Hmgcs1 inhibitors for cancer therapy

Abstract

The present disclosure relates to compounds comprising an aryl ring core having (S)-1-cyano-N-((R)-1-(4-(prop-2-yn-1- ylcarbamoyl and pyrrolidine-3-carboxamide substituents, that covalently bind the catalytic site of HMGCS1 and exhibit highly potent and selective inhibition thereof, encompassing compositions and methods thereof, for treating cancers that are associated with aberrant activity of 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), or associated with, e.g., characterized or mediated by hyperactive mTORC1 signaling.

Description

Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOHMGCS1 INHIBITORS FOR CANCER THERAPYRELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 63 / 755,511, filed February 7, 2025 and U.S. Provisional Application No: 63 / 906,160, filed October 27, 2025, each of which are incorporated herein by reference in their entireties.BACKGROUND

[0002] Enzymes involved in the first two stages of cholesterol synthesis, which convert acetyl CoA into squalene, are referred to as the mevalonate pathway (MVP). The enzymes in the mevalonate pathway not only produce cholesterol for membrane biology but also produce key isoprenoids for cell signaling, thus playing essential roles in various aspects of cellular physiology (Goldstein, et al., Nature, 1990, 343, 425). While the mevalonate pathway is critical to producing essential non-sterol isoprenoids, excess production of mevalonate and sterol products contributes to the onset of human diseases such as cardiovascular disease and cancer progression (Clendening et al. PNAS, 2010, 107, 15051). Specifically, cancer cells show increased mevalonate pathway flux, promoting tumor cell fitness, potentially assisting glucose uptake, and activating various signaling pathways involving protein prenylation. Previous studies indicate that inhibiting the mevalonate pathway leads to apoptosis in cancer cells, which can be rescued by the supplementation of geranylgeraniol, the downstream non-sterol metabolite of mevalonate (Juarez, et al., Trends in Cancer, 2021, 7, 525). As a result, several enzymes within this pathway, such as HMGCR, FDPS, GGDPS1, FTase, and GGTase, have emerged as promising therapeutic targets in the past decades.

[0003] Historically considered a rate-limiting enzyme due to its extensive regulation at the transcriptional, translational, and post-translational levels, the field has focused primarily on HMGCR as an exploratory target for an anti cancer therapeutic (Longo, et al., Clin Cancer Res, 2020, 26, 5792). Despite extensive research, current strategies and inhibitors of HMGCR have not demonstrated substantial clinical anti-cancer efficacy (Jiang, et al., J Exp Clin Cancer Res, 40, 241). For example, repurposing statins as anti-cancer agents has drawn significant interest in the field. However, the results of various epidemiologic studies have been inconsistent, mainly1AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WObecause a 1000-fold higher dose of statins is needed to inhibit HMGCR compared to the U.S. Food and Drug Administration (F.D.A.) approved dosage. This huge discrepancy arises from the different mechanisms of HMGCR inhibition, that while sufficient to lower blood cholesterol levels, do not effectively inhibit cell proliferation. Statins lower blood cholesterol by inducing the LDL receptor (LDLR) on the cell surface via SREBP2-mediated transcription as a feedback mechanism, in addition to inhibiting HMGCR, which facilitates the uptake of blood cholesterol into cells. However, the anti-proliferative effect of statins primarily comes from the direct inhibition of HMGCR activity and blockade of mevalonate synthesis. Accumulating research shows that HMGCR inhibition by statins induces approximately a 200-fold increase in HMGCR levels, thereby hindering efficient and consistent downregulation of its activity.

[0004] HMGCS1, the initial enzyme in the mevalonate pathway and upstream of HMGCR, possesses distinct characteristics that set it apart from HMGCR and other related enzymes, including a catalytic cysteine within the active site. (Yi, etal., Mol Cell. 2024, 84, 2166). A recent study reports that hymeglusin, a natural product purified from fungi in the 1980s, inhibits HMGCS1 but exhibits poor serum stability, leading to a significant loss of effectiveness after 8 hours across different cell lines, which limits its use in cellular and in vivo studies (Yi, et al., J. Biol. Chem, 2025, 301, 110660). Other recent reports of multiple cysteome profiling studies with various electrophilic small molecules failed to identify small molecules that react with HMGCSl's catalytic cysteine, despite its presence in the hydrophobic binding pocket (Kuljanin et al., Nat. Biotechnol., 2021, 39, 630, and Vinogradova et al., Cell, 2020, 182, 1009).

[0005] Accordingly, there is a need for more effective inhibitors of MVP for purposes of treating cancer.SUMMARY

[0006] Its unique features relative to other MVP enzymes aside, HMGCS1 has been largely overlooked as a target for therapeutic intervention for cancer treatment. The present disclosure, which provides, inter alia, compounds that covalently bind the catalytic pocket of HMGCS1 and achieve potent and selective inhibition of this enzyme in MVP, may provide a solution to this need.

[0007] Accordingly, a first aspect of the present disclosure is directed to a compound having a structure represented by formula I:2AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOpharmaceutically acceptable salt or stereoisomer thereof,wherein:Ri is hydrogen, methyl, ethyl, isopropyl, hydroxyl, or halo;Ring A is C > or Cio aryl or 5- or 6-membered heteroaryl with one or two heteroatoms selected from nitrogen, oxygen and sulfur, wherein the aryl and heteroaryl may be (i.e., optionally) substituted by one or two identical or different groups selected from methyl, hydroxyl, halo, and trifluoromethyl;Ring A’ is absent, Ce aryl, or pyridinyl, wherein the aryl and pyridinyl may be substituted by one or two identical or different groups selected from methyl, hydroxyl, halo, and trifluoromethyl, provided that a) when Ring A is Cio aryl, Ring A’ is absent, and that b) when Ring A’ is absent, R2 is bonded to Ring A; andONR R, wherein R’ is CH2CCH or Ci-Ce alkyl optionally substituted with -OH, -NH2, -NHMe, or -NMe2; R” is hydrogen or R’ and R”, together with the nitrogen atom to which they are bound, form a piperazine ring optionally substituted with methyl or hydroxymethyl.

[0008] A second aspect of the present disclosure is directed to a compound having a structure represented by formula I’ :(!’), or a pharmaceutically acceptable salt or stereoisomer thereof,wherein:Ri is hydrogen, methyl, ethyl, isopropyl, hydroxyl, or halo;Ring A is Ce or Cio aryl or 5- or 6-membered heteroaryl with one or two heteroatoms selected from nitrogen, oxygen and sulfur, wherein the aryl and heteroaryl may be (i.e., optionally) substituted by one or two identical or different groups selected from methyl, hydroxyl, halo, and trifluoromethyl;3AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WORing A’ is absent, Ce aryl, or pyridinyl, wherein the aryl and pyridinyl may be substituted by one or two identical or different groups selected from methyl, hydroxyl, halo, and trifluoromethyl, provided that a) when Ring A is Cio aryl, Ring A’ is absent, and that b) when Ring A’ is absent, R2 is bonded to Ring A;OR2 is, wherein R’ is CH2CCH or Ci-Ce alkyl optionally substituted with -OH, -NH2, -NHMe, or -NMe2; R” is hydrogen or R’ and R”, together with the nitrogen atom to which they are bound, form a piperazine ring optionally substituted with methyl or hydroxymethyl; and R3 is Ci-Ce alkyl or phenyl.

[0009] A third aspect of the present disclosure is directed to a pharmaceutical composition containing the compound of formula I or I’, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0010] Another aspect of the present disclosure is directed to a method of treating a cancer that is associated with, e.g., is characterized or mediated by, aberrant activity of 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), comprising administering to a subject in need thereof the compound of formula I or I’, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the compound of formula I or I’ or a pharmaceutically acceptable salt thereof.

[0011] In some embodiments, the cancer is a gastric cancer, cervical cancer, liver cancer, lung cancer, breast cancer, hematological cancer, or colon cancer. In some embodiments, the cancer is associated with, e.g., is characterized or mediated by hyperactive mTORCl signaling.

[0012] Further aspects of the present disclosure are directed to methods of making the compounds.

[0013] Working examples below demonstrate that inventive compounds such as CNP7 and CNP9, validated by multiple chemoproteomic, chemical biology, and cell biological experiments in human cell lines, are first-in-class inhibitors of HMGCS1. They covalently bind the catalytic site of HMGCS1 and exhibit highly potent and selective inhibition of this enzyme. They may provide a compelling alternative to statins and inhibition of HMGCR for purposes of treatment of cancer.BRIEF DESCRIPTION OF THE DRAWINGS4AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0014] FIG. 1A-FIG. 1J show the assessment of the binding affinity of cyanopyrrolidine derivatives to HMGCS1 in vitro and in cells. FIG. 1A shows workflow for labeling recombinant HMGCS 1 protein with Hymeglusin-fluorescein (HG-FL) activity -based probe following treatment with cyanopyrrolidine (CNF) derivatives. FIG. IB shows in-gel fluorescence assay of recombinant HMGCS 1 (1 pM), showing the dose-dependent labeling efficiency of eight CNP derivatives. FIG.1C shows the quantification graph of the in-gel fluorescence assay result in FIG. IB and data are represented as means ± s.d. (n=3~5). FIG. ID shows ICso values of GK16S and eight CNPs to saturate HMGCS 1 were calculated based on the curves in FIG 1C. FIG. IE (top) scheme of the HG-FL labeling assay for HMGCS 1 labeling by CNP in cells; (bottom) profiling the reactive catalytic cysteine of HMGS1 after treatment with the indicated small molecules confirms that CNP7 exhibits the highest labeling efficiency of HMGCS 1 in HEK293T cells. FIG. IF shows HEK293T cells were treated with increasing concentrations of CNP7 for 4 hours, followed by cell lysis, HG-FL treatment, and in-gel fluorescence analysis for HMGCS 1 cysteine activity profiling. The quantification graph is shown in the bottom (means ± s.d. of biological triplicates). FIG. 1G shows DMEM media supplemented with or without 10% FBS were pre-incubated with CNP7 or HG for 2 or 16 h. Then, HEK293T cells were incubated with the media for 2 h, followed by lysis and in vitro reaction with the HGFL probe. The in-gel fluorescent analysis shows the prolonged efficacy of CNP7 after 16 hours of preincubation with the media, whereas HG lost its efficacy after incubation with serum-containing media. FIG. 1H shows in vitro incubation of HG-TMR with recombinant HMGC SI (1 pg) orBSA (1 pg) produces a red fluorescence signal only in lanes where HMGCS1 was loaded. FIG. II shows HEK293T and HCT116 cells were treated with CNP7 (0.5pM) for the indicated time points, followed by lysis and reaction with the HG-FL probe. Subsequently, in-gel fluorescence analysis and immunoblotting with HMGCS 1 antibody were performed. Quantification of relative fluorescence intensity from three replicates is presented (means ± s.d.). FIG. 1J shows the HG-FL-based in-gel fluorescence assay of MOLM-13, Jurkat, MM. IS, and HepG2 cells treated with CNP7 (0.5 pM) for the indicated time points.

[0015] FIG. 2A-FIG. 2J shows dual activity -based profiling of CNP derivatives for their reactivity in cells. FIG. 2A shows the workflow of orthogonal in-gel fluorescence analysis. Cells treated with vehicle (DMSO) or CNP molecule were lysed and divided into two parts; one was incubated with HG-FL to assess HMGCS 1 labeling potency, and the other part was subjected to click chemistry by incubating with tetramethyl rhodamine (TMR) or fluorescein (FL)-azide to test5AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOtheir reactivity toward the proteome (selectivity). FIG. 2B shows an orthogonal in gel fluorescence assay of 293T cells treated with indicated compounds (0.5 pM) for 4 hours. Arrows: potential CNP-labeled proteins; asterisks: non-specific bands. FIG. 2C shows orthogonal in gel fluorescence assay of 293T cells treated with CNP7 (0.5 pM) for the indicated time points. FIG. 2D shows 293T cells treated with CNP7 (0.5 pM) for 1 hour were lysed and subjected to click reaction with TMR-azide, followed by in-gel fluorescence assay and immunoblotting with HMGCS1 antibody. FIG.2E shows chemical structures of CNP7 derivatives. FIG. 2F and FIG. 2G shows the HG-FL based in-gel fluorescence assay of 293T cells treated with the indicated compounds (0.5 pM) for 4 hours. FIG. 2H shows 293T cells treated with indicated CNPs (0.5 pM), or left untreated, for 4 hours were subjected to orthogonal in-gel fluorescence assay for activity profiling and immunoblotting with HMGCS1 antibody. Left: lysates clicked with FL-azide for global reactivity profiling, right: lysates incubated with HG-FL to compare the HMGCS1 occupancy rate. FIG. 21 shows the HG-FL-based activity profiling of 293 T cells treated with the indicated concentrations of CNP7 and CNP9 for 4 hours. FIG. 2J shows the quantification of relative fluorescence intensity from three replicates is presented (means ± s.d., n = 3 biological replicates for each concentration).

[0016] FIG. 3A-FIG. 3J show the proteome-wide reactivity of CNP7 and CNP9 through complementary chemoproteomics. FIG. 3 A shows a workflow of direct affinity precipitation-mass spectrometry (AP-MS) following click chemistry. 293T cells treated with vehicle, CNP7, or CNP9 (0.5 pM) for 1 hour were lysed and subjected to a copper-mediated click reaction with biotinazide. After streptavidin bead enrichment, the eluates were analyzed through tandem-mass-tag (TMT)-based proteomic analysis. FIG. 3B shows volcano plots of the -loglO-transformed p-value versus the log2 -transformed ratio of CNP7 / DMSO (left) or CNP9 / DMSO (right) treated cells prepared as presented in FIG. 3A. n = 3 biological replicates, p-values were calculated by two-sided Welch’s t-test (adjusted to 1% FDR for multiple comparisons, S0=0.585). FIG. 3C shows a workflow of competitive AP-MS. Lysates of 293T cells pre-treated with CNP7 or CNP9 (0.5 pM) for 1 hour were incubated with CNP7-biotin or CNP9-biotin (5 pM) for 1 hour, respectively. After streptavidin beads enrichment, the eluates were analyzed through TMT-based proteomic analysis. FIG. 3D shows volcano plots of the -loglO-transformed p-value versus the log2 -transformed ratio of DMSO / CNP7 (left) or DMSO / CNP9 (right) pre-treated cells prepared as presented in FIG. 3C. n = 3 biological replicates, p-values were calculated by two-sided Welch’s t-test (adjusted to 1% FDR for multiple comparisons, S0=0.585). FIG. 3E and FIG. 3F show 293T cells treated as 6AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOdescribed in FIG. 3C and FIG. 3G were probed with the indicated antibodies. Rep: replicate. FIG.3G shows a workflow of competitive scavenging MS. Lysates of 293T cells pre-treated with CNP7 or CNP9 (0.5 pM) for 1 hour were incubated with CNP7-biotin or CNP9-biotin (5 pM) for 1 hour, respectively. The lysates were taken before (input) and after (flow-through) streptavidin enrichment and subjected to TMT-based proteomic analysis. FIG. 3H shows volcano plots of the -loglO-transformed p-value versus the log2 -transformed ratio of CNP7 / DMSO (left) or CNP9 / DMSO (right) pre-treated cells prepared as presented in FIG. 3G. TMT signal of flow-through (FT) of each protein was normalized to that of input, n = 3 or 4 biological replicates, p-values were calculated by two-sided Welch’ s t-test (adjusted to 1% FDR for multiple comparisons, SOO.585). FIG. 31 and FIG. 3J are Venn diagrams showing significant interactors of CNP7 (FIG.31) or CNP9 (FIG. 31) identified through three proteomics approaches.

