Compositions and methods of use thereof

Abstract

Disclosed herein are compositions comprising a REST (repressor element-1 silencing transcription factor) inhibitor; and a fatty acid metabolism inhibitor. Also disclosed herein are methods of making and use of any of the compositions disclosed herein.

Description

[0001] COMPOSITIONS AND METHODS OF USE THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application 63 / 559,356 filed February 29, 2024, which is hereby incorporated herein by reference in its entirety.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with government support under Grant No. R01 GM 104896 and Grant No. R35 GM148356 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] The sequence listing submitted on February 25, 2025, as an .XML file entitled “10046- 558W01_ST26.xml” created on February 25, 2025, and having a file size of 41,623 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(eX5).

[0008] BACKGROUND

[0009] Improved compounds and methods for treating cancer, such as glioblastoma, are needed. The compositions and methods discussed herein address these and other needs.

[0010] SUMMARY

[0011] In accordance with the purposes of the disclosed compositions and methods as embodied and broadly described herein, the disclosed subject matter relates to compositions and methods of use thereof.

[0012] For example, disclosed herein are compositions comprising a REST (repressor element-1 silencing transcription factor) inhibitor; and a fatty acid metabolism inhibitor.

[0013] In some examples, the composition is synergistic (e.g. LDJO of each active ingredient is lower than when used alone).

[0014] In some examples, the composition exhibits limited hepatotoxicity.

[0015] In some examples, the fatty acid metabolism inhibitor comprises Triacsin C, TVB- 2640 / Denifanstat, avasimible, 2-fluoropalmitic acid, their homologues, or a combination thereof. In some examples, the fatty acid metabolism inhibitor comprises a pan-ACSL inhibitor. In some examples, the fatty acid metabolism inhibitor comprises Triacsin C.

[0016] In some examples, the REST inhibitor targets SCP1. In some examples, the REST inhibitor covalently interacts with SCP1.

[0017] In some examples, the REST inhibitor comprises a compound described by Medellin et al. J Med. Chem, 2022, 65, 507-519, or a pharmaceutically acceptable salt thereof.

[0018] In some examples, the REST inhibitor comprises a compound defined by Formula I, or a pharmaceutically acceptable salt thereof: wherein

[0019] R1, R2, R3, and R4are each independently H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 alkoxy, NRxRy. or wherein, as valence permits, R1and R2, R2and R3, or R3and R4, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms;

[0020] Rxand Ryare independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl;

[0021] R5is H, OH, halogen, or substituted or unsubstituted C1-C20 alltyl;

[0022] R6is substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, or substituted or unsubstituted C1-C20 alkoxy;

[0023] Q is C(O) or 8(0)2: and

[0024] A is a cyclic hydrophobic group, such as a substituted or unsubstituted aromatic group.

[0025] In some examples, the REST inhibitor is selected from the group consisting of:

[0026]

[0027] , pharmaceutically acceptable salts thereof, and combinations thereof.

[0028] In some examples, the REST inhibitor is selected from the group consisting of:

[0029] pharmaceutically acceptable salts thereof, and combinations thereof. In some examples, the REST inhibitor comprises: or a pharmaceutically acceptable salt thereof.

[0030] Also disclosed herein are pharmaceutical compositions comprising any of the compositions disclosed herein. In some examples, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, or a combination thereof.

[0031] Also disclosed herein are methods of making any of the compositions disclosed herein.

[0032] Also disclosed herein are methods of use of any of the compositions disclosed herein.

[0033] Also disclosed herein are methods of treating a disease in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of any of the compositions disclosed herein. In some examples, the disease comprises cancer.

[0034] Also disclosed herein are methods of treating cancer in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of any of the compositions disclosed herein.

[0035] In some examples, the methods further comprise co-administering an anticancer agent to the subject.

[0036] In some examples, the cancer has an upregulated REST level. In some examples, the cancer comprises glioblastoma multiforme, low-grade glioma, neuroblastoma, medullablastoma, or a combination thereof. In some examples, the cancer comprises glioblastoma.

[0037] Also disclosed herein are methods of suppressing tumor growth in a subject in need thereof, the methods comprising contacting at least a portion of the tumor with a therapeutically effective amount of any of the compositions disclosed herein.

[0038] Additional advantages of the disclosed compositions and methods will be set forth in part in the description which follows, and in part will be obvious from the description. The advantages of the disclosed compositions and methods will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed compositions and methods, as claimed.

[0039] The details of one or more embodiments of the invention are set forth in the accompanying drawings and tire description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0040] BRIEF DESCRIPTION OF THE FIGURES

[0041] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects of the disclosure, and together with the description, serve to explain the principles of the disclosure.

[0042] Figure 1A. Boxplot of REST mRNA expression in TCGA-LGG (low-grade glioma), TCGA-GBM (glioblastoma) samples compared to normal brain samples from TCGA and GTEx datasets (*p < 0.001). Analysis was performed using the GEPIA2 web server.

[0043] Figure IB. Survival analysis using data from TCGA-GBM and TCGA-LGG projects. All the patients were divided into two cohorts: low-REST group (in blue) with REST mRNA level < median; high-REST group (in red) with REST mRNA level > median. Analysis was done using GEPIA2 web server.

[0044] Figure 1C. Basal REST protein amount (left) and representative Western blot replicate (right) in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs. SVGpl2 was tested using ANOVA with post-hoc pairwise comparisons. **p < 0.01: *p < 0.05; ns - not significant.

[0045] Figure ID. Proliferation assay of Glioblastoma multiforme cell lines performed by counting cell numbers every 24 h. Shown is one representative replicate (left) and quantification of proliferation doubling time (right) based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post-hoc pairwise comparisons. **p< 0.01.

[0046] Figure IE. Western blot confirmation of the lack of REST protein in homozygous REST- KO clones of T98G (left) and HEK293 (right). Dashed lines indicate that adjacent blots were processed on different days.

[0047] Figure IF. Proliferation of WT and REST-KO T98G cells were examined by counting cell numbers every 24 h. Shown is one representative replicate (left) and quantification of proliferation doubling time (right) based on 3-4 independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post-hoc pairwise tests. ***p< 0.001; **p < 0.01; *p<0.05.

[0048] Figure 1G. Transient REST overexpression (OE) in REST-KO cells rescues cell growth. Proliferation of T98G WT and REST-KO CIO cells (transfected with empty vector pLPC vs REST OE) was examined by counting cell numbers every 24 h. Shown is one representative replicate (left) and quantification of proliferation doubling time (right) based on 3 independent experiments. Statistical difference between groups was tested using two-tailed paired t-test. **p< 0.01.

[0049] Figure 2A. Chemical structures of lead SCP1 inhibitors.

[0050] Figure 2B. Competitive assay against the pNPP substrate was performed with preincubation of GR-28 compound and SCP1 enzyme for 30, 60, 120, 180, 300, and 1260 min. Time-lapse ICso curves were obtained.

[0051] Figure 2C. The ICso data of the pNPP substrate was converted to In % remaining activity against time at different concentrations of GR-28 (0, 0.3125, 0.625, 1.25 and 2.5 μM).

[0052] Figure 2D. The rate of inactivation (koi*) was plotted against inhibitor concentration and fitted to the equation: kob> = kin*t x [I] / (Ki+[I]). Each data point was obtained from three replicates, and the error bars indicate SD.

[0053] Figure 2E. SCP1 WT was incubated overnight with GR-28 compound and then analyzed by MALDI-TOF mass spectrometry. The blue trace represents the DMSO control, while the orange trace represents the GR-28 treatment.

[0054] Figure 2F. pNPP assay of phosphatase activity of SCP1 WT (orange) or SCP1 C181A mutant (blue) upon incubation with GR-28 for 5 hours. For each data point, n = 3, error bars indicate SD from three replicates.

[0055] Figure 2G. SCP1 C181 A w as incubated overnight with GR-28 compound and then analyzed by MALDI-TOF mass spectrometry.

[0056] Figure 2H. Model of covalent inhibition of SCP1 by GR28 (PDB Code: 3PGL). A magnesium ion is shown in the active site of SCP1 as a green sphere. Key residues predicted to interact with GR28 are shown as sticks. GR-28 is shown as sticks colored by atoms with carbon atoms in violet, oxygen in red and nitrogen in blue. Side chain and main chain interactions between SCP1 and GR28 are shown in dashed lines.

[0057] Figure 3A. RNA-seq analyses showing gene upregulation and downregulation (highlighted in red, log2FC cut-off = 0.58, padj < 0.05) in REST KO cells compared to corresponding control (T98G- left, HEK293 - right). Representative volcano plots were built using ‘Enhanced Volcano’ Bioconductor package.

[0058] Figure 3B. Venn diagrams of deregulated genes shared between three “slow” T98G REST-KO clones (CIO, F7, and G2): upregulated genes are shown on the left, downregulated genes are shown on the right.

[0059] Figure 3C. GO (gene ontology) categories enriched among upregulated genes (top) and downregulated genes (bottom) in T98G REST-KO vs control.

[0060] Figure 3D. Overly) of upregulated genes in glioblastoma (left, shared between three “slow'” clones, in green) and HEK293 (right, shared between two clones, in green) and a published subset of genes with REST binding sites in human embryonic stem cells (in yellow) (24).

[0061] Figure 3E. A list of representative REST-target genes (n = 6) based on Tag-Seq and TCGA-GBM data analysis (shown are correlation coefficients of gene expression with REST mRNA).

[0062] Figure 3F. Validation of REST-target genes by qPCR assay in REST-KO cells. Shown are Fold Changes (FC) vs T98G control cells derived from 3 independent biological replicates. Gene expression was measured using ddCt method and normalized by ACTB expression. Comparison vs control was performed using one-tailed t-tests. *** / ?< 0.001; **p<0.01; *p< 0.05.

