See PCT International Publication No. WO 2023 / 023664, the disclosure of which is incorporated by reference in its entirety. Based on results with a biochemical assay, the EC50 of TH220 for PFKL activation was 52.4 pM. To improve the potency of the compounds for PFKL, a series of additional SuTEx ligands was designed incorporating a kinase binding element that is the same or similar in structure to that in TH211 and TH220. Figures 3A and 3B, for example, show that increasing the length of the ethylene moiety linking the kinase binding element and the triazole moiety improves EC50. Figure 3B shows that compounds where the ethylene moiety is replaced by propylene (XJ-3-9), butylene (XJ-3-42), or pentylene (XJ-3- 65) reduced the EC50 to under 5 pM. Additional analogs with longer chains between the kinase binding element and triazole were prepared. See, e.g., Figures 4A and 17. Exemplary variations on the kinase binding element are shown in Figure 15B. The group on the side of the sulfonyl group opposite the triazole was also varied, e.g., to various substituted and unsubstituted aryl and heteroaryl groups. See, for example, Figure 15A, as well as the compounds in Figures 4A and 17.

[0522] Results of the kinase assay with various SuTEx compounds are shown in Figures 4B, 16, 18, 19, 20, 21, and 22. Based on these results, several of the compounds with the longer alkylene group acted as activators for PFKL.

[0523] Based on these results, further studies were performed on select compounds, e.g., XJ-3-71, XJ- 4-65 and XJ-4-85. For example, the PFKL binding site of XH-3-71 was determined to be lysine 677 (K677), a lysine site in the regulatory domain, by studying the adduct of purified His-tagged PFKL and XJ-3-71 using liquid chromatography-tandem mass spectrometry (LC-MS / MS). See Figure 5. Activity assays were used to determine that the EC50 of XJ-4-65 was 712.4 nM (see Figure 6), while that of XJ- 4-85 was 746.3 nM. See Figures 9A and 9C. Additional EC50 data is shown in Table 1, below.

[0524] Table 1

[0525] PFKL EC50 values of Select Compounds

[0526] Recombinant PFKL, but not PFKP or PFKM assembles into fdaments. The asparagine at residue 702 (N702) in the PFKL regulatory domain is reported to be involved in mediating fdament assembly. N702 is adjacent to K677. To determine if filaments influence SuTEx ligand activity, the activity of XJ-4-65 in wild-type PFKL versus a N702T PFKL mutant was studied. See Figure 7. The EC50 of XJ-4-65 with the N702 mutant was 181.6 nM, i.e., lower than the EC50 of 712.4 nm in WT PFKL. In contrast, PFKL activation by XJ-4-65 was reduced in a mutant PFKL where the lysine at residue 315 is replaced by arginine (K315R). See Figures 8A and 8B. K315 is important for the activity of PFKL activators on recombinant PFKL. These findings showcased the different profiles of PFKL activators on endogenous PFKL and recombinant PFKL.

[0527] Additional studies with XJ-4-85 were performed. Using gel- and liquid chromatographytandem mass spectrometry (LC-MSZMS)-based chemical proteomics it was confirmed that XJ-4-85 covalently binds K677 in the regulatory domain of PFKL with good cellular potency, blocking SuTEx probe TH211. See Figures 9A-9D. As noted above, in a biochemical assay with purified His-tagged PFKL protein, XJ-4-85 activates PFKL with an EC50 of -740 nM. See Figure 9C. The impact of these findings is development of first-in-class activators of glycolysis that covalently bind lysine sites on PFKL.

[0528] Of particular note, XJ-4-85 exhibits PFK isoform- and proteome-wide-selectivity. The PFK1 enzymes are highly conserved (see Figure 10A), so it is challenging to selectively target this protein family. To investigate the isoform-specificity of XJ-4-85, biochemical assay studies were performed using purified His-tagged PFKL, PFKM, PFKP. It was demonstrated that XJ-4-85 activates PFKL but not related PFKM and PFKP, which provides evidence for isoform-specificity. See Figures 10C and 10D. TMT-based quantitative chemical proteomics data indicate that XJ-4-85 possesses excellent proteome-wide selectivity and principally and statistically significantly engages PFKL K677 across >3000 probe-modified sites evaluated across multiple cell lines. See e.g., Figures 11A-11C.