[0017] FIG. 4A-FIG. 41 show that proteome integral solubility alteration (PISA) assay detects the interactors of CNP7 and CNP9. FIG. 4A is a schematic diagram of the thermal stability assay. FIG. 4B shows the workflow applied to the PISA analysis. HEK293T or HCT116 cell lysates were left untreated or treated with CNP7 (5pM) or CNP9 (5 pM) for 15 minutes. Subsequently, the samples were divided into 12 tubes, and a heat gradient was applied. The samples were then pooled, centrifuged, digested, and labeled with TMTpro for analysis by mass spectrometry. FIG.4C-FIG. 4D are volcano plots of the -loglO-transformed p-value versus the log2 -transformed ratio of CNP7 / DMSO (left) or CNP9 / DMSO (right), n = 6, 5, 5 biological replicates, p-values were calculated by two-sided Welch’s t-test (adjusted to 1% FDR for multiple comparisons, S0=0.285). FIG. 4E is a Venn diagram showing the overlapping hits for CNP7 between HEK293T and HCT116. Proteins with a Log2 ratio greater than 0.3, or less than -0.3 with p<0.001, were curated. FIG. 4F and FIG. 4G are immunoblots to verify the thermal stability changes of HMGCS1, PDE6D, and RI0K2 after incubating the lysates with CNP7 or CNP9.

[0018] FIG. 5A-FIG. 5C shows structural study of CNP7-bound to human HMGCS1. FIG. 5 A is the CNP7-bound HMGCS1 dimer structure solved by cryo-EM analysis in 2.3 A resolution. FIG.5B is a surface plot showing that CNP7 is in the cavity of HMGCSl’s catalytic pocket. FIG. 5C shows the detailed view of CNP7-bound HMGCS1, with several hydrogen bonds highlighted.

[0019] FIG. 6A-FIG. 6K show HMGCS1 inhibition by CNP7 blocks the mevalonate pathway. FIG. 6A is a schematic illustrating the mevalonate pathway flux. The level of two metabolites (HMG-CoA and GGPP) within this pathway can be measured through immunoblotting of7AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOHMGylated FASN and prenylated proteins. FIG. 6B shows HCT116 cells were treated as indicated, and the cell extracts were probed with an anti-HMG antibody to detect HMGylated FASN. FIG. 6C shows lysates of HCT116 cells treated as indicated were incubated with HG-FL for 1 hour, followed by an in-gel fluorescence assay. The proteins on the gel were then transferred to a PVDF membrane and probed with the indicated antibodies. FIG. 6D-FIG. 6F show HCT116 cells were treated as indicated, and the cell extracts were probed with a RhoA antibody. FIG. 6G shows 293T cells were treated with increasing concentrations of Hymeglusin (HG), CNP7, and Simvastatin (Statin) for 48 hours, followed by cell viability assay using flow-cytometry. Means ± s.d. of biological triplicates. FIG. 6H shows HMGCS1 inhibition by HG or CNP7 inhibits the colony-forming activity of HCT116 cells. FIG. 61 shows colony numbers are presented as means ± s.d. (n = 6 biological replicates for each treatment). FIG. 6J shows reduced viability of HCT116 cells by CNP7 was rescued by the supplementation of geranylgeraniol (GGOH). Cell viability was evaluated 48 hours after the treatment. Means ± s.d. of biological triplicates. FIG. 6K shows that supplementation of GGOH in the culture media reversed the increase in unprenylated RhoA by CNP7. HCT116 cells treated with CNP7 and GGOH for 48 hours were subjected to immunoblotting analysis.

[0020] FIG. 7A-FIG. 7E shows evaluating cell line-specific response to CNP7. FIG. 7A and FIG.7B show the 18 cancer cell lines that were evaluated for their viability after 72 hours of treatment with hymeglusin, CNP7, or simvastatin. Response was categorized as resistant to both CNP7 and simvastatin, or sensitive to CNP7 and resistant to simvastatin. FIG. 7C shows three resistant cell lines (HCT116, HT29, and HepG2) and three sensitive cell lines (RPE1, MFE296, andMOLM13) that were evaluated for expression of HMGCS1 after 24 hours of treatment with 1 pM CNP7. Resistant cell lines showed higher basal HMGCS1 expression than sensitive cells, which further increased after CNP7 treatment. FIG. 7D shows HCT116 and MFE296 cells were tested for their HMGCS1 levels at varying concentrations of CNP7. HCT116 cells showed increased HMGCS1 expression at lower doses of the compound compared to MFE296. FIG. 7E shows quantification of FIG. 7D. Means ± s.d. of biological duplicates.

[0021] FIG. 8A-FIG. 8G show increased demand on HMGCS1 during cell proliferation. FIG.8A shows MFE296 cells exhibited hyperactive mTORCl signaling due to PTEN gene deletion and activating mutations on PI3K. FIG. 8B shows TMT -based proteomic comparison of MFE296 cells after treatment with DMSO or mTORCl inhibitor RM6 (16 hrs, 4 nM). Volcano plots of the8AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO-loglO-transformed p-value versus the log2-transformed ratio of DMS0 / RM6. n = 3 biological replicates. FIG. 8C shows quantification data of approximately 20 proteins in the mevalonate pathway are curated from panel b and displayed as a heatmap. FIG. 8D is a heatmap showing the relative expression of the MVP enzymes quantified by TMT-based proteomics after culturing the indicated cell lines in DMEM or HPLM for three weeks. FIG. 8E shows cell lysates treated as in FIG. 8D were blotted for mTORCl substrates, phospho-S6K and 4EBP1, indicating significantly different mTORCl activity caused by the different nutrient levels. FIG. 8F and FIG. 8G show immunoblotting analysis of WT and PTEN- / - 22Rvl cells or WT and TSC2- / - HEK293T cells using the indicated antibodies.

[0022] FIG. 9A-FIG. 9E show binding affinity of cyanopyrrolidine derivatives to HMGCS1. FIG. 9A is the sequence of the expected tryptic peptide that includes the catalytic cysteine of human HMGCS1. FIG. 9B shows the structure of Hymeglusin-Fluorescein (HG-FL). FIG. 9C shows (top) workflow of HG-FL-based activity profiling using recombinant HMGC SI and GK16S and (bottom) an in-gel fluorescence analysis indicates a concentration-dependent decrease in fluorescent signaling, suggesting that GK16S labels the catalytic cysteine of HMGCS1. FIG. 9D shows HEK293T cells ectopically expressing WT or C129A mutant HMGCS1-V5 were incubated with GK16S (1 pM, 24h). Then, the cell lysates were subjected to click chemistry with TMR-azide, followed by enrichment of HMGCS1-V5 using anti-V5 beads. The eluates were analyzed by in-gel fluorescence for TMR signal, and then by immunoblotting with an anti-HMGCSl antibody. FIG. 9E is a series of schematics showing the two substrates of HMGCS1.

[0023] FIG. 10A shows HEK293T cells were treated with CNP9 for increasing durations at 0.5 pM concentration (left) or at different concentrations for 4 hours (right), followed by dual activitybased profiling using HG-FL or click-chemistry via the alkyne group on the CNP9 molecule. FIG.10B shows assessment of cellular HMGCS1 occupation by CNP9, which was performed by treating HEK293T cells with CNP9 over increasing durations, followed by HG-FL (top) or HG-TMR (bottom) treatment and subsequent immunoblotting.

[0024] FIG. 11 is a 2D-correlation plot of CNP7 and CNP9 PISA.

[0025] FIG. 12 depicts that treatment of MFE296 cells with GGTi298 shows induction of RhoA protein.

[0026] FIG. 13A-FIG. 13E show cell line-specific response to CNP7. FIG. 13A shows cell viability analysis on various cell lines after treatment with Hymeglusin, CNP7 or Simvastatin for9AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO72 hours. FIG. 13B shows activity profiling of the indicated cell lines using HG-FL after CNP7 treatment, followed by immunoblotting analysis. FIG. 13C shows HCT116 or MFE296 cells were treated with the indicated concentrations of CNP7 or simvastatin (St, 10 pM), followed by analysis of HMGCS1 and RhoA via immunoblotting. FIG. 13D and FIG. 13E show immunoblotting for SREBP2 was performed after treating HCT116 and MFE296 cells with increasing concentrations of CNP7 to evaluate the appearance of cleaved SREBP2.

[0027] FIG. 14A-FIG. 14H show global proteome changes upon the inhibition of HMGCS1 by CNP7 or HMGCR by Simvastatin. FIG. 14A is a workflow of TMTpro-based global proteome analysis of HCT116 cells treated with CNP7 (5 pM), Simvastatin (10 pM), and / or GGOH (10 pM) for 24 hours. FIG. 14B shows that the extracts treated as in panel (a) were immunoblotted using the indicated antibodies. FIG. 14C is a volcano plot of-loglO-transformed p-value versus the log2-transformed ratio of CNP7 / untreated. n = 3 biological replicates, p-values were calculated by two-sided Welch’s t-test (adjusted to 1% FDR for multiple comparisons, S0=0.585). FIG. 14D is a linear regression plot visualizing the relationship between proteome changes by CNP7 (x-axis) and Simvastatin (y-axis). FIG. 14E shows gene ontology analyses of the statistically upregulated proteins (31 proteins) with more than 2-fold changes are shown. FIG. 14F shows 13 quantified proteins in the mevalonate / sterol pathway were curated, and their fold changes in expression are presented as a heat map. FIG. 14G shows a hierarchical clustering of isoprenylation targets. Proteins that increase upon NCP7 treatment and are reversed by GGOH supplementation are labeled in red, while those that decrease upon CNP7 treatment and are reversed by GGOH are in blue. FIG. 14H shows the selected proteins in panel 9 plotted as bar graphs. Means ± s.d. of biological triplicatesDETAILED DESCRIPTION

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated in order to facilitate the understanding of the present disclosure.

[0029] As used in the description and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Therefore, for example,10AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOreference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor” includes mixtures of two or more such inhibitors, and the like.

[0030] Unless stated otherwise, the term “about” means within 10% (e.g., within 5%, 2%, or 1%) of the particular value modified by the term “about.”

[0031] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. When used in the context of the number of heteroatoms in a heterocyclic structure, it means that the heterocyclic group has the minimum number of heteroatoms. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the disclosure.

[0032] With respect to compounds of the present disclosure, and to the extent the following terms are used herein to further describe them, the following definitions apply.

[0033] As used herein, the term “alkyl” refers to a saturated linear or branched-chain monovalent hydrocarbon radical. In some embodiments, and to the extent not disclosed otherwise for any one or more groups of the compounds disclosed herein), the alkyl is a Ci-Ce, C2-C6, C3-C6, C4-C6, Cs-C6, C6, C1-C5, C2-C5, C3-C5, C4-C5, C5, C1-C4, C2-C4, C3-C4, C4, C1-C3, C2-C3, C3, C1-C2, C2, or Ci group. Examples of alkyl groups include methyl, ethyl, 1 -propyl, 2-propyl, i-propyl, 1 -butyl, 2-methyl-1 -propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3 -methyl- 1 -butyl, 2-methyl-l -butyl, 1 -hexyl, 2-hexyl, 3 -hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.

[0034] As used herein, the term "aryl" refers to a radical derived from an aromatic hydrocarbon by the removal of one hydrogen atom (e.g., phenyl). The term "aryl" may be used interchangeably with the term "aryl ring". In one embodiment, aryl includes monocyclic, bicyclic or tricyclic ring systems having 6-14 carbon atoms (C6-C14). Representative examples of aryl groups include phenyl, naphthyl, and anthracenyl.

[0035] As used herein, the term "heteroaryl" refers to a monocyclic, bicyclic or tricyclic ring system having 5 to 12 ring atoms, wherein at least one ring is aromatic and contains at least one heteroatom. In one embodiment, heteroaryl includes 5- to 6-membered monocyclic aromatic11AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOgroups with one or more ring atoms selected from nitrogen, oxygen, and sulfur. Representative examples of heteroaryl groups include thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, imidazopyridyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, tetrazolo[l,5-b]pyridazinyl, purinyl, deazapurinyl, benzoxazolyl, benzofuryl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoimidazolyl, 2,3-dihydrobenzofuryl, indolinyl, indolyl, l,3-thiazol-2-yl, l,3,4-triazol-5-yl, l,3-oxazol-2-yl, l,3,4-oxadiazol-5-yl, l,2,4-oxadiazol-5-yl, l,3,4-thiadiazol-5-yl, lH-tetrazol-5-yl, and l,2,3-triazol-5-yl.

[0036] As used herein, the term “halogen” (or “halo” or “halide”) refers to fluorine, chlorine, bromine, or iodine.

[0037] In a first aspect, compounds of the disclosure are represented by formula I:or a pharmaceutically acceptable salt or stereoisomer thereof,wherein:Ri is hydrogen, methyl, ethyl, isopropyl, hydroxyl, or halo;Ring A is Ce or Cio aryl or 5- or 6-membered heteroaryl with one or two heteroatoms selected from nitrogen, oxygen and sulfur, wherein the aryl and heteroaryl may be substituted by one or two identical or different groups selected from methyl, hydroxyl, halo, and trifluoromethyl;Ring A’ is absent, Ce aryl, or pyridinyl, wherein the aryl and pyridinyl may be substituted by one or two identical or different groups selected from methyl, hydroxyl, halo, and trifluoromethyl, provided that a) when Ring A is Cio aryl, Ring A’ is absent, and that b) when Ring A’ is absent, R2 is bonded to Ring A; andOR2 is, wherein R’ is CH2CCH or C1-C6 alkyl optionally substituted with -OH, -NH2, -NHMe, or -NMe2; R” is hydrogen or R’ and R”, together with the nitrogen atom to which they are bound, form a piperazine ring optionally substituted with methyl or hydroxymethyl.

[0038] In some embodiments,12AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0039] In some embodiments, the compounds of formula (I) are represented by formula la or lb:pharmaceutically acceptable salt thereof.

[0040] In some embodiments, Ri is hydrogen.

[0041] In some embodiments, Ri is methyl.

[0042] In some embodiments, Ri is ethyl. In some embodiments, Ri is isopropyl. In some embodiments, Ri is hydroxyl. In some embodiments, Ri is halo.

[0043] In some embodiments, Ring A is Ce aryl.

[0044] In some embodiments, Ring A is Cio aryl.

[0045] In some embodiments, Ring A’ is Ce aryl.

[0046] In some embodiments,.O "

[0047] In some embodiments, lb is, wherein R’ is Ci-Ce alkyl, wherein said alkyl is optionally substituted with -OH, -NH2, -NHMe, or -NMe2; and R” is hydrogen.O"

[0048] In some embodiments, R2 is;wherein R’ and R”, together with the nitrogen atom to which they are bound, form an optionally substituted piperazine ring.

[0049] The disclosed compounds also embrace combinations of the specific Ri, R2, Ring A, and Ring A’ groups disclosed above.

[0050] In a second aspect, compounds of the disclosure are represented by formula I’:or a pharmaceutically acceptable salt or stereoisomer thereof,wherein:Ri is hydrogen, methyl, ethyl, isopropyl, hydroxyl, or halo;13AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WORing A is Ce or Cio aryl or 5- or 6-membered heteroaryl with one or two heteroatoms selected from nitrogen, oxygen and sulfur, wherein the aryl and heteroaryl may be substituted by one or two identical or different groups selected from methyl, hydroxyl, halo, and trifluoromethyl;Ring A’ is absent, Ce aryl, or pyridinyl, wherein the aryl and pyridinyl may be substituted by one or two identical or different groups selected from methyl, hydroxyl, halo, and trifluoromethyl, provided that a) when Ring A is Cio aryl, Ring A’ is absent, and that b) when Ring A’ is absent, R2 is bonded to Ring A;O"wherein R’ is CH2CCH or Ci-Ce alkyl, wherein said alkyl is optionally substituted with -OH, -NH2, -NHMe, or -NMe2; R” is hydrogen or R’ and R”, together with the nitrogen atom to which they are bound, form a piperazine ring optionally substituted with methyl or hydroxymethyl; andR3 is Ci-Ce alkyl or phenyl.