[0063] Figure 4A. Effect of GR-28 on REST protein level in Al 72 cells (4 μM for 24 h) and T98G cells (10 μM for 48 h). Shown is one representative WB replicate (left) and quantification (right) from 3-4 independent cell treatments (mean±SEM). Comparison vs DMSO was performed using paired one-tailed t-tests. Dashed lines indicate that adjacent blots were processed on different days. *p < 0.05.

[0064] Figure 4B. Effect of GR-28 on mRNA level of REST-target genes in Al 72 cells (4 μM for 18 h, left) and T98G cells (10 μM for 36 h, right). Shown are Fold Changes (FC) vs DMSO derived from 3-4 independent biological replicates. Gene expression was measured using ddCt metiiod and normalized by ACTB expression. Comparison vs DMSO was performed using paired one-tailed t-tests. Dashed line indicates FC = 1.5. *p < 0.05.

[0065] Figure 4C, Survival rates (72 h) of high-REST Glioblastoma multiforme cell lines (Al 72 and T98G) and control cells (HepG2) under single drug treatment with GR-28. Shown are viability rates (mean±SEM) normalized to that of solvent-control wells derived from 3-4 independent experiments.

[0066] Figure 4D. Sensitivity (72 h) of high-REST Glioblastoma multiforme cell lines (Al 72 and T98G) and control cells (HepG2) to GR-28. Shown are LD50s (lethal doses 50) with 95%- confidence intervals calculated from 3-4 independent biological replicates using ‘drc’ R package. TrC = Triacsin C (sensitization at 0.625 and 2.5 μM in T98G and Al 72, respectively).

[0067] Figure 5 A. Proliferation rate of “fast” D4 REST-KO clone does not differ from that of control T98G cells. Left, Western blotting confirms lack of REST protan in D4 cells. Proliferation of WT and D4 cells was examined by counting cell numbers every 24 h. Shown is one representative replicate ( / nzdtiZe) and quantification of proliferation doubling time (right) based on 3-4 independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post-hoc pairwise tests, ns - not significant.

[0068] Figure 5B. Venn diagram showing overlap between upregulated differentially expressed genes in “slow” clones and upregulated differentially expressed genes in “fast” D4 cells.

[0069] Figure 5C. Gene ontology categories significantly enriched among D4-specific upregulated differentially expressed genes.

[0070] Figure 5D. Gene network for “Fatty acid metabolic process” (00:0006631) as a top- significant category from Figure 5C. Network was built using STRING v.11.5 database.

[0071] Figure 5E. Gene expression otACSLl anAACSLS in T98G WT, “slow”, and ‘Tast” REST-KO T98G cells. CRISPR Control data points are in left most column in each lot. REST KO CIO data points are in the middle column in each plot. REST KO D4 data points are in the right most column in each plot. Gene expression was measured using ddCt method and normalized by ACTB expression. Statistical difference between groups was tested using t-test. Shown are mean±SEM from 3 biological replicates. *p < 0.05.

[0072] Figure 5F. Proliferation of D4 cells, in the presence of DMSO or pan-ACSL inhibitor Triacsin C (500 nM) in the media, was examined by counting cell numbers every 24 h. Shown is one representative replicate (left) and quantification of proliferation doubling time (right) based on 3 independent experiments. Statistical difference between groups was tested using two-tailed paired t-test. **p < 0.01.

[0073] Figure 5G. Proliferation of D4 cells in the presence of Triacsin C (500 nM) does not differ from that of “slow” REST-KO clones. Comparison was performed using ANOVA with post-hoc pairwise tests, ns - not significant.

[0074] Figure 5H. Sensitivity of T98G WT and REST-KO cells to pan-ACSL inhibitor Triacsin C (treatment duration, 72 h). Shown are LD50s (lethal doses 50) with 95%-confidence intervals calculated from 3-4 independent biological replicates using ‘drc’ R package. LD50s were compared vs T98G control using ratio test from the same package. *p < 0.05.

[0075] Figure 6A. Drug combination landscapes (72 h) in glioblastoma multiforme (T98G). Shown is the representative landscape having maximal synergy score closest to its mean value. Landscapes were built using ‘synergyfindef R package (Bliss model).

[0076] Figure 6B. Drug combination landscapes (72 h) in glioblastoma multiforme (Al 72). Shown is the representative landscape having maximal synergy score closest to its mean value. Landscapes were built using ‘synergyfinder’ R package (Bliss model).

[0077] Figure 6C. Drug combination landscapes (72 h) in HepG2 cells. HepG2 cells were treated under the same doses as Al 72. Shown is the representative landscape having maximal synergy score closest to its mean value. Landscapes were built using ‘synergy finder’ R package (Bliss model).

[0078] Figure 6D. Low-toxic dose of Triacsin C sensitizes high-REST glioblastoma multiforme cells to GR-28. Shown are viability rates (mean±SEM) normalized to that of solvent-control wells derived from 3 independent experiments. Statistical difference was tested using two-tailed unpaired t-tests. ***p < 0.001; *p < 0.05.

[0079] Figure 6E. Maximal synergy scores (mean±SEM) extracted from 3 independent drug combination landscapes (GR-28 / Triacsin C) in Al 72 (left most column), T98G (carter column), and HepG2 cells (right most column). Statistical difference was tested using a t-test. *p < 0.05.

[0080] Figure 6F. Selective effect of GR-28 / Triacsin C drug combination on glioblastoma multiforme cells. Plotted are viabilities (mean±SEM, 3 independent experiments, 72 h) normalized to that of solvent-control wells under single drug treatments and combination treatments. Statistical difference was tested using multiple t-tests. **p < 0.01 ; *p < 0.05.

[0081] Figure 6G. Simultaneous targeting of REST and fatty acid metabolism results in synergistic cell death in Glioblastoma multiforme cells. Model was created using BioRender software.

[0082] Figure 7A. Western blots confirming absence of REST protein band in REST-KO clones (three independent biological replicates per cell line) for T98G cells. Dashed lines indicate that adjacent blots were processed on different days.

[0083] Figure 7B. Western blots confirming absence of REST protein band in REST-KO clones (three independent biological replicates per cell line) for HEK293 cells. Dashed lines indicate that adjacent blots were processed on different days.

[0084] Figure 7C. Design of PCR reactions to identify the CRISPR / Cas9-repair outcomes in target cells. Yielded PCR products (where the bands were detected) were purified and analyzed with Sanger sequencing.

[0085] Figure 7D. Transient REST overexpression (OE) in REST-KO CIO cells.

[0086] Figure 8A. Survival rates (72 h) of high-REST Glioblastoma multiforme cell lines (Al 72 and T98G) under single drug treatment with T-65 compound. Shown are viability rates (mean±SEM) normalized to that of solvent-control wells derived from 3-4 independent experiments.

[0087] Figure 8B. Sensitivity (72 h) of high-REST Glioblastoma multiforme cell lines (Al 72 and T98G) to T-65. Shown are LD50s (lethal doses 50) with 95%-confidence intervals calculated from 3-4 independent biological replicates using "drc’ R package.

[0088] Figure 8C. T-65 treatment (4 μM for 24 h) did not change REST protein amount in high- REST glioblastoma cells (Al 72). Shown are representative blot and mean ± SEM from 3 independent biological replicates. Statistical comparison vs DMSO was performed using one- tailed t-test. ns - not significant.

[0089] Figure 9A. RNA-seq shows gene upregulation and downregulation (highlighted in red, logzFC cutoff = 0.58, padj < 0.05) in REST-KO cells compared to corresponding control. Volcano plots were built using ‘Enhanced Volcano’ Bioconductor package.

[0090] Figure 9B. Hcatmaps of rlog-normalizcd RNA-Scq counts across sequenced cell lines (T98G, Ze / ?; HEK293, right). Duplicate replicates of every cell line cluster together. Heatmap was built using ‘DESeq2’ R package.

[0091] Figure 10. Validation of Tag-Seq data using qPCR assay for selected deregulated genes (T98G, left; HEK293, right). Shown are Fold Changes (FC) vs respective CRISPR control cells derived from 3 independent biological replicates. Gene expression was measured using ddCt method and normalized by ACTB expression. Comparison vs control was performed using one-tailed t-tests. ***p < 0.001; **p < 0.01; *p < 0.05; ns - not significant.

[0092] Figure 11A. Venn diagrams of deregulated genes shared between two HEK293 REST-KO clones (D10&E6): upregulated genes are shown on the left, downregulated genes are shown on the right.

[0093] Figure 1 IB. Gene ontology categories enriched among upregulated genes (n = 106) in HEK293 REST-KO cells. Gene ontology analysis was performed using ‘clusterProfiler’ Bioconductor package.

[0094] Figure 12A. Dot plot of mRNA and protein levels of REST across tested cell lines. Shown are mean values obtained from 3-4 independent biological replicates. Correlation was assessed using Pearson r coefficient, line of best fit represents linear regression line. REST gene expression was normalized by ACTS expression. REST protein level was assessed with Western blot and normalized by P-tubulin amount

[0095] Figure 12B. The rationale for inclusion of specific genes in a REST-predictive gene panel (n = 9). Shown is overlap (indicated by red arrow) between three gene subsets: 1) top- 100 anti- correlated with REST mRNA genes in TCGA-GBM dataset (yellow); 2) genes with REST binding site(s) in their genomic sequence based on analysis from Rockowitz et al 2015 [PMID 25990720] (green); 3) up-regulated genes in both REST-KO T98G and HEK293 (blue).

[0096] Figure 13A. Sensitivity of Glioblastoma multiforme cell lines (A172, T98G) and hepatocareinoma cells (HepG2) to single treatment with Triacsin C (72 h). Shown are viability rates (mean±SEM) normalized to that of solvent-control wells derived from 3 independent experiments.

[0097] Figure 13B. Drug combination landscapes (1-3 biological replicates, 72 h) in A172, T98G, and HepG2 cells. Landscapes were built using ‘synergyfinder' R package (Bliss model).

[0098] DETAILED DESCRIPTION

[0099] The compositions and methods described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples included therein.

[0100] Before the present compositions and methods are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0101] Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0102] General Definitions

[0103] In tins specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings.