[0529] The cellular activity of the PFKL activator XJ-4-85 and matching PFKL-inactive negative control XJ-4-97 (see Figure 10B) was further assessed. Preliminary data show XJ-4-85 but not XJ-4- 97 activate glycolysis in THP-1 cells as determined by the stably expressed HYlight FBP sensor (Koberstein et al., 2022), and by evaluation of these cells using the Seahorse glycolytic flux assay. See Figures 12A and 12B. The increased glycolytic activity detected in XJ-4-85-treated THP-1 cells also correlated with increased cytokine production (TNF-a and IL-ip) in response to lipopolysaccharide stimulation (LPS). See Figure 12C. We also found that XJ-4-85 potentiated TCR activation in CD3 / CD28-activated Jurkat cells as measured by phospho-ERK levels. See Figure 12C. Importantly, XJ-4-85 treatment does not enhance but paradoxically shows a moderate blockade of tumor cell growth. See Figure 13. Additional studies with other SuTEx compounds with activity similar to XJ-4-85, i.e., XJ-3-129 and XJ-4-65 also increased TNF-a (see Figure 23) and reduced ROS. See Figure 14.

[0530] Antibody-drug conjugates (ADCs) are an important therapeutic modality in cancer with demonstrated efficacy across a wide range of malignancies (Dumontet et al., 2023). The rapidly growing interest in ADCs in the clinic stems from the ability to selectively deliver cytotoxic payloads to cancer cells, increasing the therapeutic index (Drago et al., 2021; Tsuchikama et al., 2024). ADC development, however, faces several significant challenges including limited access to cell surface proteins, high manufacturing costs and complexity, and the potential for immunogenic responses (Beck et al., 2017; Dumontet et al., 2023). A fully small molecule counterpart to ADCs could overcome many of these limitations including the ability to access intracellular targets. Disclosed herein is an electrophile-drug conjugate (EDC) strategy that targets glycolytically enhanced tumor cells using a selective covalent activator of PFKL to induce metabolic stress coupled to delivery of a cytotoxic leaving group.

[0531] The development of covalent activators in contrast with inhibitors is less common due to challenges with targeting allosteric regions on proteins (Wagner et al., 2016). An initial clue to the distinct activating mechanism of XJ-4-85 included the striking PFK1 isoform selectivity despite high conservation of the modified lysine site on PFKL. The cryo-EM structure of PFKL bound to XJ-4-85 revealed covalent and non-covalent interactions in the allosteric activating region engaged by ADP and NA-11 (Amara et al., 2021). These structural studies present two models for explaining the observed selectivity: (i) non-covalent binding of XJ-4-85 positions the SuTEx electrophile in proximity for K677 modification and / or (ii) covalent adduction at K677 facilitates binding of the departed leaving group in the allosteric pocket (Figures 28A-28H). A direct outcome from the distinct binding mode of XJ-4-85 is the exquisite proteome-wide selectivity observed in treated cells (Figures 27A-27I). Covalent PFKL activators in contrast with reversible ligands (Amara et al., 2021) provide prolonged activation (Figure 29H), which can be particularly beneficial in therapeutic contexts where boosting glycolysis is desired (Gleeson et al., 2016; Cai et al., 2019; Nemkov et al., 2024).