[0051] In some embodiments, the compounds of formula I’ are represented by formula I’a orpharmaceutically acceptable salt thereof.

[0052] In some embodiments, Ri is hydrogen.

[0053] In some embodiments, Ri is methyl.

[0054] In some embodiments, Ri is ethyl. In some embodiments, Ri is isopropyl. In some embodiments, Ri is hydroxyl. In some embodiments, Ri is halo.

[0055] In some embodiments, Ring A is Ce aryl.

[0056] In some embodiments, Ring A is Cio aryl.

[0057] In some embodiments, Ring A’ is Ce aryl.

[0058] In some embodiments,14AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOO NR'R"

[0059] In some embodiments, R2 is, wherein R’ is Ci-Ce alkyl, wherein said alkyl is optionally substituted with -OH, -NH2, -NHMe, or -NMe ; and R” is hydrogen.O"

[0060] In some embodiments, R2 is, wherein R’ and R”, together with the nitrogen atom to which they are bound, form an optionally substituted piperazine ring.

[0061] In some embodiments, Rs is C1-C3 alkyl. In some embodiments, R? is methyl. In some embodiments, R3 is phenyl.

[0062] The disclosed compounds also embrace combinations of the specific Ri, R2, R3, Ring A, and Ring A’ groups disclosed above.

[0063] In some embodiments, the compounds of the present disclosure are represented by any of the following structures:15AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOstereoisomer thereof.

[0064] Compounds of the present disclosure may be in the form of a free acid or free base, or a 16AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOpharmaceutically acceptable salt. Pharmaceutically acceptable salts and common methodology for preparing them are well known in the art. See, for example, Stahl, P., et al., Handbook of Pharmaceutical Salts: Properties, Selection and Use, (VCHA / Wiley-VCH, 2002); Gould, P. L., “Salt selection for basic drugs,” International Journal of Pharmaceutics, 33:201-217 (1986); Bastin, R. J., etal., “Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,” in Organic Process Research and Development, 7:427-435 (2000); and Berge, S. M., et al., “Pharmaceutical Salts,” in Journal of Pharmaceutical Sciences, 66:1-19 (1977).

[0065] Compounds of the present disclosure may have at least one chiral center and thus may be in the form of a stereoisomer, which as used herein, embraces all stereoisomers of individual compounds that differ only in the orientation of their atoms in space. The term stereoisomer includes mirror image isomers (enantiomers which include the (R-) or (S-) configurations of the compounds), mixtures of mirror image isomers (physical mixtures of the enantiomers, and racemates or racemic mixtures) of compounds, geometric (cis / trans or E / Z, R / S) isomers of compounds and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereoisomers). Additionally, the skilled artisan will appreciate that additional chiral centers may be created in the compounds of the invention by the selection of certain variables. The present disclosure contemplates all individual enantiomers or diastereomers, as well as mixtures of the enantiomers and diastereomers of said compounds including racemates. The skilled artisan will also appreciate that the Cahn-Ingold-Prelog (R) or (S) designations for all chiral centers will vary depending on the substitution patterns of the compound. The single enantiomers or diastereomers may be prepared beginning with chiral reagents or by stereoselective or stereospecific synthetic techniques. Alternatively, the single enantiomers or diastereomers may be isolated from mixtures by standard chiral chromatographic or crystallization techniques at any convenient point in the synthesis of compounds of the disclosure.

[0066] In some embodiments, a compound of the present disclosure is an isotopic derivative in that it has at least one desired isotopic substitution of an atom, at an amount above the natural abundance of the isotope, i.e., enriched. As used herein, the term “hydrogen”, i.e., H, refers to all isotopes of hydrogen, including protium (1H) and deuterium (2H). As used herein, the term “compound” embraces isotopic derivatives.

[0067] Compounds of the present disclosure may also be in the form of N-oxides, crystalline forms (also known as polymorphs), co-crystals, active metabolites of the compounds having the17AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOsame type of activity, prodrugs, tautomers, and unsolvated as well as solvated (e.g, hydrated) forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, of the compounds. As used herein, the term “compound” embraces all these forms.Methods of Synthesis

[0068] In some aspects, the present disclosure is directed to a method for making a compound of formula I or I’, or a pharmaceutically acceptable salt or stereoisomer thereof. Broadly, the compounds or pharmaceutically acceptable salts or stereoisomers thereof, may be prepared by any process known to be applicable to the preparation of chemically related compounds. The compounds of the present disclosure will be better understood in connection with the synthetic schemes that described in a working example (below) that illustrates non-limiting methods by which the compounds of the disclosure may be prepared.

[0069] The compounds of formula I / T can be prepared by methods known by those skilled in the art. In one non-limiting example the disclosed compounds can be made by scheme I.Scheme 1. Representative synthetic procedure for compounds of formula I / F .Pharmaceutical Compositions

[0070] Another aspect of the present disclosure is directed to a pharmaceutical composition that includes the compound of formula I or I’, or a pharmaceutically acceptable salt or stereoisomer 18AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOthereof, and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier,” as known in the art, refers to a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present disclosure to mammals. Suitable carriers may include, for example, liquids (both aqueous and non-aqueous alike, and combinations thereof), solids, encapsulating materials, gases, and combinations thereof (e.g., semi-solids), and gases, that function to carry or transport the compound from one organ, or portion of the body, to another organ, or portion of the body. A carrier is “acceptable” in the sense of being physiologically inert to and compatible with the other ingredients of the formulation and not injurious to the subject (also referred to herein as “patient”). Depending on the type of formulation, the composition may also include one or more pharmaceutically acceptable excipients.

[0071] Broadly, compounds of formula I and I’, and their pharmaceutically acceptable salts may be formulated into a given type of composition in accordance with conventional pharmaceutical practice such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping and compression processes (see, e.g., Remington: The Science and Practice of Pharmacy (A. Gennaro, et. al., eds., 22nd ed., Loyd V., ed., Pharmaceutical Press, 2012), each of which is incorporated herein by reference in its entirety. The type of formulation depends on the mode of administration which may include enteral e.g., oral, buccal, sublingual and rectal), parenteral (e.g., subcutaneous (s.c.), intravenous ( / .v.), intramuscular (i.mf, and intrasternal injection, or infusion techniques, intra-ocular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, interdermal, intravaginal, intraperitoneal, mucosal, nasal, intratracheal instillation, bronchial instillation, and inhalation) and topical (e.g., transdermal).

[0072] In general, the most appropriate route of administration will depend upon a variety of factors including, for example, the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and / or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). For example, parenteral (e.g., intravenous) administration may also be advantageous in that the compound may be administered relatively quickly such as in the case of a single-dose treatment and / or an acute condition.

[0073] Pharmaceutical compositions according to the invention may take a form suitable for parenteral (e.g., z.v.), oral, buccal, nasal, topical, ophthalmic or rectal administration, or a form suitable for administration by inhalation or insufflation.19AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0074] Injectable preparations for parenteral administration may include sterile aqueous solutions or oleaginous suspensions. They may be formulated according to standard techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. The effect of the compound may be prolonged by slowing its absorption, which may be accomplished by the use of a liquid suspension or crystalline or amorphous material with poor water solubility. Prolonged absorption of the compound from a parenterally administered formulation may also be accomplished by suspending the compound in an oily vehicle.

[0075] For oral administration, the pharmaceutical compositions may take the form of, for example, tablets, lozenges or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methyl cellulose); fdlers (e.g. lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium glycollate); or wetting agents (e.g. sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles or preservatives. The preparations may also contain buffer salts, flavoring agents, coloring agents or sweetening agents, as appropriate.20AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0076] Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

[0077] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0078] The compounds of formula I or I’ may be formulated for parenteral administration by injection, e.g. by bolus injection or infusion. Formulations for injection may be presented in unit dosage form, e.g. in glass ampoules or multi-dose containers, e.g. glass vials. The compositions for injection may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, preserving and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.

[0079] In addition to the formulations described above, the compounds of formula I or I’ may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation or by intramuscular injection.

[0080] For nasal administration or administration by inhalation, the compounds according to the present invention may be conveniently delivered in the form of an aerosol spray presentation for pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. dichlorodifluoromethane, fluorotrichloromethane, di chlorotetrafluoroethane, carbon dioxide or other suitable gas or mixture of gases.

[0081] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack or dispensing device may be accompanied by instructions for administration.

[0082] For topical administration the compounds of use in the present invention may be conveniently formulated in a suitable ointment containing the active component suspended or dissolved in one or more pharmaceutically acceptable carriers. Particular carriers include, for example, mineral oil, liquid petroleum, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax and water. Alternatively, the compounds of use in the present invention may be formulated in a suitable lotion containing the active component suspended or dissolved in one or more pharmaceutically acceptable carriers. Particular carriers include, for example, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, benzyl alcohol, 2-octyldodecanol and water.21AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0083] For ophthalmic administration the compounds of use in the present invention may be conveniently formulated as micronized suspensions in isotonic, pH-adjusted sterile saline, either with or without a preservative such as a bactericidal or fungicidal agent, for example phenylmercuric nitrate, benzylalkonium chloride or chlorhexidine acetate. Alternatively, for ophthalmic administration compounds may be formulated in an ointment such as petrolatum.

[0084] For rectal administration the compounds of use in the present invention may be conveniently formulated as suppositories. These can be prepared by mixing the active component with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and so will melt in the rectum to release the active component. Such materials include, for example, cocoa butter, beeswax and polyethylene glycols.Dosage Amounts

[0085] As used herein, the term, "therapeutically effective amount" refers to an amount of a compound of formula I or I’, or a pharmaceutically acceptable salt or a stereoisomer thereof; or a composition including a compound of formula I or I’, or a pharmaceutically acceptable salt or a stereoisomer thereof, effective in producing the desired therapeutic response in a particular subject in need thereof. Therefore, the term "therapeutically effective amount" includes the amount of a compound of formula I or I’, or a pharmaceutically acceptable salt or a stereoisomer thereof, that when administered, induces a positive modification in the cancer to be treated, or is sufficient to prevent development or progression of the cancer, or alleviate to some extent, one or more of the symptoms of the cancer being treated in a subject, or reduces the amount ofHMGCSl in cancerous cells.

[0086] The compounds of the present disclosure may be effective over a dosage range. The amount of the compound administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the selected compound or compounds administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. See, for example, Goodman and Gilman ’s The Pharmacological Basis of Therapeutics, 10th Edition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001.

[0087] Compounds of formula I and I’, and their pharmaceutically acceptable salts and stereoisomers may be effective over a wide dosage range. In some embodiments, the daily dosages22AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOmay range from around 10 ng / kg to 1000 mg / kg, typically from 100 ng / kg to 100 mg / kg, e.g. around 0.01 mg / kg to 40 mg / kg.Methods of Use

[0088] In some aspects, the present disclosure provides a method of treating a cancer that is associated with (e.g., characterized or mediated by) aberrant HMGCS1 activity, comprising administering to a subject in need thereof a compound of formula I or I’ or a pharmaceutically acceptable salt or stereoisomer thereof.

[0089] In a related aspect, the present disclosure provides a compound of formula I or I’, or a pharmaceutically acceptable salt or stereoisomer thereof, for use in treating a cancer that is associated with (e.g., characterized or mediated by) aberrant HMGCS1 activity.

[0090] In another related aspect, the present disclosure provides a compound of formula I or I’, or a pharmaceutically acceptable salt or stereoisomer thereof, for use in therapy, in particular for treating a cancer that is associated with (e.g., characterized or mediated by) aberrant HMGCS1 activity.

[0091] In yet another related aspect, the present disclosure provides the use of a compound of formula I or I’, or a pharmaceutically acceptable salt or stereoisomer thereof, in the manufacture of a medicament for treating a cancer that is associated with (e.g., characterized or mediated by) aberrant HMGC SI activity.

[0092] The term “subject” (or “patient”) as used herein includes all members of the animal kingdom prone to or suffering from the indicated disease or disorder. In some embodiments, the subject is a mammal, preferably a human.

[0093] In some embodiments, the cancer is a gastric cancer, cervical cancer, liver cancer, lung cancer, breast cancer, hematological cancer, or colon cancer.

[0094] In some embodiments, the cancer is associated with, e.g., further characterized or mediated by, hyperactive mTORCl signaling. In some embodiments, the hyperactive mTORCl signaling is caused by a double mutation in the PTEN-PI3K-mTOR pathway. In some embodiments, the double mutation comprises a PTEN deletion and a PI3K activating mutation. Representative examples of such cancer associated with hyperactive mTORCl signaling include endometrial cancer, lung, gastric, colorectal, renal, urinary bladder cancer, prostate, breast, head and neck squamous cell carcinoma. See, e.g., Tian et al., Int. J. Mol. Sci., 2019, 20(3):755 and Kim etal., Oncogene, 201736:2191-2201.23AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0095] Compounds of formula I and I’ may be administered to a cancer patient as a monotherapy or by way of combination therapy. Therapy may be "front / first-line", i.e., as an initial treatment in patients who have undergone no prior anti-cancer treatment regimens, either alone or in combination with other treatments; or "second-line", as a treatment in patients who have undergone a prior anti-cancer treatment regimen, either alone or in combination with other treatments; or as "third-line", "fourth-line", etc. treatments, either alone or in combination with other treatments. Therapy may also be given to patients who have had previous treatments which were unsuccessful or partially successful but who became unresponsive or intolerant to the particular treatment. Therapy may also be given as an adjuvant treatment, z.e., to prevent reoccurrence of cancer in patients with no currently detectable disease or after surgical removal of a tumor. Thus, in some embodiments, the compounds may be administered to a patient who has received another therapy, such as chemotherapy, radioimmunotherapy, surgical therapy, immunotherapy, radiation therapy, targeted therapy or any combination thereof.Combination Therapy

[0096] The compounds of formula I and T and their pharmaceutically acceptable salts and stereoisomers may be used in combination or concurrently with at least one other active agent, e.g., anti-cancer agent or regimen, in treating cancer. The terms “in combination” and “concurrently” in this context mean that the agents are co-administered, which includes substantially contemporaneous administration, by way of the same or separate dosage forms, and by the same or different modes of administration, or sequentially, e.g., as part of the same treatment regimen, or by way of successive treatment regimens. Thus, if given sequentially, at the onset of administration of the second compound, the first of the two compounds is in some cases still detectable at effective concentrations at the site of treatment. The sequence and time interval may be determined such that they can act together (e.g., synergistically) to provide an increased benefit than if they were administered otherwise. For example, the therapeutics may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they may be administered sufficiently close in time so as to provide the desired therapeutic effect, which may be in a synergistic fashion. Thus, the terms are not limited to the administration of the active agents at exactly the same time.

[0097] In some embodiments, the treatment regimen may include administration of a compound of formula I or I’ in combination with one or more additional therapeutics known for use in treating24AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOcancer. The dosage of the additional therapeutic may be the same or even lower than known or recommended doses. See, Hardman, eta!., eds., Goodman & Gilman's the Pharmacological Basis of Basis of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; Physician's Desk Reference 60th ed., 2006. Anti-cancer agents that may be suitable for use in combination with the compounds are known in the art. See, e.g., U.S. Patent 9,101,622 (Section 5.2 thereof) and U.S. Patent 9,345,705 B2 (Columns 12-18 thereof). Representative examples of additional anti-cancer agents and treatment regimens include radiation therapy, chemotherapeutics (e.g., mitotic inhibitors, angiogenesis inhibitors, anti-hormones, autophagy inhibitors, alkylating agents, intercalating antibiotics, growth factor inhibitors, anti-androgens, signal transduction pathway inhibitors, antimicrotubule agents, platinum coordination complexes, HDAC inhibitors, proteasome inhibitors, and topoisomerase inhibitors), immunomodulators, therapeutic antibodies (e.g., mono-specific and bispecific antibodies) and CAR-T therapy.