[0104] Throughout the description and claims of tins specification the word “comprise” and other forms of tire word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps. 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. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like.

[0105] “Optional” or “optionally” means that tire subsequently described event or circumstance can or cannot occur, and that tire description includes instances where tire event or circumstance occurs and instances where it does not.

[0106] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 5% of the value, e.g., within 4, 3, 2, or 1% of tiie value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0107] “Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes.

[0108] Values can be expressed herein as an “average” value. “Average” generally refers to the statistical mean value.

[0109] By “substantially” is meant within 5%, e.g., within 4%, 3%, 2%, or 1%.

[0110] It is understood that throughout this specification the identifiers “first” and “second” are used solely to aid in distinguishing the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to inply any particular order, amount, preference, or importance to the components or steps modified by these terms.

[0111] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0112] A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0113] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, C A, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0114] As used herein, by a “subject” is meant an individual. Thus, the “subject” can indude domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0115] The term “inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This can also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0116] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

[0117] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevailed, but something that is reduced could also be prevented. Likewise, something could be prevailed but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. For example, the terms “prevent” or “suppress’7can refer to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms.

[0118] As used herein, “treat” or other forms of the word, such as “treated” or “treatment,” refers to administration of a composition or performing a method in order to reduce, prevent, inhibit, or eliminate a particular characteristic or event (e.g., microbe growth or survival). The term “control” is used synonymously with the term “treat.”

[0119] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0120] The term “therapeutically effective amount” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0121] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0122] As used herein, “molecular weight” refers to number average molecular weight as measured by NMR spectroscopy, unless indicated otherwise.

[0123] As used herein, the term “delivery” encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery). As used herein, the term "encapsulation / ’ or grammatical equivalent, refers to the process of confining an individual nucleic acid molecule within a nanoparticle.

[0124] As used herein, “expression” of a mRNA refers to translation of an mRNA into a peptide (e.g., an antigen), polypeptide, or protein (e.g., an enzyme) and also can include, as indicated by context, the post-translational modification of the peptide, polypeptide or fully assembled protein (e.g., enzyme). In this application, the terms “expression” and “production,” and grammatical equivalent, are used inter-changeably.

[0125] As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one peptide, polypeptide or protein. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl- cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5- methylcytidine); chemically modified bases: biologically modified bases (e.g., methylated bases): intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N- phosphoramidite linkages).

[0126] As used herein, the term “nucleic acid,” in its broadest sense, refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, “nucleic add” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide drain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA and / or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms indude nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. In some examples, the term “nucleic acid” as used herein means natural and synthetic DNA, RNA, oligonucleotides, oligonucleosides, and derivatives thereof. For ease of discussion, such nucleic acids are at times collectively referred to herein as “constructs,” “plasmids,” or “vectors.”

[0127] The term “gene” as used in this specification refers to a segment of deoxyribonucleotides (DNA) possessing the information required for synthesis of a functional biological product such as a protein or ribonucleic acid (RNA).

[0128] The term “genetic engineering” is used to indicate various methods involved in gene manipulation including isolationjoining, introducing of gene(s) as well as methods to isolate select organisms containing the manipulated gene(s).

[0129] As specified herein, the term “DNA construct” refers to a sequence of deoxyribonucleotides including deoxyribonucleotides obtained from one or more sources.

[0130] The term “gene expression” refers to efficient transcription and translation of genetic information contained in concerned genes.

[0131] The term “recombinant” cells or population of cells refers to cells or population of cells into which an exogenous nucleic acid sequence is introduced using a delivery vehicle such as a plasmid.

[0132] Chemical Definitions

[0133] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0134] The organic moieties mentioned when defining variable positions within the general formulae described herein (e.g., the term “halogen”) are collective terms for the individual substituents encompassed by the organic moiety. The prefix Cn-Cm preceding a group or moiety indicates, in each case, the possible number of carbon atoms in the group or moiety that follows.

[0135] The term “ion,” as used herein, refers to any molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom that contains a charge (positive, negative, or both at the same time within one molecule, cluster of molecules, molecular complex, or moiety (e.g., zwitterions)) or that can be made to contain a charge. Methods for producing a charge in a molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom are disclosed herein and can be accomplished by methods known in the art, e.g., protonation, deprotonation, oxidation, reduction, alkylation, acetylation, esterification, de-esterification, hydrolysis, etc.

[0136] The term “anion” is a type of ion and is included within the meaning of the term “ion.” An “anion” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom that contains a net negative charge or that can be made to contain a net negative charge. The term “anion precursor” is used herein to specifically refer to a molecule that can be converted to an anion via a chemical reaction (e.g., deprotonation).

[0137] The term “cation” is a type of ion and is included within the meaning of the term “ion. A “cation” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom, that contains a net positive charge or that can be made to contain a net positive charge. The term “cation precursor” is used herein to specifically refer to a molecule that can be converted to a cation via a chemical reaction (e.g., protonation or alkylation).

[0138] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0139] “Z1,” “Z2,” “Z3,” and “Z4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when tiiey are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

[0140] The term “aliphatic” as used herein refers to a non-aromatic hydrocarbon group and includes branched and unbranched, alkyl, alkenyl, or alkynyl groups.

[0141] As used herein, the term “alley 1” refers to saturated, straight-chained or branched saturated hydrocarbon moieties. Unless otherwise specified, C1-C24 (e.g., C1-C22, C1-C20, Ci-Cis. Ci-Cie, C1-C14, C1-C12, C1-C10, Ci-Cs, Ci-Ce, or C1-C4) alkyl groups are intended. Examples of alkyl groups include methyl, ethyl, propyl, 1 -methyl -ethyl, butyl, 1-methyl-propyl, 2-methyl- propyl, 1,1-dimethyl-ethyl, pentyl, 1-methyl-butyl, 2-methyl-butyl, 3-methyl-butyl, 2,2- dimethyl-propyl, 1-ethyl-propyl, hexyl, 1,1-dimethyl-propyl, 1,2-dimethyl-propyl, 1 -methyl - pentyl, 2-methyl-pentyl, 3-methyl-pentyl, 4-methyl-pentyl, 1,1-dimethyl-butyl, 1,2-dimethyl- butyl, 1,3-dimethyl-butyl, 2,2-dimethyl-butyl, 2,3-dimethyl-butyl, 3,3-dimethyl-butyl, 1 -ethyl- butyl, 2-ethyl-butyl, 1,1,2-trimethyl-propyl, 1,2,2-trimethyl-propyl, 1 -ethyl- 1-methyl-propyl, 1- ethyl-2-methyl-propyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Alkyl substituents may be unsubstituted or substituted with one or more chemical moieties. The alkyl group can be substituted with one or more groups including, but not limited to, hydroxyl, halogen, acyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, cyano, carboxylic add, ester, ether, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below, provided that the substituents are sterically compatible and the rules of chemical bonding and strain energy are satisfied.

[0142] Throughout tiie specification “alkyl” is generally used to refer to both unsubstituted allcyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substiluent(s) on the alkyl group. For example, the term “halogenated alkyl” specifically refers to an alkyl group that is substituted with one or more halides (halogens; e.g., fluorine, chlorine, bromine, or iodine). The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “alkylamino” specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. When “alkyl” is used in one instance and a specific term such as “alkylalcohol” is used in another, it is not meant to imply that tiie term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like.

[0143] This practice is also used for other groups described herein. That is, while a term such as cycloalltyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalltyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g. , a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalltyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

[0144] As used herein, tiie term “alkenyl” refers to unsaturated, straight-chained, or branched hydrocarbon moieties containing a double bond. Unless otherwise specified, C2-C24 (e.g., C2-C22, C2-C20, C2-C18, C2-C16, C2-C14, C2-C12, C2-C10, C2-C8, C2-C6, or C2-C4) alkenyl groups are intended. Alkenyl groups may contain more than one unsaturated bond. Examples include ethenyl, 1 -propenyl, 2-propenyl, 1 -methyl ethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-l- propenyl, 2-methyl-l -propenyl, l-methyl-2-propenyl, 2-methyl-2-propenyl, 1 -pentenyl, 2- pentenyl, 3-pentenyl, 4-pentenyl, 1 -methyl- 1 -butenyl, 2-methyl-l -butenyl, 3-methyl-l-butenyl, l-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, l-methyl-3-butenyl, 2-methyl-3- butenyl, 3-methyl-3-butenyl, l,l-dimethyl-2-propenyl, 1,2-dimethyl-l -propenyl, l,2-dimethyl-2- propenyl, 1-ethyl-l-propenyl, l-ethyl-2-propenyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1 -methyl- 1 -pentenyl, 2-methyl-l-pentenyl, 3-methyl-l -pentenyl, 4-methyl-l- pentenyl, 1 -methyl -2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 1-methy 1-3 -pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl- 4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, l,l-dimethyl-2- butenyl, l,l-dimethyl-3-butenyl, 1,2-dimethyl-l-butenyl, 1,2-di methyl -2- butenyl, 1,2-dimethyl- 3-butenyl, 1,3-dimethyl-l -butenyl, l,3-dimethyl-2-butenyl, l,3-dimethyl-3-butenyl, 2,2- dimethyl-3-butenyl, 2,3-dimethyl-l -butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3, 3-dimethyl-l -butenyl, 3,3-dimethyl-2-butenyl, 1 -ethyl- 1 -butenyl, l-ethyl-2-butenyl, l-ethyl-3- bulenyl, 2-ethyl-l -butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, l,l,2-trimethyl-2-propenyl, 1- ethyl-l-methyl-2-propenyl, l-ethyl-2-methyl-l -propenyl, and l-ethyl-2-methyl-2-propenyl. The term “vinyl” refers to a group having the structure -CFbCFh; 1 -propenyl refers to a group with the structure -CH=CH-CH3; and 2-propenyl refers to a group with the structure -CH2-CH=CH2. Asymmetric structures such as (Z’Z^CK^CZ’Z4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. Alkenyl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below, provided that the substituents are sterically compatible and the rules of chemical bonding and strain energy are satisfied.