[0532] The high PFK1 isoform- and proteome-wide selectivity of XJ-4-85 supported its use as a chemical probe to elucidate metabolic and signaling impact of PFKL activation in cancer cells. Singlecell, real-time analyses revealed treatment with XJ-4-85 confers rapid production of FBP and a concomitant increase in both glycolytic flux and glycolytic metabolites. Importantly, XJ-4-85 was found to mediate metabolic reprogramming and alter cytokine production and global phosphorylation, revealing a broader role for PFKL in tumor cell biology. Notably, the prominent accumulation of key glycolytic metabolites with XJ-4-85 treatments indicated metabolic stress that is supported by broad perturbations to the phosphoproteome (Figures 29A-29H and 31A-3 ID).

[0533] The data presented herein identified XJ-4-85 as an antitumor agent that exerts its effects through a coupled mechanism involving: (i) metabolic imbalance through PFKL activation, and (ii) release of a leaving group that itself exhibits cytotoxic activity (Figures 30A-30G and 31A-31D). The enhanced antitumor activity of XJ-4-85 compared with XJ-4-119 at the same dose provides in vivo evidence in support of an EDC mechanism (Figures 30A-30G). Antitumor activity using a PFKL activator, while initially surprising given the Warburg effect, is supported by reports that excessive accumulation of intracellular FBP can disrupt glycolytic flux, induce energy stress, and thus inhibit tumor growth (Snaebjomsson et al., 2025). Additionally, FBP analogs have been shown to inhibit phosphoglycerate mutase 1 (PGAM1) activation, thereby suppressing cancer cell proliferation (Zhang et al., 2024). PFKL is also reported to regulate lipid metabolism and targeting this moonlighting function could affect tumor cell proliferation (Meng et al., 2024).

[0534] Summarily, disclosed herein are covalent, allosteric PFKL activators (e.g., XJ-4-85) loaded with a cytotoxic leaving group as an anticancer modality. The representative compound XJ-4-85 site- specifically bound K677 and its leaving group interacted with the nucleotide effector site to stabilize the active R-state tetramer of PFKL as determined by a cryo-electron microscopy structure (3.2 A resolution). Quantitative chemoproteomics revealed excellent PFK1 isoform and proteome-wide selectivity of XJ-4-85 in cells. XJ-4-85 treatments resulted in glycolytic reprogramming and reduced tumor burden in vivo compared with the free leaving group, presenting a rationale for electrophile-drug conjugates (EDCs) as a payload delivery strategy targeting intracellular metabolic proteins and vulnerabilities.

[0535] REFERENCES

[0536] All references listed in the instant disclosure, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (including but not limited to UniProt, EMBL, and GENBANK® biosequence database entries and including all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, and / or teach methodology, techniques, and / or compositions employed herein. The discussion of the references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.

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[0602] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMSWhat is claimed is:

1. A compound having a structure of Formula (I):wherein:X, Y, and Z are independently CH or N, subject to the proviso that at least one of X, Y, and Z is N;Ri is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl;Li is -(CTLjn- or -C(=0)-(CH2)ni -, wherein n is an integer selected from 3, 4, and 5; and R’ is selected from H,wherein:— is a double or single bond;A is selected from methylene, NH, O, and S;Ai is selected from CH and N;A2 is selected from C, CH, and N, subject to the proviso that when — is a single bond, A2 is N or CH, and when — is a double bond, A2 is C;R2 and R3 are each selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; andR4, R5, R„. and R7 are each independently selected from the group consisting of H and alkyl; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, where two of X, Y, and Z are N, optionally wherein Z and Y are each N and X is CH.

3. The compound of claim 1 or claim 2, wherein Ri is selected from lower alkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, wherein substituted aryl and substituted heteroaryl are aryl or heteroaryl groups substituted with one or more substituent selected from the group consisting of halo, cyano, alkyl, alkoxy, haloalkyl, haloalkoxy, perhaloalkyl, perhaloalkoxy,cycloalkyl, aralkyl, aryl, amino, and amido, or wherein two substituents together form a divalent group, optionally alkylene, halo-substituted alkylene, alkylenedioxy, or halo- substituted alkylenedioxy group.