[0098] In some embodiments, a compound of formula I or F and the additional (e.g., anticancer) therapeutic may be administered less than 5 minutes apart, less than 30 minutes apart, less than 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours part. The two or more (e.g., anti cancer) therapeutics may be administered within the same patient visit.

[0099] When the active components of the combination are not administered in the same pharmaceutical composition, it is understood that they can be administered in any order to a subject in need thereof. For example, a compound of the present disclosure can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the25AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOadministration of the additional therapeutic, to a subject in need thereof. In various aspects, the therapeutics are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart. In one example, the (e.g., anticancer) therapeutics are administered within the same office visit. In another example, the combination anticancer therapeutics may be administered at 1 minute to 24 hours apart.

[0100] In some embodiments, a compound of formula I or I’ and the additional anti-cancer agent or therapeutic are cyclically administered. Cycling therapy involves the administration of one anticancer therapeutic for a period of time, followed by the administration of a second anticancer therapeutic for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one or both of the anticancer therapeutics, to avoid or reduce the side effects of one or both of the anti cancer therapeutics, and / or to improve the efficacy of the therapies. In one example, cycling therapy involves the administration of a first anticancer therapeutic for a period of time, followed by the administration of a second anticancer therapeutic for a period of time, optionally, followed by the administration of a third anticancer therapeutic for a period of time and so forth, and repeating this sequential administration, i.e., the cycle in order to reduce the development of resistance to one of the anticancer therapeutics, to avoid or reduce the side effects of one of the anti cancer therapeutics, and / or to improve the efficacy of the anticancer therapeutics.

[0101] In some embodiments, the compounds of the present disclosure may be used in combination with other anti-cancer agents, examples of which include Etoposide (e.g., lymphomas, and non-lymphocytic leukemia), Vincristine (e.g., leukemia), Daunorubicin (e.g., acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), and Kaposi's sarcoma), Rituximab (e.g., non-Hodgkin's lymphoma), Alemtuzumab (e.g., chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL) and T-cell lymphoma), Bortezomib (e.g., multiple myeloma and mantle cell lymphoma), Pegaspargase (e.g., acute lymphoblastic leukemia), Keytruda® (e.g., Hodgkin lymphoma), and dexamethasone (e.g., acute multiple myeloma).26AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0102] In some embodiments, the additional anti-cancer agent is a statin (e.g., Simvastatin). See, Duarte et al., Biomed. Pharmacother., 2021, 141.111858.

[0103] Other aspects of the present disclosure will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the disclosure but are not intended to limit its scope, as defined by the claims.EXAMPLES

[0104] Example 1: Synthesis of Compounds

[0105] General Details

[0106] All reagents were purchased from commercial sources and used as received unless stated otherwise. Analytical TLC has been performed on Silicycle SiliaPlate TLC plates and visualized under UV light (254 nm) or by staining with potassium permanganate. Flash chromatography was carried out on Sigma silica gel (200-300 mesh). All compounds were characterized by UPLC-SQD-LC-MS (Waters). (4.6 mm *150 mm 5 pm C18 column; 10 L injection; 10-100% CH3CN / H2O, linear-gradient, with constant 0.1% v / v TFA additive; 8 min run; ESI; positive ion mode; UV detection at 190-500 nm).

[0107] Synthesis of (S)-l-cyano-N-((3'-(prop-2-yn-l-y1carbamoyl)-[l,l'-biphenyl]-3-

[0108] To a solution of 3'-(((te / 7-butoxycarbonyl)amino)methyl)-[l,l'-biphenyl]-3-carboxylic acid (50 mg, 0.15 mM) in DMF (5 mL) was added N,N-diisopropylethylamine (DIPEA, 58 mg, 0.45 mM) and hexafluorophosphate azabenzotri azole tetramethyl uronium (HATU, 85 mg, 0.225 mM) and the reaction mixture stirred for 30 min at room temperature (rt). Propargylamine (9 mg, 0.165 mM) was then added, and the reaction mixture was stirred overnight. The reaction was quenched with water and extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a residue which was purified by CombiFlash using ethyl acetate / hexane (gradient wash from 0% to 50% ethyl acetate) to give the product as a white solid (40 mg, 68%). H NMR (500 MHz, CDCh) 87.97 (s, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.68 (d,27AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOJ = 7.8 Hz, 1H), 7.52 - 7.42 (m, 3H), 7.39 (t, J = 7.6 Hz, 1H), 7.28 (d, J = 7.4 Hz, 1H), 6.72 (s, 1H), 5.04 (s, 1H), 4.36 (d, J = 6.1 Hz, 2H), 4.26 (dd, J = 5.2, 2.6 Hz, 2H), 2.28 (t, J = 2.6 Hz, 1H), 1.46 (s, 9H);13C NMR (126 MHz, CDC13) 5 167.16, 156.03, 141.45, 140.51, 139.67, 134.34, 130.42, 129.19, 129.06, 126.86, 126.21, 125.95, 125.91, 79.57, 77.31, 77.05, 76.80, 71.86, 60.42, 44.67, 29.82, 28.43, 14.20.

[0109] / c / 7-Butyl ((3'-(prop-2-yn-l-ylcarbamoyl)-[l,r-biphenyl]-3-yl)methyl)carbamate (30 mg, 0.11 mM) was added to a solution of DCM:TFA (3 mb, 3:1) and stirred for 30 min at rt. The reaction mixture was concentrated under reduced pressure and the residue was dissolved in DMF (3 mL). DIPEA (42 mg, 0.45 mM) and HATU (62 mg, 0.165 mM) were added and the reaction mixture stirred for 30 min at rt. (S)-l-(te / 7-butoxycarbonyl)pyrrolidine-3 -carboxylic acid (26 mg, 0.12 mM) was then added, and the reaction mixture stirred overnight. The reaction was quenched with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a residue which was purified by CombiFlash using ethyl acetate / hexane (gradient wash from 0% to 100% ethyl acetate) to give the product as a white solid (30 mg, 53%). 'H NMR (500 MHz, CDCh) 87.87 (d, J = 13.9 Hz, 1H), 7.65 (d, J = 6.5 Hz, 1H), 7.53 (t, J = 7.2 Hz, 1H), 7.40 - 7.22 (m, 5H), 7.19 - 7.09 (m, 1H), 6.90 (t, J = 5.9 Hz, 1H), 4.34 (dd, J = 14.1, 5.8 Hz, 2H), 4.13 (dd, J = 5.4, 2.6 Hz, 2H), 3.46 (q, J = 10.0 Hz, 1H), 3.40 -3.27 (m, 2H), 3.14 (dd, J = 29.4, 7.5 Hz, 1H), 2.83 (p, J = 8.2 Hz, 1H), 2.16 (t, J = 2.6 Hz, 1H), 2.10 - 2.01 (m, 1H), 1.39 - 1.35 (m, 1H), 1.33 (s, 9H).13C NMR (126 MHz, CDCI3) 6 171.21, 154.41, 141.14, 140.53, 138.98, 134.37, 130.27, 129.22, 129.01, 126.25, 126.04, 79.73, 79.48, 77.33, 77.08, 76.82, 71.53, 60.41, 48.71, 44.67, 43.53, 29.69, 29.32, 28.54, 28.47, 21.05, 14.19, 1.02.

[0110] / c' / V-Butyl (S)-3-(((3'-(prop-2-yn-l-ylcarbamoyl)-[l,l'-biphenyl]-3-yl)methyl)carbamoyl)pyrrolidine-l -carboxylate (30 mg, 0.11 mM) was added to a solution of DCM:TFA (3 mL, 3:1) and stirred for 30 min at rt. The reaction mixture was concentrated under 28AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOreduced pressure and the residue was dissolved in DCM (3 mL) on ice. Potassium carbonate (22 mg, 0.26 mmol) and a 3 M solution of cyanogen bromide in DCM (15 pL, 0.08 mmol) were added to the reaction mixture and stirred overnight. The reaction was quenched with water and extracted with ethyl acetate. The organic layer was concentrated under reduced pressure and the residue was purified via CombiFlash to give the title compound (16 mg, 53%). 'H NMR (500 MHz, DMSO) 8 9.06 (t, J = 5.5 Hz, 1H), 8.62 (t, J = 5.9 Hz, 1H), 8.14 (s, 1H), 7.90 - 7.78 (m, 2H), 7.67 - 7.55 (m, 3H), 7.47 (t, J = 7.6 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 4.45 - 4.32 (m, 2H), 4.10 (dd, J = 5.5, 2.6 Hz, 2H), 3.56 (t, J = 8.6 Hz, 1H), 3.49 - 3.42 (m, 2H), 3.41 - 3.35 (m, 1H), 3.15 (t, J = 2.6 Hz, 1H), 3.07 (p, J = 7.5 Hz, 1H), 2.17 - 2.07 (m, 1H), 2.05 - 1.94 (m, 1H).13C NMR (126 MHz, DMSO) 8171.88, 166.28, 140.65, 140.51, 140.00, 134.98, 130.06, 129.59, 129.57, 127.16, 126.94, 126.29, 125.94, 125.90, 117.75, 81.75, 73.43, 52.96, 50.54, 43.70, 42.75, 40.51, 40.44, 40.35, 40.27, 40.18, 40.10, 40.01, 39.93, 39.85, 39.68, 39.51, 30.02, 29.01. HRMS w / zfor C23H23N4O2 ([M+H]+) calculated: 387.1821, found: 387.1825.

[0111] The syntheses of other cyanopyrrolidine derivatives were performed in a similar manner as above.

[0113] 'H NMR (500 MHz, DMSO) 8 8.89 (t, J = 5.6 Hz, 1H), 8.61 (t, J = 5.9 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 4.34 (d, J = 4.6 Hz, 2H), 4.05 (dt, J = 3.9, 2.0 Hz, 2H), 3.56 (dd, J = 9.3, 7.8 Hz, 1H), 3.47 - 3.41 (m, 2H), 3.41 - 3.36 (m, 1H), 3.33 (t, 1H), 3.12 (t, J = 2.5 Hz, 1H), 3.06 (p, J = 7.4 Hz, 1H), 2.14- 2.06 (m, 1H), 2.02 - 1.94 (m, 1H).13CNMR(126 MHz, DMSO) 8 171.94, 171.86, 166.16, 143.34, 132.88, 127.85, 127.46, 117.74, 81.84, 73.29, 52.95, 50.52, 43.67, 43.62, 42.38, 42.26, 40.51, 40.44, 40.35, 40.27, 40.18, 40.01, 39.84, 39.68, 39.51, 29.94, 28.92, 28.81. HRMS m,z for CnHwN^ ([M+H]+) calculated: 311.1511, found: 311.1508.29AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0115] XH NMR (500 MHz, DMSO) 88.93 (t, J = 5.6 Hz, 1H), 8.61 (t, J = 6.0 Hz, 1H), 7.79 -7.69 (m, 2H), 7.43 (d, J = 7.0 Hz, 2H), 4.37 - 4.30 (m, 2H), 4.05 (dd, J = 5.6, 2.5 Hz, 2H), 3.60 -3.51 (m, 1H), 3.47 - 3.35 (m, 3H), 3.12 (t, J = 2.5 Hz, 1H), 3.06 (p, J = 7.5 Hz, 1H), 2.10 (td, J = 12.5, 7.3 Hz, 1H), 2.04 - 1.92 (m, 1H).13C NMR (126 MHz, DMSO) 8 171.89, 166.35, 139.99, 134.41, 130.70, 128.86, 126.84, 126.10, 117.74, 81.80, 73.29, 52.92, 50.52, 43.67, 42.54, 40.51, 40.34, 40.26, 40.17, 40.10, 40.01, 39.84, 39.67, 39.51, 29.99, 28.96. HRMS zw zfor C17H19N4O2 ([M+H]+) calculated: 311.1512, found: 311.1508.

[0116] (S)-l -cyano-N-((S)-l-(4-(prop-2-yn-l-ylcarbamoyl)phenyl)ethyl)pyirolidine-3-carboxamide (CNP3)

[0117] 1H NMR (500 MHz, CDCh) 57.95 (t, J = 8.2 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 6.83 (s, 1H), 6.03 (d, J = 7.8 Hz, 1H), 5.19 - 5.08 (m, 1H), 4.64 (t, J = 2.9 Hz, 1H), 3.61 - 3.52 (m, 3H), 3.49 - 3.38 (m, 1H), 2.89 (p, J = 7.7 Hz, 1H), 2.80 (s, 1H), 2.40 (s, 2H), 2.26-2.11 (m, 2H), 1.56 - 1.45 (m, 3H).13C NMR (126 MHz, DMSO) 6 171.86, 143.32, 132.86, 127.84, 127.44, 117.74, 81.81, 73.30, 52.93, 50.51, 43.66, 43.61, 42.24, 40.49, 40.41, 40.32, 40.25, 40.16, 40.08, 39.99, 39.91, 39.82, 39.66, 39.49, 29.93, 28.91, 28.80. HRMS m'z for CISH2IN4O2+([M+H]+) calculated: 325.1668, found: 325.1665.

[0118] (S)-l -cyano-N-((R)-l-(4-(prop-2-yn-l-ylcarbamoyl)phenyl)ethyl)pyrrolidine-3-carboxamide (CNP4)

[0119] 'H NMR (500 MHz, DMSO) 88.87 (t, J = 5.7 Hz, 1H), 8.55 (dd, J = 15.9, 7.7 Hz, 1H), 7.84 (dd, J = 29.7, 8.0 Hz, 2H), 7.41 (dd, J = 30.2, 8.6 Hz, 2H), 4.94 (p, J = 7.0 Hz, 1H), 4.05 (dd, J = 5.5, 2.5 Hz, 2H), 3.57 - 3.49 (m, 1H), 3.44 - 3.35 (m, 3H), 3.30 (s, 1H), 3.12 (t, J = 2.5 Hz, 1H), 3.04 (p, J = 7.4 Hz, 1H), 2.69 (s, 2H), 2.15 - 2.00 (m, 1H), 1.94 - 1.82 (m, 1H), 1.36 (d, J = 7.1 Hz, 3H).13C NMR (126 MHz, DMSO) 8 171.03, 166.20, 148.60, 132.77, 127.85, 126.20, 117.75, 81.84, 73.27, 52.86, 50.50, 48.33, 43.54, 40.51, 40.34, 40.27, 40.18, 40.10, 40.01, 39.93,30AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO39.84, 39.67, 39.51, 38.72, 29.98, 28.91, 22.71. HRMS m / z for CisH2iN4O2+([M+H]+) calculated: 325.1664, found: 325.1665.[001201 ( )-l -cyano-N-((7-(prop-2-yn-l-ylcarbamoyl)naphthalen-2-yl)methyl)pyrrolidine-3-carboxamide (CNP5)

[0121] 'HNMR (600 MHz, DMSO) 59.14 (t, J = 5.5 Hz, 1H), 8.65 (t, J = 5.6 Hz, 1H), 8.59 (s, 1H), 8.04 (d, J = 8.7 Hz, 1H), 7.97 (d, J = 10.3 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 6.9 Hz, 1H), 4.93 - 4.78 (m, 2H), 4.15 (dd, J = 5.6, 2.5 Hz, 2H), 3.61 - 3.53 (m, 1H), 3.48 (td, J = 9.2, 6.0 Hz, 2H), 3.43 - 3.36 (m, 1H), 3.31 (s, 1H), 3.17 (t, J = 2.5 Hz, 1H), 3.11 (p, J = 7.4 Hz, 1H), 2.16 - 1.98 (m, 2H).13C NMR (151 MHz, DMSO) 6 171.99, 171.91, 166.51, 166.43, 136.18, 136.16, 135.04, 131.65, 131.60, 130.44, 129.18, 127.74, 127.71, 125.95, 125.93, 124.58, 123.62, 117.78, 81.76, 81.74, 73.50, 52.97, 50.53, 43.64, 43.59, 40.66, 40.53, 40.40, 40.26, 40.13, 39.99, 39.85, 39.71, 39.57, 31.17, 30.08, 29.08, 28.96. HRMS m / z for C2iH2iN4O2+([M+H]+) calculated: 361.1665, found: 361.1667.