[0145] As used herein, the term “alkynyl” represents straight-chained or branched hydrocarbon moieties containing a triple bond. Unless otherwise specified, C2-C24 (e.g., C2-C24, C2-C20, C2- Cis, C2-C16, C2-C14, C2-C12, C2-C10, C2-C8, C2-C6, or C2-C4) alkynyl groups are intended. Alkynyl groups may contain more than one unsaturated bond. Examples include C2-C6-alkynyl, such as ethynyl, 1-propynyl, 2-propynyl (or propargyl), 1-butynyl, 2-butynyl. 3-butynyl, 1- methyl-2-propynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 3-methyl- 1-butynyl, 1- methyl-2-butynyl, l-methyl-3-butynyl, 2-methyl-3-butynyl, l,l-dimethyl-2-propynyl, l-ethyl-2- propynyl, 1 -hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 3-methyl-l-pentynyl, 4- metiiyl- 1-pentynyl, l-methyl-2-pentynyl, 4-methyl-2-pentynyl, l-methyl-3-pentynyl, 2-methyl- 3-pentynyl, l-methyl-4-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, l,l-dimethyl-2- butynyl, l,l-dimethyl-3-butynyl, l,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 3,3-dimethyl- 1-butynyl, l-ethyl-2-butynyl, l-ethyl-3-butynyl, 2-ethyl-3-butynyl, and l-ethyl-l-methyl-2- propynyl. Alkynyl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyd, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

[0146] As used herein, the term “aryl,” as well as derivative terms such as aryloxy, refers to groups that include a monovalent aromatic carbocyclic group of from 3 to 50 carbon atoms. Aryl groups can include a single ring or multiple condensed rings. In some examples, aryl groups include Ce-Cio aryl groups. Examples of aryl groups include, but are not limited to, benzene, phenyl, biphenyl, naphthyl, tetrahydronaphthyl, phenylcyclopropyl, phenoxybenzene, and indanyl. The term “aryl” also includes “heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term “non-heteroaryl,” which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain a heteroatom The aryl substituents may be unsubstituted or substituted with one or more chemical moieties. Examples of suitable substituents include, for example, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein. The tom “biaryl” is a specific type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

[0147] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term “heterocycloalkyl” is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

[0148] The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one double bound, / .e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadirayl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above and is included within tire meaning of tire term “cycloalkenyl,” where at least one of tire carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, acyl, aldehyde, amino, cyano, carboxylic acid, ester, ether, halide, hydroxyl, ketone, nitro, phosphonyl, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

[0149] The term “cyclic group” is used herein to refer to either aryl groups, non-aryl groups (z.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems (e.g., monocyclic, bicyclic, tricyclic, polycyclic, etc.) that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.

[0150] The term “acyl” as used herein is represented by the formula -C(O)Z1where Z1can be a hydrogen, hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. As used herein, the term “acyl” can be used interchangeably with “carbonyl.” Throughout this specification “C(O)” or “CO” is a shorthand notation for C=O.

[0151] The term “acetal” as used herein is represented by the formula (Z1Z2)C(=OZ3X=OZ4), where Z1, Z2, Z3, and Z4can be, independently, a hydrogen, halogen, hydroxyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0152] The term “alkanol” as used herein is represented by tire formula ZtoH, where Z1can be an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloallyl, or heterocycloalkenyl group described above.

[0153] As used herein, the term “alkoxy” as used herein is an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group can be defined as to a group of the formula Z^O-, where Z1is unsubstituted or substituted alkyl as defined above. Unless otherwise specified, alkoxy groups wherein Z1is a C1-C24 (e.g., C1-C22, C1-C20, Ci-Cis, Ci-Cie, C1-C14, Ci- C12, Ci-Cio, Ci-Cs, Ci-Ce, or C1-C4) alkyl group are intended. Examples include methoxy, ethoxy, propoxy, 1 -methyl -ethoxy, butoxy, 1 -methyl -propoxy, 2-methyl-propoxy, 1,1 -dimethyl- ethoxy, pentoxy, 1-methyl-butyloxy, 2-methyl-butoxy, 3-methyl-butoxy, 2,2-di-methyl-propoxy, 1-ethyl-propoxy, hexoxy, 1,1-dimethyl-propoxy, 1,2-dimethyl-propoxy, 1-methyl-pentoxy, 2- methyl-pentoxy, 3-methyl-pentoxy, 4-methyl-penoxy, 1,1 -dimethyl -butoxy, 1,2-dimethyl- butoxy, 1,3-dimethyl-butoxy, 2,2-dimethyl-butoxy, 2,3-dimethyl-butoxy, 3,3-dimethyl-butoxy, 1-ethyl-butoxy, 2-ethylbutoxy, 1,1,2-trimethyl-propoxy, 1,2,2-trimethyl-propoxy, 1 -ethyl- 1- methyl-propoxy, and l-ethyl-2-methyl-propoxy.

[0154] The term “aldehyde” as used herein is represented by the formula — C(O)H. Throughout this specification “C(O)” is a shorthand notation for C=O.

[0155] The term “amino” as used herein are represented by the formula — NZ^Z3, where Z1, Z2, and Z3can each be substitution group as described herein, such as hydrogen, an allyl, alkenyl, alkynyl, aryl, heteroaiyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0156] The terms “amide” or “amido” as used herein are represented by the formula — CXOJNZ^2, where Z1and Z2can each be substitution group as described herein, such as hydrogen, an alkyl, alkenyl, alkynyl, aryl, heteroaiyl, cycloallyl, cycloalkenyl, heterocycloallyl, or heterocycloalkenyl group described above.

[0157] The term “anhydride” as used herein is represented by the formula Z*C(O)OC(O)Z2where Z1and Z2, independently, can be an alkyl, alkenyl, allynyl, aryl, heteroaiyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0158] The term “cyclic anhydride” as used herein is represented by the formula: where Z1can be an alkyl, alkenyl, allynyl, aryl, heteroaiyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0159] The term “azide” as used herein is represented by the formula -N=N=N.

[0160] The term “carboxylic acid” as used herein is represented by the formula — C(O)OH. A “carboxylate” or “carboxyl” group as used herein is represented by the formula

[0161] — C(O)O"

[0162] The term “cyano” as used herein is represented by the formula — CN.

[0163] The term “ester” as used herein is represented by the formula — (DC(O)Z1or — CCOJOZ1, where Z1can be an allyl, alkenyl, allynyl, aryl, heteroaiyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0164] The term “ether” as used herein is represented by the formula Z^Z2, where Z1and Z2can be, independently, an allyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0165] The term “epoxy” or “epoxide” as used herein refers to a cyclic ether with a three atom ring and can represented by the formula: where Z1, Z2, Z3, and Z4can be, independently, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0166] The term “ketone” as used herein is represented by the formula Z’QO) / 2, where Z1and Z2can be, independentiy, an alkyl, alkenyl, alkynyl, aryl, heteroaiyl, cycloallyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0167] The term “halide” or “halogen” or “halo” as used herein refers to fluorine, chlorine, bromine, and iodine.

[0168] The term “hydroxyl” as used herein is represented by the formula — OH.

[0169] The term “nitro” as used herein is represented by the formula — NO2.

[0170] The term “phosphonyl” is used herein to refer to the phospho-oxo group represented by the formula — P(OXOZ1)2, where Z1can be hydrogen, an allyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0171] The term “silyl” as used herein is represented by the formula — SiZ^Z3, where Z1, Z2, and Z3can be, independently, hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloallyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0172] The term “sulfonyl” or “sulfone” is used herein to refer to the sulfo-oxo group represented by the formula — SCOJiZ1, where Z1can be hydrogen, an allyl, alkenyl, alkynyl, aryl, heteroaryl, cycloallyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0173] The term “sulfide” as used herein comprises the formula •S-

[0174] The term “thiol” as used herein is represented by the formula — SH.

[0175] “R1,” “R 22,„” u “R3,” “Rn,” etc., where n is some integer, as used herein can, independentiy, possess one or more of the groups listed above. For example, if R1is a straight chain allyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an amino group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within a second group or, alternatively, the first group can be pendant (i.e., attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.

[0176] As used herein, Me refers to a methyl group; OMe refers to a methoxy group; and z-Pr refers to an isopropyl group.

[0177] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible stereoisomer or mixture of stereoisomer (e.g„ each enantiomer, each diastereomer, each meso compound, a racemic mixture, or scalemic mixture).

[0178] Compounds

[0179] Disclosed herein are compounds and methods of making and use thereof. For example, disclosed herein are compounds defined by Formula I, or a pharmaceutically acceptable salt thereof: wherein

[0180] R1, R2, R3, and R4are each independently H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alky l, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 alkoxy, NRxRy, or wherein, as valence permits, R1and R2, R2and R3, or R3and R4, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms;

[0181] Rxand Ryare independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl; R5is H, OH, halogen, or substituted or unsubstituted C1-C20 alkyl;

[0182] R6is substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, or substituted or unsubstituted C1-C20 alkoxy;

[0183] Q is C(O) or S(O)2; and

[0184] A is a cyclic hydrophobic group, such as a substituted or unsubstituted aromatic group.

[0185] In some examples of Formula I, R6is substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted C3-C20 aryl. In some examples of Formula I, R6is substituted or unsubstituted Ci-Cs alkyl or substituted or unsubstituted benzyl. In some examples of Formula I, R6is substituted or unsubstituted Ci-Ce alkyl.

[0186] In some examples of Formula I, Q is S(O)2. In some examples of Formula I, Q is C(O).

[0187] In some examples of Formula I, Q is S(O)2 and R6is substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted C3-C20 aryl. In some examples of Formula I, Q is S(O)2 and R6is substituted or unsubstituted Ci-Ce alkyl or substituted or unsubstituted benzyl. In some examples of Formula I, Q is S(O)z and R6is substituted or unsubstituted Ci-Ce alky l.