4. The compound of any one of claims 1-3, wherein Ri is selected from substituted phenyl or substituted pyridinyl, optionally wherein said substituted phenyl or substituted pyridinyl is phenyl or pyridinyl substituted with at least one alkoxy or haloalkoxy group or wherein the substituted phenyl is a phenyl group substituted with two substituents that together form a divalent group, optionally an alkylenedioxy group or halo-substituted alkylenedioxy group, further optionally -O(CH2)O-, -O(CF2)O-, or -O(CH2)2O-.

5. The compound of any one of claims 1-4, wherein n is 4 and Li is butylene.

6. The compound of any one of claims 1-5, wherein R’ is H oroptionally wherein A is methylene, further optionally wherein R4, R5, Re, and R7 are each H.

7. A compound of any one of claims 1-5, wherein the compound of Formula (I) has a structure of Formula (II) or Formula (III):wherein:— is a double or single bond;X, Y, Z, Li, Ai, A2, and R1-R7 are as defined for Formula (I); or a pharmaceutically acceptable salt thereof.

8. The compound of claim 7, wherein R2 and / or R3 is substituted aryl or substituted heteroaryl, wherein said substituted aryl or substituted heteroaryl is aryl or heteroaryl substituted with one or more substituents selected from halo, nitro, cyano, carboxyl, ester, formyl, alkyl, halo- substituted alkyl, alkoxy, haloalkoxy, aryl, and heteroaryl.

9. The compound of claim 8, wherein R2 and / or R3 is halo-substituted phenyl, optionally fluorosubstituted phenyl, further optionally mono- or di-fluoro-substituted phenyl.

10. The compound of any one of claims 1-9, wherein R4, R5, Rs. and R7 are each independently H or methyl, optionally wherein two, three, or four of R4, Rs, Rs. and R7 are H.

11. The compound of any one of claims 1-10, wherein the compound of Formula (I) has a structure of Formula (IV):wherein:X, Y, and Z are independently CH or N, subject to the proviso that at least one of X, Y, and Z is N, optionally wherein Y and Z are N and X is CH;Ri is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, optionally substituted aryl or substituted heteroaryl;Li is -(CH2)n-, wherein n is an integer selected from 3, 4, and 5, optionally 4;R2 and R3 are each selected from aryl, substituted aryl, heteroaryl, and substituted heteroaryl, optionally fluoro-substituted phenyl; andR4, R5, R6, and R7 are each independently selected from the group consisting of H and alkyl, optionally H or methyl; or a pharmaceutically acceptable salt thereof.

12. The compound of claim 1, wherein the compound is selected from the group consisting of:XJ-3-129 XJ-3-71and pharmaceutically acceptable salts thereof.

13. A pharmaceutical composition comprising a compound of any one of claims 1-12 or a pharmaceutically acceptable salt thereof.

14. A method of selectively activating the liver isoform of phosphofructokinase (PFKL), wherein the method comprises contacting a sample comprising PFKL with a compound of any one of claims 1-12 or a pharmaceutical composition of claim 13.

15. The method of claim 14, wherein the sample comprising PFKL is selected from a cell extract, a biological fluid, a cell, a tissue, an organ, or an organism.

16. The method of claim 15, wherein the sample comprising PFKL is a mammal, optionally a human, and activating PFKL in the sample provides increased glycolysis and / or T cell activation, optionally tumor-infiltrating T cell (TIL) reactivation.

17. A method of treating a cancer and / or a tumor in a subject in need thereof, and / or for inhibiting growth of a cell associated therewith, the method comprising administering to the subject an effective amount of a compound of any one of claims 1-12 or a pharmaceutical composition of claim 13.

18. The method of claim 17, wherein the compound is selected from the group consisting of XJ-3- 41, XJ-4-27, XJ-3-129, XJ-3-71, XJ-4-65, XJ-4-85, XJ-4-89, XJ-4-97, XJ-3-9, XJ-3-65, and pharmaceutically acceptable salts thereof.