[0122] (S)-l-cyano-N-((6-(prop-2-yn-l-ylcarbamoyl)naphthalen-l-yl)methyl)pyrrolidine-3-carboxamide (CNP6)<

[0123] ‘HNMR (500 MHz, DMSO) 69.08 (t, J = 5.7 Hz, 1H), 8.69 (t, J = 6.1 Hz, 1H), 8.45 (s, 1H), 8.05 - 7.89 (m, 3H), 7.80 (s, 1H), 7.49 (d, J = 8.4 Hz, 1H), 4.48 (d, J = 6.1 Hz, 2H), 4.17 -4.05 (m, 2H), 3.57 (d, J = 9.2 Hz, 1H), 3.52 - 3.36 (m, 3H), 3.20 - 3.03 (m, 2H), 2.18 - 2.07 (m, 1H), 2.07- 1.96 (m, 1H).13C NMR (126 MHz, DMSO) 8 171.98, 166.44, 139.33, 134.63, 131.67, 131.37, 129.60, 128.19, 127.96, 127.09, 125.56, 124.81, 117.76, 81.81, 73.37, 53.01, 50.55, 43.73, 42.83, 40.51, 40.34, 40.27, 40.18, 40.01, 39.84, 39.67, 39.51, 29.97, 29.48, 29.07. HRMS m / z for C2iH2iN4O2+([M+H]+) calculated: 361.1665, found: 361.1663.

[0124] (S)-l-cyano-N-((4'-(prop-2-yn-l-ylcarbamoyl)-[ 1, 1 '-biphenyl]-2-yl)methyl)pyrrolidine-3-carboxamide (CNP8)31AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0125] ’H NMR (500 MHz, DMSO) 5 8.99 (t, J = 5.6 Hz, 1H), 7.93 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 8.3 Hz, 2H), 7.44 - 7.32 (m, 3H), 7.26 (d, J = 7.2 Hz, 1H), 4.28 - 4.15 (m, 2H), 4.12 - 4.05 (m, 2H), 3.52 - 3.45 (m, 1H), 3.43 - 3.34 (m, 3H), 3.13 (t, J = 2.5 Hz, 1H), 2.99 (p, J = 7.4 Hz, 1H), 2.07 - 1.98 (m, 1H), 1.93 - 1.84 (m, 1H).13C NMR (126 MHz, DMSO) 8 171.62, 143.87, 140.51, 136.48, 132.99, 130.12, 129.48, 128.63, 128.45, 127.72, 127.62, 117.72, 81.81, 73.31, 61.53, 52.88, 50.48, 43.50, 43.46, 40.51, 40.44, 40.35, 40.27, 40.18, 40.01, 39.84, 39.68, 39.51, 29.91, 29.00, 28.89. HRMS w zfor C23H23N4O2+([M+H]-) calculated: 387.1821, found: 387.1824.

[0126] (S)-l-cyano-N-((R)-l-(3'-(prop-2-yn-l-ylcarbamoyl)-[l,P-biphenyl]-3-

[0127] 'H NMR (500 MHz, CDC13) 67.98 (s, 1H), 7.71 (dd, J = 8.4, 7.3 Hz, 2H), 7.57 - 7.45 (m, 3H), 7.42 (t, J = 7.9 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 6.75 (t, J = 5.3 Hz, 1H), 6.37 (d, J = 7.8 Hz, 1H), 5.12 (t, J = 7.2 Hz, 1H), 4.26 (d, J = 5.2 Hz, 2H), 3.63 - 3.49 (m, 3H), 3.40 - 3.31 (m, 1H), 2.90 (q, J = 7.6 Hz, 1H), 2.29 (s, 1H), 2.16 - 2.04 (m, 2H), 1.51 (d, J = 7.0 Hz, 3H). HRMS m / z for C24H2SN4O2+([M+H]+) calculated: 401.1978, found: 401.1980.

[0128] (S)-l-cyano-N-((S)-l-(3'-(prop-2-yn-l-ylcarbamoyl)-[l, 1 '-biphenyl] -3-

[0129] ' H NMR (600 MHz, DMSO) 89.07 (t, J = 5.6 Hz, 1H), 8.55 (d, J = 8.0 Hz, 1H), 8.13 (s, 1H), 7.85 (dd, J = 14.1, 7.9 Hz, 2H), 7.66 (s, 1H), 7.64 - 7.56 (m, 2H), 7.46 (t, J = 7.7 Hz, 1H), 7.34 (d, J = 7.7 Hz, 1H), 5.01 (p, J = 7.1 Hz, 1H), 4.10 (dd, J = 5.6, 2.5 Hz, 2H), 3.56 - 3.50 (m, 1H), 3.43 (td, J = 8.4, 5.4 Hz, 1H), 3.40 - 3.35 (m, 2H), 3.31 (s, 1H), 3.16 (t, J = 2.5 Hz, 1H), 3.0532AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO(p, J = 7.4 Hz, 1H), 2.14 - 2.06 (m, 1H), 2.03 - 1.94 (m, 1H), 1.42 (d, J = 7.1 Hz, 3H).13C NMR (151 MHz, DMSO) 8 170.97, 166.32, 145.81, 140.80, 140.03, 134.96, 130.16, 129.56, 129.52, 126.91, 125.98, 125.85, 125.78, 125.04, 117.74, 81.76, 73.43, 52.92, 50.53, 48.66, 43.63, 40.52, 40.40, 40.26, 40.13, 39.99, 39.85, 39.71, 39.57, 29.99, 29.01, 23.13. HRMS m / zfor C2.1H25N.1O2 ([M+H]+) calculated: 401.1978, found: 401.1982.

[0130] (S)-l-cyam-3-methyl-N-((3'-(prop-2-yn-l-ylcarbamoyl)-[ 1. l'-biphenyl ]-3-

[0131] ’H NMR (500 MHz, DMSO) 89.06 (t, J = 5.5 Hz, 1H), 8.49 (t, J = 5.9 Hz, 1H), 8.13 (s, 1H), 7.83 (dd, J = 21.4, 7.8 Hz, 2H), 7.66 - 7.54 (m, 3H), 7.46 (t, J = 7.7 Hz, 1H), 7.27 (d, J = 7.6 Hz, 1H), 4.40 (d, J = 6.0 Hz, 2H), 4.10 (dd, J = 5.5, 2.5 Hz, 2H), 3.77 (d, J = 9.4 Hz, 1H), 3.52 -3.45 (m, 1H), 3.41 - 3.34 (m, 1H), 3.20 - 3.13 (m, 2H), 2.35 - 2.25 (m, 1H), 1.87 - 1.79 (m, 1H), 1.32 (s, 3H).13C NMR (126 MHz, DMSO) 8 174.21, 166.29, 140.91, 140.68, 139.93, 135.00, 129.99, 129.60, 129.53, 126.91, 126.79, 125.96, 125.94, 125.76, 117.79, 81.75, 73.42, 58.83, 49.79, 49.71, 42.80, 40.51, 40.44, 40.34, 40.27, 40.18, 40.10, 40.01, 39.93, 39.84, 39.68, 39.51, 35.75, 29.01, 22.48. HRMSm / z for C24H25N4O2+([M+H] ) calculated: 401.1978, found: 401.1979.

[0132] ( 3S, 4R)-l-cyano-4-phenyl-N-( ( 3 '-(prop-2-yn-l-ylcarbamoyl)-[l, 1 '-biphenyl]-3-

[0133] 'HNMR (500 MHz, DMSO) 89.06 (t, J = 5.5 Hz, 1H), 8.61 (t, J = 5.9 Hz, 1H), 8.09 (s, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.58 (t, J = 7.7 Hz, 2H), 7.44 (s, 1H), 7.38 - 7.24 (m, 5H), 7.21 (d, J = 7.3 Hz, 1H), 6.95 (d, J = 8.1 Hz, 1H), 4.43 (dd, J = 15.4, 6.6 Hz, 1H), 4.19 (dd, J = 15.3, 5.2 Hz, 1H), 4.11 (dd, J = 5.5, 2.5 Hz, 2H), 3.90 - 3.78 (m, 2H), 3.68 - 3.58 (m, 1H), 3.57 - 3.42 (m, 2H), 3.31 (s, 1H), 3.27 - 3.19 (m, 1H), 3.15 (t, J = 2.5 Hz, 1H).13C NMR (126 MHz, DMSO) 8 170.35, 166.29, 140.67, 140.17, 139.97, 138.65, 134.94, 130.06, 129.53, 129.38, 128.97, 128.10, 127.65, 126.89, 126.81, 126.09, 125.98, 125.83, 117.36, 81.76, 73.41, 60.22, 56.54, 53.89, 50.97, 48.46, 42.65, 40.51, 40.44, 40.34, 40.27, 40.18, 40.10, 40.01, 39.84,33AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO39.68, 39.51, 29.02, 21.23, 14.56. HRMS m / z for C29H27N4O2+([M+H]+) calculated: 463.2134, found: 463.2127.

[0134] (3R,4S)-l-cyano-4-phenyl-N-((3'-(prop-2-yn-l-ylcarbamoyl)-[ 1, 1 '-biphenyl J-3-

[0135] ‘HNMR (500 MHz, DMSO) 89.06 (s, 1H), 8.61 (s, 1H), 8.08 (s, 1H), 7.86 (s, 1H), 7.72 (s, 1H), 7.57 (s, 2H), 7.44 (s, 1H), 7.38 - 7.14 (m, 6H), 6.95 (s, 1H), 4.43 (d, J = 22.0 Hz, 1H), 4.19 (d, J = 16.6 Hz, 1H), 4.11 (s, 2H), 3.84 (d, J= 16.1 Hz, 2H), 3.63 (d, J = 9.2 Hz, 1H), 3.58 -3.43 (m, 2H), 3.23 (d, J = 7.6 Hz, 1H), 3.15 (s, 1H).13C NMR (126 MHz, DMSO) 8 170.35, 166.29, 140.67, 140.17, 139.97, 138.65, 134.94, 130.07, 129.53, 129.38, 128.97, 128.10, 127.65, 126.90, 126.81, 126.09, 125.99, 125.83, 117.36, 81.76, 73.41, 56.54, 53.89, 50.97, 48.46, 42.66, 40.51, 40.34, 40.18, 40.01, 39.84, 39.68, 39.51, 29.02. HRMS m / z for C29H27N ([M+H]+) calculated: 463.2134, found: 463.2127.

[0137] To a solution of sodium ascorbate (16.1 mg, 0.081 mmol, 1.5 equiv.) and CuSO4'5H2O (26.9 mg, 0.108 mmol, 2 equiv.) in water (1 mL, 0.07 M) was added a solution of (S)-l-cyano-N-((3'-(prop-2-yn-l-ylcarbamoyl)-[l,l'-biphenyl]-3-yl)methyl)pyrrolidine-3-carboxamide (10.0 mg, 0.026 mmol, 1 equiv.) and N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-5-((4S)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanamide (10.3 mg, 0.026 mmol, 1 equiv.) in THF (1 mL) and the reaction was stirred overnight at rt. The reaction mixture was washed with brine, and extracted with 20% methanol in DCM. The combined organic layers were dried over sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography (0- 34AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO30% methanol in DCM) to give the product as a white solid (3.8 mg, 19%).rH NMR (500 MHz, MeOD) 87.44 (t, J = 1.8 Hz, 1H), 7.32 (s, 1H), 7.20 - 7.09 (m, 2H), 6.95 - 6.84 (m, 3H), 6.77 (t, J = 7.7 Hz, 1H), 6.63 (d, J = 7.6 Hz, 1H), 4.00 (s, 2H), 3.90 (t, J = 5.0 Hz, 2H), 3.77 (d, J = 8.9 Hz, 3H), 3.58 (dd, J = 7.9, 4.4 Hz, 1H), 3.21 (t, J = 5.0 Hz, 2H), 2.98 - 2.83 (m, 7H), 2.81 - 2.72 (m, 3H), 2.59 (t, J = 5.6 Hz, 2H), 2.50 - 2.40 (m, 2H), 2.24 - 2.17 (m, 1H), 2.00 (d, J = 12.7 Hz, 1H), 1.56 - 1.42 (m, 7H), 1.06 - 0.81 (m, 4H), 0.70 (p, J = 7.5 Hz, 2H), 0.61 (s, 1H).13C NMR (151 MHz, DMSO) 8 172.60, 172.56, 170.96, 166.50, 163.16, 145.93, 140.74, 140.06, 135.27, 129.96, 129.49, 126.91, 125.98, 125.72, 125.70, 124.89, 123.86, 117.77, 69.93, 69.86, 69.60, 69.21, 61.49, 59.64, 55.88, 52.88, 50.51, 49.74, 48.62, 43.59, 40.52, 40.40, 40.26, 40.13, 39.99, 39.85, 39.71, 39.57, 38.87, 35.55, 35.36, 30.04, 28.65, 28.50, 25.72, 23.16, 14.56. HRMS zfor C39H5INIO06S+([M+H]+) calculated: 787.3714, found: 787.3711.

[0139] CNP9-Biotin was synthesized in a similar manner to CNP7-Biotin.1H NMR (600 MHz, DMSO) 89.19 (t, J = 5.9 Hz, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H), 7.97 (s, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 7.9 Hz, 2H), 7.64 (s, 1H), 7.58 (q, J = 8.1 Hz, 2H), 7.45 (t, J = 7.7 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 6.42 (s, 1H), 6.36 (s, 1H), 5.00 (p, J = 6.9 Hz, 1H), 4.55 (d, J = 5.7 Hz, 2H), 4.50 (t, J = 5.4 Hz, 2H), 4.34 - 4.28 (m, 1H), 4.11 (d, J = 12.2 Hz, 1H), 3.80 (t, J = 5.3 Hz, 2H), 3.57 - 3.50 (m, 4H), 3.47 - 3.38 (m, 4H), 3.31 (s, 3H), 3.17 (d, J = 5.0 Hz, 2H), 3.07 (d, J = 26.8 Hz, 2H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.11 - 2.02 (m, 3H), 1.93 - 1.85 (m, 2H), 1.57 - 1.37 (m, 7H), 1.33 - 1.22 (m, 3H).13C NMR (151 MHz, DMSO) 8 172.60, 172.56, 170.96, 166.50, 163.16, 145.93, 140.74, 140.06, 135.27, 129.96, 129.49, 126.91, 125.98, 125.72, 125.70, 124.89, 123.86, 117.77, 69.93, 69.86, 69.60, 69.21, 61.49, 59.64, 55.88, 52.88, 50.51, 49.74, 48.62, 43.59, 40.52, 40.40, 40.26, 40.13, 39.99, 39.85, 39.71, 39.57, 38.87,35AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO35.55, 35.36, 30.04, 28.65, 28.50, 25.72, 23.16, 14.56. HRMS m z for C4OH53NIO06S+([M+H]+) calculated: 801.3870, found: 801.3869.