[0188] In some examples of Formula I, Q is C(O) and R6is substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted C3-C20 aryl. In some examples of Formula I, Q is C(O) and R6is substituted or unsubstituted Ci-Ce alkyl or substituted or unsubstituted benzyl. In some examples of Formula I, Q is C(O) and R6is substituted or unsubstituted Ci-Ce alkyl.

[0189] In some examples of Formula I, A is a substituted or unsubstituted heteroaryl group or a substituted or unsubstituted non-heteroaryl group. In some examples of Formula I, A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula I, A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula I, A is a substituted or unsubstituted benzyl group.

[0190] In some examples of Formula I, R6is substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted C3-C20 aryl and A is a substituted or unsubstituted heteroaryl group or a substituted or unsubstituted non-heteroaryl group. In some examples of Formula I, R6is substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted C3-C20 aryl and A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula I, R6is substituted or unsubstituted Ci-Ce alkyl or substituted or unsubstituted benzyl and A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof, In some examples of Formula I, R6is substituted or unsubstituted Ci-Cs alkyl and A is a substituted or unsubstituted benzyl group.

[0191] In some examples of Formula I, Q is S(O)2 and A is a substituted or unsubstituted heteroaiyl group or a substituted or unsubstituted non-heteroaryl group. In some examples of Formula I, Q is S(O)z and A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula I, Q is S(O)z and A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula I, Q is S(O)z and A is a substituted or unsubstituted benzyl group.

[0192] In some examples of Formula I, Q is C(O) and A is a substituted or unsubstituted heteroaiyl group or a substituted or unsubstituted non-heteroaryl group. In some examples of Formula I, Q is C(O) and A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula I, Q is C(O) and A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula I, Q is C(O) and A is a substituted or unsubstituted benzyl group.

[0193] In some examples of Formula I, Q is 8(0)2; R6is substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted C3-C20 aryl; and A is a substituted or unsubstituted heteroaiyl group or a substituted or unsubstituted non-heteroaiyl group. In some examples of Formula I, Q is S(O)2; R6is substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted C3-C20 aryl; and A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula I, Q is 8(0)2; R6is substituted or unsubstituted Ci-Ce alkyl or substituted or unsubstituted benzyl; and A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof, In some examples of Formula I, Q is 8(0)2; R6is substituted or unsubstituted Ci-Ce alkyl; and A is a substituted or unsubstituted benzyl group.

[0194] In some examples of Formula I, Q is C(O); R6is substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted C3-C20 aryl; and A is a substituted or unsubstituted heteroaiyl group or a substituted or unsubstituted non-heteroaiyl group. In some examples of Formula I, Q is C(O); R6is substituted or unsubstituted C1-C20 allyl, or substituted or unsubstituted C3-C20 aryl; and A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula 1, Q is C(O); R6is substituted or unsubstituted Ci-Ce alkyl or substituted or unsubstituted benzyl; and A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula I, Q is C(O); R6is substituted or unsubstituted Ci-Ce alkyl; and A is a substituted or unsubstituted benzyl group.

[0195] In some examples of Formula I, R5is hydrogen or halogen. In some examples of Formula I, R5is hydrogen.

[0196] In some examples of Formula I, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula I, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 6 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula I, RT-R4are each independentiy H, halogen, OCH3, CHs, or wherein R2and R3, together with the atoms to which they are attached form a benzyl group. In some examples of Formula I, at least one of R:-R4is not hydrogen. In some examples of Formula I, R^R4are all hydrogen.

[0197] In some examples, the compound is defined by Formula II, or a pharmaceutically acceptable salt thereof: wherein n is an integer from 0 to 2.

[0198] In some examples of Formula II, Q is S(O)2. In some examples of Formula II, Q is C(O).

[0199] In some examples of Formula II, n is 0.

[0200] In some examples of Formula II, A is a substituted or unsubstituted heteroaryl group or a substituted or unsubstituted non-heteroaryl group. In some examples of Formula n, A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula II, A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula II, A is a substituted or unsubstituted benzyl group.

[0201] In some examples of Formula II, Q is S(O)2 and n is 0. In some examples of Formula II, Q is C(O) and n is 0.

[0202] In some examples of Formula II, Q is S(O)z and A is a substituted or unsubstituted heteroaryl group or a substituted or unsubstituted non-heteroaryl group. In some examples of Formula II, Q is S(O)z and A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula II, Q is S(O)i and A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula II, Q is S(O)2 and A is a substituted or unsubstituted benzyl group.

[0203] In some examples of Formula II, Q is C(O) and A is a substituted or unsubstituted heteroaryl group or a substituted or unsubstituted non-heteroaryl group. In some examples of Formula II, Q is C(O) and A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula II, Q is C(O) and A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula II, Q is C(O) and A is a substituted or unsubstituted benzyl group.

[0204] In some examples of Formula II, n is 0 and A is a substituted or unsubstituted heteroaryl group or a substituted or unsubstituted non-heteroaryl group. In some examples of Formula II, n is 0 and A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula II, n is 0 and A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula II, n is 0 and A is a substituted or unsubstituted benzyl group.

[0205] In some examples of Formula II, Q is S(O)z, n is 0, and A is a substituted or unsubstituted heteroaryl group or a substituted or unsubstituted non-heteroaryl group. In some examples of Formula II, Q is S(O)z, n is 0, and A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula II, Q is S(O)2, n is 0, and A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula 11, Q is S(O)2, n is 0, and A is a substituted or unsubstituted benzyl group.

[0206] In some examples of Formula II, Q is C(O), n is 0, and A is a substituted or unsubstituted heteroaryl group or a substituted or unsubstituted non-heteroaryl group. In some examples of Formula II, Q is C(O), n is 0, and A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula II, Q is C(O), n is 0, and A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula II, Q is C(O), n is 0, and A is a substituted or unsubstituted benzyl group.

[0207] In some examples of Formula II, R5is hydrogen or halogen. In some examples of Formula II, R5is hydrogen.

[0208] In some examples of Formula II, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula n, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted Ci-Ce alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 6 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula n, Rx-R4are each independently H, halogen, OCH3, CHa, or wherein R2and R3, together with the atoms to which they are attached form a benzyl group. In some examples of Formula II, at least one of Rx-R4is not hydrogen. In some examples of Formula II, Rx-R4are all hydrogen.

[0209] In some examples, the compound is defined by Formula HI: or a pharmaceutically acceptable salt thereof.

[0210] In some examples of Formula in, n is 0. In some examples of Formula Ill, A is a substituted or unsubstituted heteroaryl group or a substituted or unsubstituted non-heteroaryl group. In some examples of Formula III, A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula HI, A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula III, A is a substituted or unsubstituted benzyl group.

[0211] In some examples of Formula III, n is 0 and A is a substituted or unsubstituted heteroaryl group or a substituted or unsubstituted non-heteroaryl group. In some examples of Formula III, n is 0 and A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula III, n is 0 and A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula III, n is 0 and A is a substituted or unsubstituted benzyl group.

[0212] In some examples of Formula III, R5is hydrogen or halogen. In some examples of Formula in, R5is hydrogen.

[0213] In some examples of Formula III, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula III, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 6 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula III, R*-R4are each independently H, halogen, OCH3, CH3, or wherein R2and R3, together with the atoms to which they are attached form a benzyl group. In some examples of Formula III, at least one of R^R4is not hydrogen. In some examples of Formula III, R:-R4are all hydrogen. In some examples, the compound is defined by Formula IV: or a pharmaceutically acceptable salt thereof.

[0214] In some examples of Formula IV, A is a substituted or unsubstituted heteroaryl group or a substituted or unsubstituted non-heteroaiyl group. In some examples of Formula IV, A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group. In some examples of Formula IV, A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof. In some examples of Formula IV, A is a substituted or unsubstituted benzyl group.

[0215] In some examples of Formula IV, R5is hydrogen or halogen. In some examples of Formula IV, R5is hydrogen.

[0216] In some examples of Formula IV, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula IV, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 6 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula IV, R*-R4are each independently H, halogen, OCH3, CHa, or wherein R2and R3, together with the atoms to which they are attached form a benzyl group. In some examples of Formula IV, at least one of Rr-R4is not hydrogen. In some examples of Formula IV, Rx-R4are all hydrogen. In some examples, the compound is defined by Formula V, or a pharmaceutically acceptable salt thereof: wherein

[0217] R7, R8, R9, R10, and R11are each independently H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 alkoxy, or NRxRy, or wherein, as valence permits, R7and R8, R* and R9, R9and R10, or R10and Ru, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms; and

[0218] Rxand Ryare independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl.

[0219] In some examples of Formula V, R7, R8, R9, R10, and R11are each independently H, OH, nitro, substituted or unsubstituted Ci-Ce allyl, substituted or unsubstituted Cs-Cs aryl, substituted or unsubstituted C4-C10 alkylaryl, substituted or unsubstituted C3-C10 heteroaryl, substituted or unsubstituted Ci-Ce acyl, substituted or unsubstituted Ci-Cs alkoxy, or wherein, as valence permits, R7and R8, R8and R9, R9and R10, or R10and R11, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms.

[0220] In some examples of Formula V, R7, R8, R9, R10, and R11are each independently H, NO2, CH3, OCH3, substituted or unsubstituted tetrazole, substituted or unsubstituted benzyl, or wherein, as valence permits, R9and R10, together with the atoms to which they are attached, form a substituted or unsubstituted benzyl.

[0221] In some examples of Formula V, R7, R8, R10, R11, or a combination thereof is hydrogen.

[0222] In some examples of Formula V, R7, R8, R10, and R11are all hydrogen.

[0223] In some examples of Formula V, R9is a substituted or unsubstituted aryl group.

[0224] In some examples of Formula V, R9is a substituted or unsubstituted benzyl group.

[0225] In some examples of Formula V, R7, R8, R10, and R11are all hydrogen and R9is a substituted or unsubstituted aryl group.