19. The method of claim 17, wherein the compound is XJ-4-85.

20. The method of any one of claims 17-19, wherein the cancer and / or the tumor is selected from a breast cancer and / or tumor, a liver cancer and / or tumor, a colorectal cancer and / or tumor, a leukemia, optionally a monocytic leukemia, a T-cell leukemia, and / or an acute myeloid leukemia; a hepatocellular carcinoma, a lung adenocarcinoma, a melanoma, a triple-negative breast cancer, and / or a neuroblastoma.

21. A method for enhancing fructose- 1,6-bisphosphate (FBP) content in a cell, the method comprising contacting the cell with an effective amount of a compound of any one of claims 1- 12 or a pharmaceutical composition of claim 13.

22. The method of claim 21, wherein the compound is selected from the group consisting of XJ-3- 41, XJ-4-27, XJ-3-129, XJ-3-71, XJ-4-65, XJ-4-85, XJ-4-89, XJ-4-97, XJ-3-9, XJ-3-65, and pharmaceutically acceptable salts thereof.

23. The method of claim 21, wherein the compound is XJ-4-85.

24. The method of claim 21, wherein the cell is a tumor cell and / or a cancer cell, or an endothelial cell associated with a tumor and / or a cancer.

25. The method of claim 24, wherein the cell is from a solid tumor and / or cancer selected from the group consisting of breast tumor and / or cancer, a liver tumor and / or cancer, and / or a colorectal tumor and / or cancer.

26. The method of claim 24, wherein the tumor cell and / or the cancer cell is a leukemia cell, optionally monocytic leukemia cell, a T-cell leukemia cell, and / or an acute myeloid leukemia cell; a hepatocellular carcinoma cell, a lung adenocarcinoma cell, a melanoma cell, a triplenegative breast cancer cell, and / or a neuroblastoma cell.

27. Use of a compound of any one of claims 1-12 or a pharmaceutical composition of claim 13 for treating a cancer and / or a tumor in a subject in need thereof, for inhibiting growth of a cell associated therewith, or for enhancing fructose- 1,6-bisphosphate (FBP) content in a cell of cancer and / or the tumor.

28. The use of claim 27, wherein the compound is selected from the group consisting of XJ-3-41, XJ-4-27, XJ-3-129, XJ-3-71, XJ-4-65, XJ-4-85, XJ-4-89, XJ-4-97, XJ-3-9, XJ-3-65, and pharmaceutically acceptable salts thereof.

29. The use of claim 27, wherein the compound is XJ-4-85.

30. The use of any one of claims 27-29, wherein the cancer and / or the tumor is selected from a breast cancer and / or tumor, optionally a triple-negative breast cancer and / or tumor; a liver cancer and / or tumor, a colorectal cancer and / or tumor, a leukemia, optionally a monocytic leukemia, a T-cell leukemia, and / or an acute myeloid leukemia; a hepatocellular carcinoma, a lung adenocarcinoma, a melanoma, and / or a neuroblastoma.

31. A compound for use in treating a cancer and / or a tumor in a subject in need thereof, for inhibiting growth of a cell associated therewith, and / or for enhancing fructose- 1 ,6-bisphosphate (FBP) content in a cell of cancer and / or the tumor wherein the compound is selected from the group consisting of XJ-3-41, XJ-4-27, XJ-3-129, XJ-3-71, XJ-4-65, XJ-4-85, XJ-4-89, XJ- 4-97, XJ-3-9, XJ-3-65, and pharmaceutically acceptable salts thereof.

32. The compound for use of claim 31, wherein the compound is XJ-4-85.

33. The compound for use of claim 31 or claim 32, wherein the cancer and / or the tumor is selected from a breast cancer and / or tumor, optionally a triple-negative breast cancer and / or tumor; a liver cancer and / or tumor, a colorectal cancer and / or tumor, a leukemia, optionally a monocytic leukemia, a T-cell leukemia, and / or an acute myeloid leukemia; a hepatocellular carcinoma, a lung adenocarcinoma, a melanoma, and / or a neuroblastoma.

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