[0140] Example 2: HMGCS 1 is a limiting enzyme for mTORCl -active cancer cell proliferation

[0141] The enzymes of the mevalonate pathway, particularly HMGCS1, are tightly regulated by mTORCl, the master regulator of cell growth, at multiple levels. mTORCl promotes sterol synthesis by activating the Sterol Regulatory Element Binding Protein 2 (SREBP2) transcription factor, resulting in increased expression of the MVP enzymes. The degradation of HMGCS1 protein is selectively regulated by mTORCl, with significant stabilization occurring only when mTOR is active. This combination of transcriptional and post-translational regulation ofHMGCSl leads to a noticeable rise in HMGCS1 levels in cells with hyperactive mTORCl signaling. Analysis of proteomic data of MFE296 endometrial cancer cells, which exert hyperactive mTORCl activity due to the PTEN deletion and PI3K activating mutations, showed that the HMGCS1 level is 5.7-fold higher when mTORCl is active compared to when it is inactive (FIG.8A-FIG. 8C). The levels of other MVP enzymes were either mildly increased or remained unchanged (FIG. 8B and FIG. 8C). Additionally, proteomic analysis of nine cancer cell lines, after culturing them in a medium with high nutrient (DMEM, mTORCl activity up) or low nutrient (HPLM, mTORC 1 activity down), also showed the most robust elevation in HMGCS 1 levels when mTORCl activity is upregulate for cell proliferation (FIG. 8D). Immunoblotting analysis of the same lysates confirmed the upregulation of mTORCl signaling in cells grown in DMEM compared to those in HPLM (FIG. 8E). Genomically modulating the mTORCl activity by deleting the PTEN gene, a negative regulator of mTORCl, using CRISPR-Cas9 in a prostate cancer line (22Rvl) resulted in a dramatic increase in HMGCS1 level (FIG. 8F). Depleting TSC2, another negative regulator of mTORCl, also resulted in increased levels of the HMGCS 1 protein in HEK293T cells (FIG. 8G). The combined data indicate a higher requirement for HMGCS1 in cell proliferation compared to other MVP enzymes, suggesting that HMGCS1 may serve as a limiting enzyme for cell proliferation in rapidly dividing cells with active mTORCl signaling.

[0142] The following experiments showcase the combined use of several orthogonal approaches to characterize inventive compounds and assess their selectivity in the global proteome, which included both covalent and non-covalent interactions. We combined conventional AP-MS or competitive AP-MS approaches, orthogonal immunoblotting (which36AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOeliminated false positives caused by the former due to the limitations of proteomics that allow comparison of only the enriched proteins, disregarding the information in input and flow-through fractions after enrichment), the scavenger-MS approach (which proved to be a highly unbiased method that encompasses a broader proteome coverage than the enrichment assay and complements the comparative AP-MS approach), and a PISA study (which unlike the other methods, evaluated non-covalent interactors of reactive small molecules as potential off-targets). The results show that the main changes in the global proteome after CNP7 treatment were mainly caused by its inhibition of HMGCS1 and the mevalonate pathway, indicating the primary functional effect.

[0143] Example 3: Developing small molecule inhibitors of HMGCS1

[0144] Publicly available cysteome profding datasets that report the engagement of reactive cysteines in the proteome with various small molecule libraries do not detect the HMGCS1 Cysl29 region because the tryptic peptide containing C129 is 46 amino acids in length (FIG. 9A). Instead, analysis of the affinity chemo-proteomic datasets of cyanopyrrolidine probes deposited by independent academic labs identified HMGCS1 as an off-target hit. An activity-based probe profiling assay was employed for HMGCS1 using Hymeglusin-Fluorescein (HG-FL), which specifically reacts with HMGCS1 C129 (FIG. 9B). This probe allows for measurement of the available catalytic cysteine levels of HMGCS1 in vitro and in cells after small molecule treatment. Incubation of recombinant HMGCS1 with GK16S for one hour diminished the following labeling of catalytic cysteine by HG-FL, indicating that the catalytic cysteine of HMGCS1 is preoccupied with GK16S, albeit low efficiency (FIG. 9C). Consistently, a cell-based assay demonstrated that GK16S covalently engaged and copurified WT HMGCS1, but not C129A mutant HMGCS1 expressed in HEK293T cells, confirming its specific reactivity toward C129 (FIG. 9D).

[0145] Subsequently, cyanopyrrolidine derivatives were developed to improve the efficiency of labeling HMGCS1. HMGCS1 has a hydrophobic binding pocket that can accommodate two different substrates, acetyl-CoA and acetoacetyl-CoA (FIG. 9E). This suggests that adding a large hydrophobic group, such as aromatic rings, to the propargyl alkane of GK16S might enhance overall binding. CNP molecules were synthesized with varying levels of hydrophobicity and compared their binding efficacy to recombinant HMGCS1 using HG-FL activity -based probe in vitro (FIG 1A-FIG. ID). Incorporating a benzyl group (CNP1 and CNP2) improved the labeling37AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOefficiency of the catalytic cysteine compared to GK16S (FIG. 1B-FIG. ID). Incorporating a methyl group at the benzylic position with the S configuration nearly abolished the binding (CNP1 vs. CNP3), whereas the R configuration maintained the HMGCS1 labeling efficiency (CNP1 vs. CNP4). Replacing the phenyl group in CNP1 with a naphthyl moiety (CNP5 and CNP6) did not enhance labeling, while replacing the phenyl group with a biphenyl group (CNP7 and CNP8) resulted in a significant increase in the engagement of the catalytic cysteine. Of the eight CNP derivatives, CNP7 displayed the highest labeling efficiency in this in vitro assay, which corresponded with the in-cell labeling efficiency assessed by incubating HEK293T cells with the CNP compounds, followed by lysis and incubation with HG-FL (FIG. IE). The results of the HG-FL assay indicate that a concentration of 0.5 pM CNP7 charges over 70% of the catalytic cysteine residue of HMGCS1 in HEK293T cells within a 4-hour time frame (FIG. IF).

[0146] Hymeglusin has inherently poor serum stability. See, Yi, et al., (2025), supra. The stability of Hymeglusin and CNP7 were compared by pre-incubating them in DMEM with or without fetal bovine serum (FBS) for 2 hours or 16 hours before adding them to HEK293T cells. The subsequent activity profiling showed that the CNP7 potently reacted with HMGCS1 C129 even after 16 hours of pre-incubation in a serum-containing medium, whereas Hymeglusin completely lost its reactivity toward HMGCS1 (FIG. 1G). CNP7 did not label recombinant BSA at even a 40 pM concentration in vitro, based on the in-gel fluorescent analysis of TMR-modified CNP7 (FIG. 1H), in contrast to Hymeglusin, which reacted with recombinant BSA. Consistently, CNP7’s labeling ofHMGCSl persisted even after 24 hours of incubation in various cell lines (FIG. II and FIG. 1 J).

[0147] Example 4: Dual activity-profiling assays allow for assessing the potency and selectivity of CNP derivatives

[0148] The disclosed compounds provide advantages over other MVP inhibitors in characterizing off-targets because they form covalent bonds with proteins, including HMGCS1. Each synthesized compound contains an alkyne group, which offers a functional handle for click chemistry-based activity profiling. After treating cells with the compounds, the cell lysates were divided into two fractions: one was treated with the HG-FL to test HMGCS 1 occupancy rate, using HG-FL as an activity-based probe (ABP). The other underwent click chemistry with tetramethyl rhodamine (TMR) azide for selectivity assessment, wherein the compounds serve as ABPs (FIG.38AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO2A). In comparisons of GK16S and CNP5-8, CNP7 demonstrated the highest potency in HEK293T cells, with a major signal around 57 Da region (FIG. 2B). Applying this workflow for CNP7 timecourse treatment, the HG-FL assay indicated that CNP7 substantially occupied the catalytic cysteine of HMGCS1 within 60 minutes. A concomitant increase in TMR signal at three distinct molecular weights was observed, suggesting possible CNP7-interactors (FIG. 2C). The primary TMR signal observed near the 57 kDa area coincided with the band reactive to the anti-HMGCSl antibody (FIG. 2D). To improve the selectivity of CNP7 toward HMGCS1, CNP7 was derivatized, specifically in the region close to the cyannopyrrolidine warhead group, by either introducing a methyl group at the benzylic position to produce CNP9 and CNP10 (FIG. 2E, left) or by introducing a substituent in the pyrrole ring (FIG. 2E, right). The potency of HMGCS1 labeling was tested by the CNP7 analogues and it was found that adding a methyl group in the R conformation on the benzylic position (CNP9) or on the pyrrolidine ring (CNP11) did not compromise the potency (FIG. 2F and FIG. 2G). In contrast, CNP9’s stereoisomer (CNF 10), or the addition of a large substituent in the pyrrolidine ring (CNP12 and CNP13), dramatically reduced the potency of HMGCS1 labeling. Dual activity-based profiling of CNP11 revealed that it further increased band intensity around the 25 kDa range, indicating enhanced off-target binding (FIG. 2H). Conversely, CNP9 reduced the intensity in the same area compared to CNP7 (FIG. 2H). In a concentration dependence assay using HEK293T cells, however, CNP9 demonstrated slightly reduced potency in HMGCS1 engagement compared to CNP7, although the difference was not statistically significant (FIG. 21, FIG. 21, FIG. 10A and FIG. 10B).

[0149] Example 5 : Analysis of covalent interactors of CNP7 and CNP9 through complementary chemoproteomics

[0150] To investigate the reactivity of CNP7 and CNP9 toward the global proteome and identify their covalent interactors, three orthogonal and comprehensive proteomic analyses were implemented: direct affinity precipitation-mass spectrometry (AP-MS) following click chemistry, competitive AP-MS utilizing CNPbiotin probes, and scavenger MS, which compares the remaining proteome after the enrichment of interactors by CNP-biotin probes. First, the CNP7 and CNP9-enriched proteome was compared using a tandem mass spectrometry (TMT) based quantification (FIG. 3A). HEK293T cells were incubated with DMSO, CNP7, or CNP9 for 1 hour. Then, the lysates underwent click chemistry with biotin-azide to label the CNP7 and CNP9-bound39AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOproteins with biotin, followed by streptavidin enrichment and TMTproteomics, which quantified the relative enrichment of 835 proteins (FIG. 3B). Nine and six proteins were enriched with statistical significance, of which HMGCS1 showed the strongest enrichment in both cases (FIG.3B). Other enriched proteins included a subfamily member of aldehyde dehydrogenases and Cathepsin Z, both of which are cysteine hydrogenases. However, this AP-MS data lacks information on the overall percentage of engagement for each protein by CNF probes, and the enrichment result can be largely affected by the protein abundance in HEK293T cells. This limitation prevents comparing the labeling efficacy of CNP molecules for individual proteins. Additionally, it was found that some of the covalent CNP-protein adducts were reversed during the click reaction, potentially through the hydrolysis of the isothiourea. These variations introduced during sample preparation can introduce artifacts, leading to false negatives and reducing consistency.

[0151] To overcome these limitations, biotin-functionalized CNP7 and CNP9 probes were synthesized and used to perform competitive AP-MS analysis (FIG. 3C). In this case, cells were pretreated with CNP7 or CNP9 or left untreated for 2 hours, followed by cell lysis and incubation with the corresponding CNP-biotin probes. This method enabled assessment of the ratio of enriched proteins in a CNP pre-treatment dependent manner. Six and five proteins showed a significant enrichment in DMSO pretreated cells compared to CNP7 or CNP9 pre-treated cells, respectively (FIG. 3D). Again, HMGCS1 was detected as one of the strongly enriched proteins in both cases. Notably, some of the earlier hits from direct AP-MS, such as ALDH9A1 and DESI1, showed little change in this competitive AP-MS analysis, suggesting that only a small portion of these proteins interacted with the CNP probes. Conversely, ERP44, BTD, and ISOCI were identified as new targets that were not detected in the direct AP-MS dataset. The hits identified in the competitive APMS analysis were validated using an orthogonal approach: immunoblotting (FIG. 3E and FIG. 3F). Importantly, the input, elute, and the flowthrough were compared side by side, which provided several additional layers of information. First, most HMGCS1 in the lysate was pulled down by CNP-biotin in DMSO pretreated cells, whereas CNP-pretreated cells showed little enrichment. ISOCI exhibited a similar pattern to HMGCS1, but with less enrichment in the elute, confirming the competitive AP-MS results for both proteins. Second, BTD, ALDH1B1, and ERP44 showed unexpectedly little enrichment under the DMSO elute condition. Since the competitive AP-MS analysis only compares proteins in the elute, their ratio in the elute was40AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOartificially elevated, despite their weak pull-down efficiency by the CNP-biotins. This emphasizes the importance of validating the chemoproteomic data with alternative methods.

[0152] Since antibodies are not available for every protein identified in AP-MS, comparing the input and flow-through fractions of the competitive affinity purification steps (competitive scavenger mass spectrometry analysis), can serve as a high-throughput and quantitative alternative method to immunoblotting (FIG. 3G). Both CNP7 and CNP9-biotin depletion assays demonstrated a strong depletion of HMGCS1, followed by ISOCI . All the other hits identified in both direct and competitive AP-MS exhibited no significant depletion, indicating that CNP probes did not effectively charge these proteins.

[0153] The comprehensive chemoproteomics analysis reveals that both CNP7 and CNP9 strongly target HMGCS1, whereas other proteins like BTD and ISOCI are labeled to a lesser degree under the same conditions (FIG. 31 and FIG. 3 J). The study also shows that combining data from the direct APMS, competition AP-MS, and scavenger chemoproteomic analyses offers a comprehensive view of the reactivity of covalent chemical probes in the global proteome.

[0154] Example 6: Thermal Proteome Profiling uncovers both covalent and non-covalent binders of CNP7 and CNP9

[0155] The chemoproteomics technique described above relies on the covalent bond-forming properties of cyanopyrrolidine derivatives. However, this approach does not identify non-covalent interactors of the compounds, a piece of information often overlooked with covalent inhibitors. To address this, thermal proteome profiling was used to identify proteins whose melting temperatures changed following treatment with CNP7 or CNP9 compared to DMSO (FIG. 4A). Specifically, the proteome integral solubility alteration (PISA) method combined with TMT-based multiplexing of proteins to analyze both HEK293T and HCT116 cells was employed (FIG. 4B). This analysis quantified 5218 soluble proteins in HEK293T and 6533 soluble proteins in HCT116 (FIG. 4C and FIG. 4D). Among these, HMGCS1 was consistently stabilized in both cell lines treated with CNP7 and CNP9. Additionally, PDE6D was stabilized in both cell lines upon CNP7 and CNP9 treatment, and RIOK2 was specifically stabilized upon CNP7 treatment but not upon CNP9 treatment. These two proteins were not identified in prior chemoproteomic analysis, implying the potential non-covalent interaction between PDE6D and CNP molecules. Immunoblotting of the TPP samples confirmed the stabilization of HMGCS1 and PDE6D (FIG. 4F and FIG. 4G).41AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0156] Aside from these hits, no other proteins exhibited significant changes in thermal stability upon CNP treatment compared to HMGCS1. In summary, four orthogonal chemoproteomic studies in two different cell lines show that CNP7 and CNP9 bind to HMGCS1 most significantly, and PDE6D and RIOK2 may also interact with the molecule through non-covalent interactions.