[0226] In some examples of Formula V, R7, R8, R10, and R11are all hydrogen and R9is a substituted or unsubstituted benzyl group.

[0227] In some examples of Formula V, R5is hydrogen or halogen. In some examples of Formula V, R5is hydrogen.

[0228] In some examples of Formula V, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula V, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted Ci-Ce alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 6 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula V, Rx-R4are each independently H, halogen, OCH3, CHa, or wherein R2and R3, together with the atoms to which they are attached form a benzyl group. In some examples of Formula V, at least one of Rx-R4is not hydrogen. In some examples of Formula V, R’-R4are all hydrogen.

[0229] In some examples, the compound is defined by Formula VI, or a pharmaceutically acceptable salt thereof: wherein

[0230] R12, R13, R14, R15, and R16are each independently H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C3-C20 aryloxy, substituted or unsubstituted C1-C20 ester, substituted or unsubstituted C1-C20 carbonate ester, substituted or unsubstituted C1-C20 sulfonate ester, substituted or unsubstituted C1-C20 sulfamate, or NR*Rb, or wherein, as valence permits, R12and R13, R13and R14, R14and R15, or R15and R16, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms; and

[0231] R* and Rbare independently selected from substituted or unsubstituted C1-C20 alltyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl.

[0232] In some examples of Formula VI, R7, R8, R10, and R11are each independently H, OH, nitro, substituted or unsubstituted Ci-Ce alltyl, substituted or unsubstituted Cs-Cs aryl, substituted or unsubstituted C4-C10 alkylaryl, substituted or unsubstituted C3-C10 heteroaryl, substituted or unsubstituted Ci-Ce acyl, substituted or unsubstituted Ci-Cs alkoxy, or wherein, as valence permits, R7and R8, or R10and R11, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms.

[0233] In some examples of Formula VI, R7, R8, R10, and R11are each independently H, NCh, CH3, OCH3, substituted or unsubstituted tetrazole, substituted or unsubstituted benzyl.

[0234] In some examples of Formula VI, R7, R8, R10, R11, or a combination thereof is hydrogen.

[0235] In some examples of Formula VI, R7, R8, R10, and R11are all hydrogen.

[0236] In some examples, the compound is defined by Formula VI-A: or a pharmaceutically acceptable salt thereof.

[0237] In some examples of Formula VI or Formula VI-A, R12, R13, R14, R15, and R16are each independently H, OH, nitro, substituted or unsubstituted Ci-Cs alkyl, substituted or unsubstituted Cs-Cs aryl, substituted or unsubstituted C4-C10 alkylaryl, substituted or unsubstituted C3-C10 heteroaryl, substituted or unsubstituted Ci-Cs acyl, substituted or unsubstituted Ci-Cs alkoxy, substituted or unsubstituted Cs-Cs aryloxy, substituted or unsubstituted Ci-Cs ester, substituted or unsubstituted C1-C10 carbonate ester, substituted or unsubstituted C1-C10 sulfonate ester, or substituted or unsubstituted C1-C4 sulfamate.

[0238] In some examples of Formula VI or Formula VI-A R12, R13, R14, R15, and R16are each independently H, halogen, nitro, CH3, tert-butyl, -OCH3, -CH(O), -C(O)OCH3, -OC(O)CH3, -

[0239] 20 OC(O)CH2CH3, -OCH2C(OKH3, -OC(O)OCH3, -OC(O)OCH2CH3, -OCCH2)2O(CH2)2OCH3,

[0240] -O(CH2)2O(CH2)2O(CH2)2OCH3,

[0241] In some examples of Formula VI or Formula VI-A, R13, R15, or a combination thereof is hydrogen. In some examples of Formula VI or Formula VI-A, R13and R13are both hydrogen.

[0242] In some examples of Formula VI or Formula VI-A, R13, R14, and R13are all hydrogen. In some examples of Formula VI or Formula VI-A, R12, R13, R15, and R16are all hydrogen.

[0243] In some examples of Formula VI or Formula VI-A, R12, R13, R14, R13, and R16are all hydrogen.

[0244] In some examples of Formula VI or Formula VI-A, R3is hydrogen or halogen. In some examples of Formula VI or Formula VI-A, R3is hydrogen.

[0245] In some examples of Formula VI or Formula VI-A, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula VI or Formula VI-A, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 6 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula VI or Formula VI-A, R^R4are each independently H, halogen, OCHa, CH3, or wherein R2and R3, together with the atoms to which they are attached form a benzyl group. In some examples of Formula VI or Formula VI-A, at least one of Rx-R4is not hydrogen. In some examples of Formula VI or Formula VI-A, R^R4are all hydrogen.

[0246] In some examples, the compound is defined by Formula Vll, or a pharmaceutically acceptable salt thereof: wherein

[0247] R14is H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, orNR*Rb; and

[0248] R* and Rbare independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl.

[0249] In some examples of Formula VII, R14is hydrogen.

[0250] In some examples of Formula VII, R3is hydrogen or halogen. In some examples of Formula VII, R3is hydrogen.

[0251] In some examples of Formula VII, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula VII, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 6 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula VII, RkR4are each independently H, halogen, OCH3, CH3, or wherein R2and R3, together with the atoms to which they’ are attached form a benzyl group. In some examples of Formula VII, at least one of RT-R4is not hydrogen. In some examples of Formula VII, R*-R4are all hydrogen.

[0252] In some examples, the compound is defined by Formula VIII, or a pharmaceutically acceptable salt thereof: wherein

[0253] R12and R16are each independently H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, or NR*Rb; and

[0254] R* and Rbare independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl.

[0255] In some examples of Formula VIII, R12is hydrogen.

[0256] In some examples of Formula VIII, R5is hydrogen or halogen. In some examples of Formula VIII, R5is hydrogen.

[0257] In some examples of Formula VIII, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted C1-C20 allyl, substituted or unsubstituted C1-C20 alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula VIII, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 6 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula VIII, R*-R4are each independently H, halogen, OCHa, CHa, or wherein R2and R3, together with the atoms to which they are attached form a benzyl group. In some examples of Formula VIII, at least one of R*-R4is not hydrogen. In some examples of Formula VIII, RT-R4are all hydrogen.

[0258] In some examples, the compound is defined by Formula IX, or a pharmaceutically acceptable salt thereof: wherein

[0259] R16is H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, or NR*Rb; and

[0260] R’ and Rbare independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl.

[0261] In some examples of Formula IX, R5is hydrogen or halogen. In some examples of Formula IX, R5is hydrogen. In some examples of Formula IX, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula IX, R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 6 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms. In some examples of Formula IX, R*-R4are each independently H, halogen, OCH3, CH3, or wherein R2and R3, together with the atoms to which they are attached form a benzyl group. In some examples of Formula IX, at least one of Rr-R4is not hydrogen. In some examples of Formula IX, Rx-R4are all hydrogen.

[0262] In some examples, the compound is selected from the group consisting of:

[0263] «n

[0264] , pharmaceutically acceptable salts thereof, and combinations thereof

[0265] In some examples, the compound is selected from the group consisting of: pharmaceutically acceptable salts thereof, and combinations thereof. In some examples, the compound comprises: or a pharmaceutically acceptable salt thereof.

[0266] Compositions

[0267] Also disclosed herein are pharmaceutical compositions comprising any of the compounds disclosed herein (e.g., of Formula I - Formula IX). In some examples, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, or a combination thereof.

[0268] Also disclosed herein are compositions comprising any of the compounds disclosed herein (e.g., of Formula I - Formula IX) or any of the pharmaceutical compositions disclosed herein. In some examples, the composition can further comprise a fatty acid metabolism inhibitor.

[0269] Also disclosed herein are compositions comprising a REST (repressor element- 1 silencing transcription factor) inhibitor and a fatty acid metabolism inhibitor.

[0270] In some examples, the REST inhibitor targets SCP1. In some examples, the REST inhibitor covalently interacts with SCP1.

[0271] In some examples, the REST inhibitor comprises a compound described by Medellin et al. J Med. Chem, 2022, 65, 507-519, or a pharmaceutically acceptable salt thereof. In some examples, the REST inhibitor comprises any of the compounds disclosed herein (e.g., of Formula I - Formula IX).

[0272] In some examples, the fatty acid metabolism inhibitor comprises Triacsin C, TVB- 2640 / Denifanstat, avasimible, 2-fluoropalmitic acid, their homologues, or a combination thereof. In some examples, the fatty acid metabolism inhibitor comprises a pan-ACSL inhibitor. In some examples, the fatty acid metabolism inhibitor comprises Triacsin C.

[0273] In some examples, the composition is synergistic (e.g. LDso is lower than when the REST inhibitor and the fatty acid metabolism inhibitor are used alone).

[0274] In some examples, the composition exhibits limited hepatotoxicity. In some examples, the compositions further comprise a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, or a combination thereof.

[0275] In some examples, the disclosed compositions optionally further comprise other therapeutic ingredients or adjuvants.

[0276] In some examples, the therapeutic agent comprises an anticancer agent. In some examples, the therapeutic agent comprises a chemotherapeutic agent, an immunotherapeutic agent, or a combination thereof.

[0277] In some examples, the therapeutic agent can comprise a chemotherapeutic agent. Chemotherapy is the treatment of cancer with one or more cytotoxic anti-neoplastic drugs (e.g., chemotherapeutic agents) as part of a standardized regimen. Chemotherapy may be given with a curative intent or it may aim to prolong life or to palliate symptoms. In some cases, it can be used in conjunction with other cancer treatments, such as radiation therapy, surgery, hyperthermia therapy, or a combination thereof. Examples of chemotherapeutic agents include, but are not limited to, 13-cis-Retinoic Acid, 2-Amino-6-Mercaptopurine, 2-CdA, 2- Chlorodeoxyadenosine, 5-fluorouracil, 6-Thioguanine, 6-Mercaptopurine, Accutane, Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alitretinoin, Alkaban-AQ, Alkeran, All-transretinoic acid, Alpha interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron, Anastrozole, Arabinosylcytosine, Aranesp, Aredia, Arimidex, Aromasin, Arsenic trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib, Busulfan, Busulfex, C225, Calcium Leucovorin, Campath, Camptosar, Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine, Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cerubidine, cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine liposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposomal, DaunoXome, Decadron, Delta-Cortef, Deltasone, Denileukin diftitox, DepoCyt, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposomal, Droxia, DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate, Eulexin, Evista, Exemestane, Fareston, Faslodex, F emar a, Filgrastim, Floxuridine, Fludara, Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron, Lupron Depot, Matulane, Maxidex, Mechlorethamine, -Mechlorethamine Hydrochlorine, Medralone, Medrol, Megace, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate Sodium, Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta, Neumega, Neupogen, Nilandron, Nilutamide, Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide acetate, Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orapred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG Interferon, Pegaspaigase, Pegfilgrastim, PEG-INTRON, PEG-L-asparaginase, Phenylalanine Mustard, Platinol, Platinol- AQ, Prednisolone, Prednisone, Prelone, Procarbazine, PROCRIT, Proleukin, Prolifeprospan 20 with Carmustine implant, Purinethol, Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alfa-2a), Rubex, Rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Saigramostim, Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin, Taxol, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide, Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab, Tretinoin, Trexall, Trisenox, TSP A, VCR, Velban, Velcade, VePesid, Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Vumon, Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zometa, Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte colony stimulating factor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, HMM, Hycamtin, Hydrea, Hydrocort Acetate, Hydrocortisone, Hydrocortisone sodium phosphate, Hydrocortisone sodium succinate, Hydrocortone phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin, Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib mesylate, Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEG conjugate), Interleukin 2, Interleukin-11, Intron A (interferon alfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine, Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, L- PAM, L-Sarcolysin, Meticortai, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX, Mustaigen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan, Isotretinoin, Kidrolase, Lanacort, L-asparaginase, LCR, FAM-HYD-1, Marizomib (NPI-0052), Lenalidomide, Carfilzomib, Panobinostat, Quisinostat, Selinexor, Oprozomib, and combinations thereof. The anticancer agent can also include biopharmaceuticals such as, for example, antibodies.

[0278] Examples of suitable immunotherapeutic agents include, but are not limited to, alemtuzumab, cetuximab (ERBITUX), gemtuzumab, iodine 131 tositumomab, rituximab, trastuzamab (HERCEPTIN), and combinations thereof. In some examples, the composition is administered to a subject. In some examples, the subject is a mammal. In some examples, the mammal is a primate. In some examples, the mammal is a human. In some examples, the human is a patient.

[0279] The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0280] Methods of Making

[0281] Also disclosed herein are methods of making any of the compounds or compositions disclosed herein.

[0282] The compounds described herein can be prepared in a variety of ways known to one skilled in the art of organic synthesis or variations thereon as appreciated by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions can vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art.

[0283] Variations on the compounds described herein include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule can be changed. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0284] The starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Katchem (Prague, Czech Republic), Aldrich Chemical Co., (Milwaukee, WI), Acres Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Lotus, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley' and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and Suppiementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical excipients disclosed herein can be obtained from commercial sources.

[0285] Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which tire reactions are carried out, z.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., or13C) infrared spectroscopy, spectrophotometry (e.g., UV- visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0286] Methods of Use

[0287] Also disclosed herein are methods of use of any of the compounds or compositions disclosed herein.

[0288] For example, also disclosed heron are methods of treating, preventing, or ameliorating a disease or a disorder in a subject in need thereof, the method comprising administering to tire subject a therapeutically effective amount of any of the compounds or compositions disclosed herein.

[0289] For example, disclosed herein are methods of treating a disease or a disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the compounds or compositions disclosed herein.

[0290] Examples of diseases and disorders include, but are not limited to, cancer.

[0291] Also disclosed herein are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any' of the compounds or compositions disclosed herein.

[0292] For example, the compounds and compositions described herein or pharmaceutically acceptable salts thereof are useful for treating cancer in humans, e.g., pediatric and geriatric populations, and in animals, e.g., veterinary applications. The disclosed methods can optionally include identifying a patient who is or may be in need of treatment of a cancer. Examples of cancer types treatable by the compounds and compositions described herein include bladder cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, and testicular cancer. Further examples include cancer and / or tumors of the anus, bile duct, bone, bone marrow, bowel (including colon and rectum), eye, gall bladder, kidney, mouth, larynx, esophagus, stomach, testis, cervix, mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid, vagina, vulva, uterus, liver, muscle, blood cells (including lymphocytes and other immune system cells). Further examples of cancers treatable by tire compounds and compositions described herein include carcinomas, Karposi’s sarcoma, melanoma, mesothelioma, soft tissue sarcoma, pancreatic cancer, lung cancer, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, and other), and lymphoma (Hodgkin’s and non-Hodgkin’s), and multiple myeloma.

[0293] In some examples, the cancer has an upregulated REST level.

[0294] In some examples, the cancer comprises glioblastoma multiforme, low-grade glioma, neuroblastoma, medullablastoma, or a combination thereof.

[0295] In some examples, the cancer comprises glioblastoma.

[0296] The methods of treatment or prevention of cancer described herein can, in some examples, further include treatment with one or more additional agents (e.g., an anti-cancer agent or ionizing radiation). For example, the compounds or compositions or pharmaceutically acceptable salts thereof as described herein can be combined into a pharmaceutical composition with an additional anticancer agent, such as a chemotherapeutic agent, an immunotherapeutic agent, or a combination thereof.

[0297] The additional anti-cancer agent can also include biopharmaceuticals such as, for example, antibodies. Many tumors and cancers have viral genome present in the tumor or cancer cells. For example, Epstein-Barr Virus (EBV) is associated with a number of mammalian malignancies. The compounds disclosed herein can also be used alone or in combination with anticancer or antiviral agents, such as ganciclovir, azidothymidine (AZT), lamivudine (3TC), etc., to treat patients infected with a virus that can cause cellular transformation and / or to treat patients having a tumor or cancer that is associated with the presence of viral genome in the cells. The compounds disclosed herein can also be used in combination with viral based treatmeits of oncologic disease.

[0298] Also described herein are methods of suppressing tumor growth in a subject. The method includes contacting at least a portion of the tumor with a therapeutically effective amount of any of the compound or compositions as described herein. In some examples, the methods further include the step of irradiating at least a portion of the tumor with a therapeutically effective amount of ionizing radiation. As used herein, the term ionizing radiation refers to radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization. An example of ionizing radiation is x-radiation. A therapeutically effective amount of ionizing radiation refers to a dose of ionizing radiation that produces an increase in cell damage or death when administered in combination with the compounds described herein. The ionizing radiation can be delivered according to methods as known in the art, including administering radiolabeled antibodies and radioisotopes.

[0299] The methods of treatment of the disease or disorder described herein can further include treatment with one or more additional agents. The one or more additional agents and the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be administered in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart. The methods can also include more than a single administration of the one or more additional agents and / or the compounds and compositions or pharmaceutically acceptable salts thereof as described herein. The administration of the one or more additional agents and the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be by the same or different routes. When treating with one or more additional agents, the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be combined into a pharmaceutical composition that includes the one or more additional agents.

[0300] The methods, compounds, and compositions as described herein are usefill for both prophylactic and therapeutic treatment. As used herein the term treating or treatment includes prevention; delay in onset; diminution, eradication, or delay in exacerbation of signs or symptoms after onset; and prevention of relapse. For prophylactic use, a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein are administered to a subject prior to onset (e.g., before obvious signs of the disease or disorder), during early onset (e.g., upon initial signs and symptoms of the disease or disorder), or after an established development of the disease or disorder. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of a disease or disorder. Therapeutic treatment involves administering to a subject a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein after the disease or disorder is diagnosed. Compositions, Formulations, Methods of Administration, and Kits

[0301] In vivo application of the disclosed compounds, and compositions containing them, can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed compounds can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrastemal administration, such as by injection. Administration of the disclosed compounds or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art.

[0302] The compounds disclosed herein, and compositions comprising them, can also be administered utilizing liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time. The compounds can also be administered in their salt derivative forms or crystalline forms.

[0303] The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington 's Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable excipient in order to facilitate effective administration of the compound. The compositions used can also be in a variety of forms. These include, for example, solid, semi- solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and application. The compositions can also include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in tire art.

[0304] Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired application, compositions disclosed herein can comprise between about 0.1% and 100% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

[0305] The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0306] Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the excipients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question.

[0307] Compounds disclosed herein, and compositions comprising them, can be delivered to a cell either through direct contact witii the cell or via a carrier means. Carrie- means for delivering compounds and compositions to cells are known in the art.

[0308] For the treatment of oncological disorders, the compounds or compositions disclosed herein can be administered to a patient in need of treatment in combination witii other antitumor or anticancer substances and / or with radiation and / or photodynamic therapy and / or with surgical treatmeit to remove a tumor. These other substances or treatments can be given at the same as or at different times from the compounds or compositions disclosed herein. For example, the compounds or compositions disclosed herein can be used in combination with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalates such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and / or other anti-cancer drugs or antibodies, such as, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.), respectively, or an immunotherapeutic such as ipilimumab and bortezomib.

[0309] In certain examples, compounds and compositions disclosed herein can be locally administered at one or more anatomical sites, such as sites of unwanted cell growth (such as a tumor site or benign skin growth, e.g., injected or topically applied to the tumor or skin growth), optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. Compounds and compositions disclosed herein can be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent, or an assimilable edible carrier for oral delivery. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient’s diet. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

[0310] The tablets, troches, pills, capsules, and the like can also contain the following: binders such as gum tragacanth, acacia, com starch or gelatin; diluents such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of Wintergreen, or cherry flavoring can be added. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir can contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound can be incorporated into sustained-release preparations and devices.