[0157] Example 7: Cryo-EM structure of CNP7-bound HMGCS1

[0158] Recombinant human HMGCS1 protein was incubated with CNP7 to form an HMGCS1-CNP7 adduct, achieving 90% labeling as determined by the HG-FL assay, prior to cryo-EM grids preparation. Single-particle ciyo-electron microscopy (cryo-EM) analysis revealed the complex structure at 2.3 A resolution (FIG. 5). Two HMGCS1 monomers formed a homodimeric complex with C2 symmetry, as previously reported, and the CNP7 appeared in the hydrophobic pocket of HMGCS1 near Cysl29, similarly to the natural substrate (Fig. 5A). The cyanamide group of CNP7 reacted with the catalytic Cysl29, as expected from our biochemical assays. The backbone amide and the hydroxide of Ser377 form an ‘oxy-anion hole’-like pocket for the imide nitrogen, which may stabilize the intermediate during the reaction between C 129 and the cyano group of CNP7. Histidine 264, part of the catalytic triad,95Glu-129Cys-264His, forms a hydrogen bond with the central oxygen of the carboxylic group in CNP7 (Fig. 5C). Hydrogen bonds that extends through the propargyl amide amine, Lys273, and Thy267 at the entrance of the catalytic pocket further stabilizes the interaction. The extended hydrophobic interactions between the propargyl and biphenyl groups on CNP7 with neighboring hydrophobic side chains were also observed. In summary, the cryo-EM structure of HMGCS1-CNP7 adduct confirms that the catalytic cysteine forms a covalent bond with CNP7, and that hydrophilic and hydrophobic interactions formed throughout the binding pocket contribute to stabilizing the HMGCS1-CNP7 interaction.

[0159] Example 8: Systematic analysis of the impact of HMGCS1 inhibition by CNP7 within the mevalonate pathway

[0160] After confirming the specificity of CNP7 and CNP9, a series of systematic analyses to assess the pharmacologic effects of HMGCS1 inhibition in cells was conducted (FIG. 6A). Initially, the impact of the compounds on cellular HMG-CoA levels, the direct product of HMGCS1, was investigated using an antibody that identifies proteins modified by a 3-hydroxy-3-42AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOmethylglutaryl (HMG) moiety. Studies showed that inhibition of HMGCR by statins causes an increase in intracellular HMG-CoA levels that leads to the formation of an isopeptide bond between the HMG moiety and nucleophilic side chains on proteins through non-enzymatic reactions (FIG. 6A) (Trub, et al., Nat. Commun., 2022, 73:2542). Particularly, the FASN protein was mainly modified by the HMG group, and the amount of HMGylated FASN was linked to the intracellular HMG-CoA levels. Indeed, the addition of Simvastatin induced an HMGylated protein band in HCT116 cells, and Hymeglusin co-treatment cancelled this (FIG. 6B). GK16S or CNP3-6 did not show any discernible effect on HMGylated protein level, but CNP7 and CNP8 treatment in the presence of Simvastatin showed a dramatic decrease in HMGylated protein level. These results conform with the HMGCS1 labeling assays by HG-FL activity -based probe (above), wherein CNP7 most potently labeled the catalytic cysteine of HMGCS1 in cells. See, FIG. 1A-FIG. II. Concentration dependence assay using CNP7 showed that a concentration of 5 pM was required to completely inhibit HMGylation over a period of 48 hours in HCT116 cells. This increased need for CNP7 was attributed to the substantial upregulation of HMGCS1, which acts as a negative feedback mechanism resulting from the statin treatment (FIG. 6C). Consistently, the HG-FL labeling assay conducted on the same cell extract indicated substantial engagement of the catalytic cysteine of HMGCS1 under 5 pM CNP7, with a minor level of free HMGCS1 still detected.

[0161] The effect of HMGCS1 inhibition on one of the end products of HMG-CoA, protein prenylation, was investigated by probing for RhoA. The prenylated RhoA migrates faster than the unprenylated form in the electrophoresis, and the inhibition of HMGCR or geranylgeranyl transferase by its inhibitor, GGTi298, leads to an increased level in the unprenylated form of RhoA (FIG. 12). Consistent with the other assays, CNP7 exhibited the most significant induction of RhoA protein, followed by CNP7 (FIG. 6D). Additionally, CNP7 led to a significantly greater accumulation of RhoA in comparison to Hymeglusin or CNP9 (FIG. 6E and FIG. 6F).

[0162] HEK293T cells started showing cell death at 5 pM of CNP7 after 48 h of treatment, which was comparable to Simvastatin, but Hymeglusin did not show any effect on cell viability (FIG. 6G). Colony formation assays also showed a significant reduction in both the number and size of colonies in the presence of CNP7, but not Hymeglusin (FIG. 6H and FIG. 61). Supplementing the media with geranylgeranyl alcohol rescued the cell death induced by CNP7, which also reversed the unprenylated RhoA level upon CNP7 treatment (FIG. 61 and FIG. 6K).43AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOCollectively, the results show that CNP7 inhibits HMGCS1, thereby blocking the synthesis of HMG-CoA and isoprenoids. This leads to the death of HKE293T cells over time, mainly caused by the lack of the cellular geranylgeranylation process, as seen with statins and genomic knockdown of HMGCS1.

[0163] Example 9: Susceptibility to CNP7 and Simvastatin is cell line-dependent

[0164] Accumulating studies indicate that the susceptibility of cells to statins varies by cell type, and the underlying mechanism of this variation is complex, requiring further investigation at the molecular level. The anti-proliferative effect of CNP7 across 18 cancer cell lines was evaluated, and compared side-by-side to Simvastatin and Hymeglusin to gain experimental insights into the cellular response upon the inhibition of HMGCS1 and HMGCR, the two initial enzymes in the mevalonate pathway. Given the wide range of responses to these drugs, we employed the area under the curve (AUC) measurement as our comparative readout (FIG. 7A and FIG. 13 A). The overview of these cell viability assays, based on nucleic acid staining-flow cytometry analysis, demonstrated three traits. First, hymeglusin showed no efficacy in these three-day culture assays, confirming its low efficacy in serum-containing media, as we reported before (FIG. 7A). Second, some cell lines resistant to CNP7 also showed resistance to Simvastatin, indicating their resistance is a broad characteristic against mevalonate pathway inhibition (FIG. 7A and FIG. 7B). Third, some cell lines, such as RPE1 and MFE296, showed sensitivity toward CNP7 but were rather resistant to Simvastatin (FIG. 7B). Lastly, MM I S and SNU449 cell lines showed similar sensitivity curves that saturated at earlier doses after treatment with CNP7 or CNP9. Overall, the data reveal a clear pattern linking CNP7 with Simvastatin, alongside a unique response characterized by sensitivity solely to CNP7. The findings, made possible by developing a selective inhibitor for HMGCS1, highlights the importance of pharmacologically examining different enzymes within the same metabolic pathway, as they may trigger varied cellular responses due to the complexity of cellular signaling pathways across various cell types.

[0165] Example 10: Correlation between the negative feedback effect and sensitivity to CNP7

[0166] The activity-based profiling and subsequent immunoblotting analysis of the CNP7-sensitive and CNP7-resistant cell lines showed that a concentration of 0.5 pM CNP7 was sufficient to label catalytic cysteine of HMGCS1 in 24 hours, consistent with the previous time course44AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOexperiment (FIG. 13B). However, the overall expression of HMGCS1 varied across the cell lines, and the extent of their negative feedback responses to CNP7 treatment also differed, with HCT116 cells showing a dramatically increased HMGCS1 level upon CNP7 treatment (FIG. 13C). This raised the question of whether the resistance to CNP7 was partly due to their inherent feedback upregulation of HMGCS1 upon its inhibition, likely mediated by SREBP2. To test this, the HMGCS1 levels were measured across CNP7-sensitive or resistant cell lines using an immunoblotting approach (FIG. 7B and FIG. 7C). Overall, the resistant cell lines compared in this study showed greater steady-state HMGCS1 levels, which were then further elevated upon CNP7 treatment (FIG. 7C). The sensitive cell lines showed lower expression to begin with, and the increase upon CNP7 was relatively less striking. Concentration-dependent assay on HCT116 and MFE296 cells showed that HMGCS1 concentration in HCT116 cells increased upon low doses of CNP7 treatment, whereas the negative feedback response in MFE296 was observable at higher doses, beyond 2.5 pM of CNP7 treatment (FIG. 7D and FIG. 7E). Cleaved SREBP2 appeared with a similar pattern, with more robust generation at low CNP7 treatment in HCT116, suggesting the negative feedback mediated by this transcription factor (FIG. 13D and FIG. 13E).

[0167] To strengthen the immunoblotting results, cell lines using CRISPR-Cas9 to express endogenous HMGCSl-mEGFP in HCT116, HT29, MFE296, and RPE1 cell lines were engineered (FIG. 7F). The GFP intensity changes were measured with flow cytometry, which allowed for a quantitative assessment of the response to CNP7 concentration. Analysis of HMGCSl-mEGFP signal upon CNP7 treatment showed an increase in green fluorescent signal at low doses of CNP7 in HCT116, and higher doses in MFE296 (FIG. 7G and FIG. 7H). Together, the data suggest that the overall expression level of HMGCS1 before and after the CNP7 treatment contributes to the resistance or sensitivity.

[0168] Example 11: Global proteomic analysis shows CNP7 primarily affects the mevalonate pathway

[0169] To gain a proteome-wide understanding of CNP7 treatment, a global proteome response analysis was performed after 24 hours in HCT116 cells (FIG. 14A). A condition wherein cells were co-treated with CNP7 and geranylgeranyl alcohol (GGOH), which is converted to geranylgeranylpyrophosphate (GGPP) in cells, was included. This control group will decouple the impact of CNP7 on the protein geranylgeranylation, which caused CNP7-induced cell death, from45AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOother possible off-target effects. The effect of Simvastatin on the global proteome was also compared, along with the condition where Simvastatin and GGOH were co-treated. Lastly, GGOH treatment alone was compared as a negative control. Immunoblotting analysis of the cell extracts confirmed the expected increase of HMGCS1 upon CNP7 or Statin treatment, which was partially reversed by the GGOH co-treatment (FIG. 14B). GGOH alone did not induce any changes in HMGCS1 or RhoA levels.

[0170] The following multiplexing proteomic analysis led to the quantification of 9277 proteins, with 167 proteins statistically significantly upregulated while 108 proteins showed downregulation (FIG. 14C). The comparison of the CNP7 and Simvastatin-treated cell extracts revealed highly correlated global proteome changes, with an r-correlation of 0.8633, supporting the major impact of CNP7 on the mevalonate pathway (FIG. 14D). Consistently, gene ontology analysis of the significant hits in the CNP7-treated cells showed enrichment in the sterol -related pathways, including cholesterol metabolic process, sterol biosynthetic process, and LDL particle binding, etc. (FIG. 14E). Several enzymes in the mevalonate pathway were upregulated upon both CNP7 and Simvastatin treatment, which were reversed by GGOH co-treatment (FIG. 14F). Substrate proteins of prenylation were enriched in both upregulated and downregulated groups (FIG. 14G). This result implies that each prenylation substrate attains different steady-state protein levels when they do not get prenylated due to reduced protein stability from prenylation defects and elevated protein synthesis from transcriptional feedback effects.

[0171] Proteins identified as potential off-targets of CNP7 in the affinity-purification proteomics or thermal stability assay did not show any significant changes in their abundance across different treatments (FIG. 14H). When comparing protein levels in cells treated with Simvastatin directly to those treated with CNP7 (FIG. 14D), ELF3 exhibited a two-fold increase only in the statin treatment, with no significant change observed in the CNP7 treatment (FIG. 141). In contrast, TYMS and USP45 showed a twofold decrease upon CNP7 treatment, which was unaffected by GGOH supplementation, suggesting a potential off-target effect of CNP7. Analyzing the global proteomics changes caused by CNP7 and comparing them to statins and GGOH supplementation helped us evaluate the on-target effects of CNP7 and identify off-target effects. This approach adds another layer of information at a functional level that complements previous chemoproteomics studies, which provided insights into molecular interactions that may not always result in changes in functional outputs.46AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0172] Example 12: Materials and Methods

[0173] Cell lines

[0174] HEK293T (human embryonic kidney, fetus, ATCC CCL-3216, RRID: CVCL 0063), HCT116 (human colorectal carcinoma, male, ATCC CCL-247, RRID: CVCL 0291), MCF7 (human breast adenocarcinoma, female, ATCC HTB-22, RRID: CVCL 0031), U2OS (human osteosarcoma, female, ATCC HTB-96, RRID: CVCL 0042), DLD1 (human colorectal adenocarcinoma, male, ATCC CCL-221, RRID: CVCL 0248), HepG2 (human liver hepatocellular carcinoma, male, ATCC HB-8065, RRID: CVCL 0027), PC-3 (human prostate adenocarcinoma, male, ATCC CRL-1435, RRID: CVCL 0035) andMFE296 (human endometrial adenocarcinoma, female, generous gift from Neal Rosen, RRID: CVCL 1406), HT-29 (human colorectal adenocarcinoma, ATCC HTB-38, RRID: CVCL 0320), BT-474 (human breast ductal carcinoma, female, ATCC HTB-20, RRID: CVCL 0179), DU145 (human prostate carcinoma, male, ATCC HTB-81, RRID: CVCL 0105), hTERT RPE-1 (human retinal pigment epithelium, ATCC CRL-4000, RRID: CVCL 4388), and HeLa (human uterine adenocarcinoma, female, ATCC CRMCCL-2, RRID: CVCL 0030) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, high glucose and pyruvate) supplemented with 10% fetal bovine serum and maintained in a 5% CO2 incubator at 37°C. Pane 05.04 (human pancreatic adenocarcinoma, female, ATCC CRL-2557, RRID: CVCL 1637), M0LM13 (human acute myeloid leukemia, RRID: CVCL 2119), lurkat cells (human acute T cell leukemia, male, ATCC TIB-152, RRID: CVCL_0367), MM. IS (human myeloma, female, ATCC CRL-2974, RRID: CVCL-8792), SNU449 (human liver hepatocellular carcinoma, ATCC CRL-2234, RRID: CVCL 0454), and LNCaP (human prostate carcinoma, male, ATCC CRL-1740, RRID: CVCL 0395) were grown in RPMI supplemented with 10% fetal bovine serum. All cell lines were found to be free of mycoplasma using the Mycoplasma Plus PCR assay kit (Agilent).

[0175] Antibodies and Chemicals

[0176] The following antibodies and reagents were used in this study: HMGCS1 (36877S, Cell Signaling Technology®), HMGCR (Ab242315, Abeam®), HMG-Lysine (ABS2108, Sigma Aldrich®), phospho-S6 ribosomal protein Ser235,236 (4858S, Cell Signaling Technology®), Tubulin (ab7291, Abeam®), IRDye 800CW Goat anti-Rabbit IgG H+L (926-32211, LI-COR®), IRDye 800CW Goat anti -Mouse IgG H+L (926-32210, LI-COR), IRDye 680 RD Goat anti-Mouse47AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOIgG H+L (926-68070, LI-COR®), Lipofectamine 3000 (L3000008, Invitrogen™), Simvastatin (11-101-5563, Fisher Scientific), Hymeglusin (11899, Cayman Chemical), dTAGvl (6914, Tocris™ Bioscience), Benzonase Nuclease HC (71205-3, Millipore®), REVERT™ Total Protein Stain kit (LI-COR®, P / N926-11010), l,l,l,3,3,3-Hexafluoro-2-propanol (52517, Sigma®), sodium dodecyl sulfate (SDS) (Fisher Scientific, PI28364), chloroacetamide (Fisher Scientific, AAA1523830), Sodium pyruvate (Gibco, 11360-070), cycloheximide (Fisher Scientific, AC357420050). TCEP (Gold Biotechnology), Formic acid (Sigma Aldrich®, 94318), TMTpro 18plex Label Reagent (Fisher Scientific, A52045), InstantBlue (Abeam®, abl 19211).

[0177] Generation of knock-in cell line using CRISPR-Cas9 system

[0178] Knock-in HCT116, HT-29, MFE296, and RPE1 cell lines containing HMGCS1-FKBP12 F36V-V5 were generated as previously demonstrated (Mol Cell). Briefly, gRNA targeting the C-terminus of human HMGCS1 (5’-GAACATTAAGATACTCTGTG-3’) (SEQ ID NO: 1) was inserted into pX459 plasmid, and the donor vector was generated by assembling FKBP12 F36V-V5 followed by P2A and blasticidin-resistant transgene into digested pSMART plasmid through Gibson assembly. Cells were transfected with pX459 with gRNA and donor vectors followed by puromycin selection for 48 h after 24 h of the transfection. Cells were either immediately sorted into 96 well plates by their GFP signal via FACS or subjected to a 2-week blasticidin selection before being plated into 96 well plates for clonal selection. Expanded single clones were screened for the integration of the transgene by immunoblotting (An etal., Nat. Cell Biol., 2018, 20:135-143).

[0179] Cell lysis and immunoblotting assay

[0180] Cells were incubated with the corresponding chemicals to -50-60% confluency. After removing the media, the cells were washed with DPBS twice, then lysed with RIPA buffer or 0.5% NP-40 buffer (50 mM HEPES HC1, 150 mM NaCl, pH 7.4) containing protease inhibitors, 200 pM TCEP, 250 mM MgCE, 20 unit / mL Benzonase (Millipore, for RIPA buffer only) were added directly onto the cells. The lysates were sonicated (with RIPA buffer only) and centrifuged, then the concentration of the supernatant was measured by Bradford assay. The whole cell lysate was denatured by the addition of LDS sample buffer supplemented with 50 mM DTT, followed by boiling at 90°C for 5 min. 20-30 pg of each lysate were loaded onto the 4-12% NuPAGE Bis-Tris gel (Thermo Fisher Scientific), followed by SDS-PAGE with MES SDS running buffer (Thermo Fisher Scientific). The proteins were electro-transferred to PVDF membranes (0.45 pm,48AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOMillipore), and then the total protein was stained by Revert total protein stain kit (LLCOR®) or Ponceau staining (Thermo Fisher Scientific). The membrane was then blocked with 5% non-fat milk, incubated with the indicated primary antibodies (4°C, overnight), washed three times with TBST (total 15 min), and further incubated with fluorescent IRDye secondary antibody (1:20000) for 1 h at rt. After a wash with TBST for 15 min, the near-infrared signal was detected using an OdysseyCLx imager (LI-COR®) or Chemidoc MP (Bio Rad) and quantified using ImageStudioLite (LI-COR®) or Image Lab.

[0181] Total proteomics analysis using TMTpro

[0182] The total proteomics analyses were performed based on previously reported method (An et al. Nature, 2020, 583, 303). Briefly, cells are plated into a 10 cm dish per condition, total 16 dishes. 24 h later, Torinl (250 nM) was added to cells and incubated for 10 h. Cells were then washed with ice-cold PBS five times. The cells were then lysed with 800 mL of RIP A buffer (50 mM HEPES, 150 mM NaCl, pH 7.6, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 10 mM glycerophosphate, 10 mM sodium pyrophosphate, protease inhibitor cocktail, Phosphatase inhibitor cocktail, pH 7.5). The lysate was then collected and sonicated three times, followed by Bradford assay to measure the protein concentration. The lysate was then adjusted to become 3 mg / mL concentration through-out the samples using RIPA buffer. 125 mg of each sample was taken, and reduced by the incubation in the presence of 5 mM TCEP at 55°C for 10 min. The lysate was then cooled down to room temperature, and fresh chloroacetamide solution (final cone. 20 mM) was added and incubated at room temperature for 15 min, followed by chloroform / methanol precipitation. Samples were resuspended in 100 mM EPPS, pH 8.5 containing 0.1% RapiGest and digested at 37°C overnight with Trypsin (100:1 protein-to-protease ratio). Tandem mass tag labeling of each sample was performed by adding 10 pL of the 20 ng / mL stock of TMTpro reagent along with acetonitrile to achieve a final acetonitrile concentration of approximately 30% (v / v). Following incubation at room temperature for 1 h, the reaction was quenched with hydroxylamine to a final concentration of 0.5% (v / v) for 15 min. The TMTpro-labeled samples were pooled together at a 1:1 ratio. The sample was vacuum centrifuged to near dryness and subjected to Cl 8 solid-phase extraction (SPE) (50 mg, Sep-Pak, Waters). Dried TMTpro-labeled sample was resuspended in 100 mL of 10 mM NH4HCO3 pH 8.0 and fractionated using BPRP HPLC. Briefly, samples were offline fractionated over a 90 min run, into 96 fractions by high pH reverse phase HPLC (Agilent LC1260) through an Aeris peptide xb-cl8 column (Phenomenex; 250 mm x 3.649AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOmm) with mobile phase A containing 5% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O, and mobile phase B containing 90% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O (both pH 8.0). The 96 resulting fractions were then pooled in a non-continuous manner into 24 fractions for mass spectrometry analysis. Fractions were vacuum centrifuged to near dryness. Each consolidated fraction was desalted via Stage Tip, dried again via vacuum centrifugation, and reconstituted in 5 % acetonitrile, 1 % formic acid for LC-MS / MS processing.

[0183] Mass spectrometry data were acquired using an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific) connected to an UltiMate 3000 RSLCnano system liquid chromatography (LC) pump (Thermo Fisher Scientific). Peptides were separated on a 100 pm inner diameter microcapillary column packed in house with ~ 30 cm of HALO Peptide ES-C18 resin (2.7 pm, 160 A, Advanced Materials Technology) with a gradient consisting of 5%-23% (0-75 min), 23-40% (75-110 min) (ACN, 0.1% FA) over a 120 min run at -500 nL / min. 3 / 10 of each fraction was loaded onto the column for analysis. Proteome analysis used Multi-Notch MS3-based TMT quantification, combined with Real Time Search analysis software, and the FAIMS Pro Interface (using previously optimized 3 CV parameters), to reduce ion interference. The scan sequence began with an MS1spectrum (Orbitrap analysis; resolution 120,000 at 200 Th; mass range 400-1500 m / z; maximum injection time 50 ms; automatic gain control (AGC) target 4xl03). For MS2analysis precursors were selected based on a cycle time of 1.25 sec / CV method (FAIMS CV=-40 / -60 / -80). MS2analysis consisted of collision induced dissociation (quadrupole ion trap analysis; Rapid scan rate; AGC LOxlO4; isolation window 0.5 Th; normalized collision energy (NCE) 35; maximum injection time 35 ms). Monoisotopic peak assignment was used, and previously interrogated precursors were excluded using a dynamic window (180s ±10 ppm). Following acquisition of each MS2spectrum, a synchronous-precursor-selection (SPS) API-MS3scan was collected on the top 10 most intense b- or y-ions matched by the online search algorithm in the associated MS2spectrum. MS3precursors were fragmented by high energy collision-induced dissociation (HCD) and analyzed using the Orbitrap (NCE 45; AGC 2.5x105; maximum injection time 200 ms, resolution was 50,000 at 200 Th). Closeout was set at two peptides per protein per fraction, so that MS3s were no longer collected for proteins having two peptide spectrum matches (PSMs) that passed quality filters.

[0184] Proteomics Data Analysis50AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0185] Mass spectra were processed using a Comet-based (2020.01 rev. 4) software pipeline. Spectra were first converted to mzXML and monoisotopic peaks were re-assigned using Monocle software. MS2spectra were matched with peptide sequences with a composite sequence database including the Human Reference Proteome (2020-01 - SwissProt entries only) UniProt database, as well as sequences of common contaminants. This database was concatenated with one composed of all protein sequences in the reversed order. Analysis was performed using a 50 ppm precursor ion tolerance. Static modifications included, TMTpro tags on lysine residues and peptide N termini (+304.207 Da) and carbamidomethylation of cysteine residues (+57.021 Da). Oxidation of methionine residues (+15.995 Da) was set as a variable modification. For phosphorylation dataset search, phosphorylation (+79.966 Da) on Serine, Threonine or Tyrosine were set as additional variable modifications. Peptide-spectrum matches (PSMs) were adjusted to a 1% false discovery rate (FDR). PSM filtering was performed using a linear discriminant analysis, while considering the following parameters, namely: Comet log expect; different sequence delta Comet log expect; missed cleavages; peptide length; charge state; precursor mass accuracy; and fraction of ions matched. For protein-level comparisons, PSMs were identified, quantified, and collapsed to a 1% peptide false discovery rate (FDR) and then collapsed further to a final protein-level FDR of 1%. To generate the smallest set of proteins required to account for all observed peptides, the principles of parsimony were applied. For TMTpro-based reporter ion quantitation, summed signal-to-noise (S:N) ratio for each TMT channel was first extracted based on the closest matching centroid to the expected mass of the TMT reporter ion (integration tolerance of 0.003 Da). Isotopic impurities of the different TMTpro reagents provided by the manufacturer specifications, were used to adjust reporter ion intensities. Proteins were quantified by summing reporter ion signal-to-noise measurements across all matching PSMs, resulting in a “summed signal-to-noise” measurement. For total proteome, PSMs with poor quality, or isolation specificity less than 0.75, or with TMT reporter summed signal-to-noise ratio that were less than 160 or had no MS3spectra were excluded from quantification. For phospho proteome, PSMs with poor quality, MS2spectra with 13 or more TMT reporter ion channels missing, or isolation specificity less than 0.8, or with TMT reporter summed signal-to-noise ratio that were less than 160 were excluded from quantification. The AScore algorithm was used to determine the localization of phosphorylation sites. AScore is a probability-based approach for high throughput protein phosphorylation site localization. Precisely, a threshold of 13 relates to 95% confidence in site localization.51AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0186] Protein or phospho-peptide quantification values were exported for further analysis in Microsoft Excel and Perseus. Each reporter ion channel was summed across all quantified proteins and normalized assuming equal protein loading of all samples. Phospho-peptides were normalized to the corresponding protein abundance value (when available and indicated in supplementary tables). The maximum and minimum TMT ratios quantifiable were capped to 100-fold. Organellar protein marker annotations were compiled using the proteins which had scored with confidence “very high” or “high” from a previously published HeLa dataset and additional entries from manually curated literature.

[0187] Cell viability assay

[0188] Cell viability was evaluated using DAPI staining (MedChemExpress) and flow cytometry. Each respective cell line was cultured as described above and plated in triplicates per drug dose in 24-well plates. After the 48 or 72-hour treatment period, cells were harvested using trypsin, resuspended in sorting buffer (IX DPBS, 1 mM EDTA, 25 mM HEPES, 1% FBS, final pH 7.3-7.5) with 0.125 ug / mL DAPI, and filtered through 35 pM cell strainer caps. Flow cytometry was performed on either the SONY SH800 (Sony Biotechnology) or the LSR Fortessa (BD Biosciences), and analysis was done using the FlowJo software. Graphs were processed in Prism after exporting cells’ median intensity values.

[0189] Colony formation assay

[0190] To evaluate the ability of single cancer cells to form a colony, cells were plated at a concentration of 1,500 cells / well onto 6 well plates (day 0). At day 5 and day 8, the cells were treated with the corresponding chemicals. At day 10, the cells were stained with crystal violet working solution containing 0.5% crystal violet and 4% paraformaldehyde in PBS. Numbers of colonies were counted for each well (n = 2 or 3) and presented as mean ± s.d.

[0191] Statistical analysis

[0192] P values in the volcano plots of the proteomics data were analyzed by two-sided Welch’s t-test which was adjusted for multiple comparisons. Comparisons of the quantified data (immunoblotting, colony formation, tumor spheroids) were performed by unpaired Student’s t-test; Statistical significance was judged based on p-values; *p < 0.05; **p < 0.01; ***p < 0.001.52AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO

[0193] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications (including any specific portions thereof that are referenced) are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0194] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.53AFSDOCS 304976302.1

Claims

Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOWhat is claimed is:

1. A compound which is of formula I:pharmaceutically acceptable salt or stereoisomer thereof,wherein:Ri is hydrogen, methyl, ethyl, isopropyl, hydroxyl, or halo;Ring A is Ce or Cio aryl or 5- or 6-membered heteroaryl with one or two heteroatoms selected from nitrogen, oxygen and sulfur, wherein the aryl and heteroaryl may be substituted by one or two identical or different groups selected from methyl, hydroxyl, halo, and trifluoromethyl;Ring A’ is absent, Ce aryl, or pyridinyl, wherein the aryl and pyridinyl may be substituted by one or two identical or different groups selected from methyl, hydroxyl, halo, and trifluoromethyl, provided that a) when Ring A is Cio aryl, Ring A’ is absent, and that b) when Ring A’ is absent, R2 is bonded to Ring A; andO "wherein R’ is CH2CCH or Ci-Ce alkyl, wherein said alkyl is optionally substituted with -OH, -NH2, -NHMe, or -NMe2; R” is hydrogen or R’ and R”, together with the nitrogen atom to which they are bound, form a piperazine ring optionally substituted with methyl or hydroxymethyl.

2. The compound of claim 1, which is of formula la:pharmaceutically acceptable salt thereof.

3. The compound of claim 1, which is of formula lb:pharmaceutically acceptable salt thereof.54AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO4. The compound of any one of claims 1-3, wherein Ri is hydrogen.

5. The compound of any one of claims 1-3, wherein Ri is methyl.

6. The compound of any one of claims 1-5, wherein Ring A is Ce aryl.The compound of any one of claims 1-5, wherein Ring A is Cio aryl.

8. The compound of any one of claims 1-5, wherein Ring A’ is Ce aryl.O9. The compound of any one of claims 1-8, wherein R2 is10. The compound of claim 1, which is55AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WOor a pharmaceutically acceptable salt or stereoisomer thereof.

11. A compound which is of formula I’:pharmaceutically acceptable salt or stereoisomer thereof,wherein:Ri is hydrogen, methyl, ethyl, isopropyl, hydroxyl, or halo;56AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WORing A is Ce or Cio aryl or 5- or 6-membered heteroaryl with one or two heteroatoms selected from nitrogen, oxygen and sulfur, wherein the aryl and heteroaryl may be substituted by one or two identical or different groups selected from methyl, hydroxyl, halo, and trifluoromethyl;Ring A’ is absent, Ce aryl, or pyridinyl, wherein the aryl and pyridinyl may be substituted by one or two identical or different groups selected from methyl, hydroxyl, halo, and trifluoromethyl, provided that a) when Ring A is Cio aryl, Ring A’ is absent, and that b) when Ring A’ is absent, R2 is bonded to Ring A;O2 is", wherein R’ is CH2CCH or Ci-Ce alkyl, wherein said alkyl is optionally substituted with -OH, -NH2, -NHMe, or -NMe2; R” is hydrogen or R’ and R”, together with the nitrogen atom to which they are bound, form a piperazine ring optionally substituted with methyl or hydroxymethyl; andR3 is Ci-Ce alkyl or phenyl.O12. The compound of claim 11, wherein R2 is.

13. The compound of claim 12, which is:pharmaceutically acceptable salt or stereoisomer thereof.57AFSDOCS 304976302.1Date of Deposit: February 6, 2026 Attorney Docket No. 046139-084001WO14. A pharmaceutical composition, comprising the compound of any one of claims 1-13, or pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.

15. The method of treating a cancer that is associated with aberrant activity of 3 -hydroxy-3 -methylglutaryl-CoA synthase 1 (HMGCS1), comprising administering to a subject in need thereof the compound pharmaceutically acceptable salt or stereoisomer thereof of any one of claims 1-13, or the pharmaceutical composition of claim 14.

16. The method of claim 15, wherein the cancer is a gastric cancer, cervical cancer, liver cancer, lung cancer, breast cancer, hematological cancer, or colon cancer.

17. The method of claim 15, wherein the cancer is further associated withmediated by hyperactive mTORCl signaling.

18. The method of claim 17, wherein the hyperactive mTORCl signaling is caused by a double mutation in the PTEN-PI3K-mTOR pathway.

19. The method of claim 18, wherein the double mutation comprises a PTEN deletion and a PI3K activating mutation.58AFSDOCS 304976302.1

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