[0311] Compounds and compositions disclosed herein, including pharmaceutically acceptable salts thereof, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

[0312] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0313] Pharmaceutical compositions disclosed herein suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some examples, the final injectable form can be sterile and can be effectively fluid for easy syringability. In some examples, the pharmaceutical compositions can be stable under the conditions of manufacture and storage; thus, they can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0314] Sterile injectable solutions are prepared by incorporating a compound and / or agent disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient presort in the previously sterile-filtered solutions.

[0315] Pharmaceutical compositions disclosed herein can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, solution, tincture, and the like. In some examples, tire compositions can be in a form suitable for use in transdermal devices. In some examples, it will be desirable to administer them topically to the skin as compositions, in combination with a dermatologically acceptable carrier, which can be a solid or a liquid. Compounds and agents and compositions disclosed herein can be applied topically to a subject’s skin. These formulations can be prepared, utilizing any of tire compounds disclosed herein or pharmaceutically acceptable salts thereof, via conventional processing methods. Useful solid carriers include finely divided solids such as talc, city, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize tire properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example.

[0316] Thickeners such as synthetic polymers, fatty adds, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and tire like, for application directly to the skin of the user.

[0317] Pharmaceutical compositions disclosed herein can be in a form suitable for rectal administration wherein the carrier is a solid. In some examples, the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition witii the softened or melted carriers) followed by chilling and shaping in molds.

[0318] In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient Compositions containing any of the compounds disclosed herein, and / or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

[0319] Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

[0320] The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary witii the age, body weight, general health, condition, sex, diet, and extent of the disease in the patient and can be determined by one of skill in the art. Other factors include the time and route of administration, rate of excretion, drug combination, and the type and severity of the particular disease or disorder. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

[0321] Also disclosed are kits that comprise a compound or composition disclosed herein in one or more containers. The disclosed kits can optionally include pharmaceutically acceptable carriers and / or diluents. In one embodiment, a kit includes one or more other components, adjuncts, or adjuvants as described herein. In one embodiment, a kit includes instructions or packaging materials that describe how to administer a compound or composition of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, a compound and / or agent disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, a compound and / or agent disclosed herein is provided in the kit as a liquid or solution. In one embodiment, the kit comprises an ampoule or syringe containing a compound and / or agent disclosed herein in liquid or solution form.

[0322] In some examples, the kit further comprises at least one agent, wherein the compound and the agent are co-formulated.

[0323] In some examples, the compound and the agent are co-packaged.

[0324] The kits can also comprise compounds and / or products co-packaged, co-formulated, and / or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and / or product and another component for delivery to a patient.

[0325] It is contemplated that the disclosed kits can be used in connection with the disclosed methods of making, the disclosed methods of using, and / or the disclosed compounds and compositions.

[0326] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0327] The examples below are intended to further illustrate certain aspects of the systems and methods described herein, and are not intended to limit the scope of the claims.

[0328] EXAMPLES

[0329] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0330] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of measuremeit conditions, e.g., component conceitrations, temperatures, pressures and other measurement ranges and conditions that can be used to optimize the described process.

[0331] Example 1 - Targeting of REST with rationally-designed small-molecule compounds exMbits synergetic therapeutic potential in human glioblastoma cells

[0332] Abstract Glioblastoma multiforme (GBM) is an aggressive brain cancer associated with poor prognosis, intrinsic heterogeneity, plasticity, and therapy resistance. In some Glioblastoma multiforme tumors, cell proliferation is fueled by a transcriptional regulator, repressor el email- 1 silencing transcription factor (REST). Using CRISPR / Cas9, Glioblastoma multiforme cell lines dependent on REST activity were identified. New small-molecule inhibitory compounds targeting small C-terminal domain phosphatase 1 (SCP1) were developed to reduce REST protein level and transcriptional activity in glioblastoma cells. SCP1 inhibitors exhibit potent cytotoxicity, reduce REST protein level and suppress its transcriptional activity. Upon the loss of REST protein, Glioblastoma multiforme cells can potentially compensate by rewiring fatty acid metabolism, enabling continued proliferation. Combining REST inhibition with the blockade of this compensatory adaptation demonstrated substantial synergetic potential without inducing hepatotoxicity. These results highlight the efficacy and selectivity of targeting REST alone or in combination as a therapeutic strategy to combat high-REST Glioblastoma multiforme.

[0333] Introduction. Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults, with an incidence rate of 3.7 per 100,000 person-years and a high mortality rate (1). Glioblastoma multiforme exhibits high resistance to conventional radiation and chemotherapy. Emerging evidence suggests that metabolic reprogramming or adaptation may contribute to therapy resistance in glioblastoma multiforme (2). Additionally, glioblastoma multiforme is characterized by great intratumoral molecular and metabolic heterogeneity, further contributing to its high lethality (2). Given these facts, new therapeutic approaches targeting deregulated cellular pathways must be explored to improve patient prognosis and eventually develop treatments for this fatal disease (2, 3).

[0334] One of the deregulated genes in glioblastoma multiforme is a repressor element- 1 silencing transcription factor (REST), a transcriptional repressor that has been identified as an oncogenic protein in various brain tumor types, including neuroblastoma, medulloblastoma, and glioblastoma (4, 5). High expression of REST was significantly associated with worse overall survival, progression-free interval, and worse disease-specific survival in glioma patients (6). Targeting REST may inhibit cancer stem cell proliferation as REST is crucial for cancer stem cell self-renewal (7). Chronologically, one of the initial studies on REST in glioblastoma stem cells (GSC) demonstrated that glioblastoma stem cells with high REST expression produced more invasive tumors compared to those with low REST expression in orthotopic mouse tumor models (8). Genetic knockdown of REST in high-REST glioblastoma stem cells resulted in increased survival of mice (8). Importantly, treatments targeting REST may' have less severe neurological side effects than conventional chemotherapy because post-mitotic neurons do not express REST (7). Therefore, reducing REST levels in high-REST glioblastoma tumors holds promising therapeutic effects.

[0335] The REST protein acts as a transcription factor, silencing the neuronal gene expression (9). Unlike enzymes, targeting transcription factors with small-molecule inhibitors has historically been challenging (10). However, prior studies have shown that REST level is post- translationally regulated by phosphorylation-dependent protein turnover (11, 12). Once phosphorylated, REST is targeted to the cytosol for degradation by the ubiquitin ligase SCF^T,cp(11, 12). Thus, the chemical modulation of the REST protein level can be achieved by regulating its phosphorylation. One potential molecular approach to reduce REST involves targeting C- terminal domain small phosphatase 1 (CTDSP1 / SCP1), which dephosphorylates REST at sites, such as Ser861 and Ser864, that function as checkpoints for REST degradation (12, 13). REST lacking phosphorylation at Ser-861 of Ser-864 becomes more stable (13), and REST stabilization can be prevented by inhibiting SCP1 ’s phosphatase activity, leading to a reduced REST protein level. To this end, a series of compounds (called the T-series) of small-molecule covalent inhibitors of SCP1 were previously designed (14). These compounds demonstrated the capability to inhibit SCP1 phosphatase activity and decrease REST protein levels in human HEK293 cells.

[0336] Herein, a well-characterized high-REST glioblastoma cell line (T98G) was used as a starting point to validate the role of REST in glioblastoma growth. CRISPR / Cas9 gene editing was used to generate homozygous REST-null single-cell clonal lines from T98G and non-neural HEK293 cells and compared their transcriptomes. It was demonstrated that REST knockout significantly impaired the proliferation of Glioblastoma multiforme cells. A new optimized chemical lead (GR-28) was developed that causes degradation of REST protein in REST- dependent glioblastoma cells via covalent inhibition of SCP1. The GR-28 compound degraded cellular REST protein, derepressed REST-silenced genes, and induced cell death in high-REST glioblastoma multiforme cells. It was also shown that some REST-null clones were able to rewire fatty add metabolism to derepress their growth, and that this compensation effect could be negated using a chemical inhibitor of fatty acid metabolism, Triacsin C. GR-28 exhibited profound synergy when combined with Triacsin C in Glioblastoma multiforme cells, but not in hepatocarcinoma cells (HepG2), allowing effective eradication of glioblastoma cells with limited hepatotoxidty in vitro.

[0337] Results

[0338] REST is upregulated in TCGA-LGG / Glioblastoma multiforme samples and select glioblastoma cell lines. To analyze the impact of REST on Glioblastoma multiforme growth, a bioinformatics analysis was conducted of The Cancer Genome Atlas database (TCGA- Glioblastoma multiforme & TCGA-LGG projects) and compared REST mRNA abundance in low-grade glioma / LGG samples (n = 518) and glioblastoma multiforme (GBM) samples (n = 163) against normal brain samples (n = 207) from two combined datasets - TCGA and GTEx (Genotype-Tissue Expression project, https: / / gtexportal.org / home / ). Significantly elevated REST gene expression were observed in both low- and high-grade glioma (p < 0.001), Figure 1 A. Consistent with other published analyses (6), higher REST expression was associated with worse overall survival in the pooled population of patients with low-grade glioma and glioblastoma (logrank p = 9.9e-l 1, Figure IB).

[0339] REST is a silencing transcription factor that suppresses the expression of neuronal genes, and its expression is ubiquitous in non-neural tissues but down-regulated in neural precursors and neurons (15). REST protein amount was measured in several well-characterized glioblastoma multiforme cell lines compared to control cell lines - non-neural HEK293 cells and glial SVGpl2 cells. Three glioblastoma multiforme cell lines were included in the study: U251, Al 72, and T98G. Two of the cell lines - Al 72 and T98G- had significantiy upregulated basal REST protein amount compared to SVGpl2 cells (p < 0.05), Figure 1C. The REST expression level was also high in HEK293 cells since REST is ubiquitously expressed in non-neural cells. Interestingly, proliferation assays showed that the two high-REST glioblastoma multiforme cell lines (Al 72 and T98G) proliferate faster than low-REST U251 cells (Figure ID). Taken together, these results suggest glioblastoma cells vary in their REST protein level.

[0340] REST promotes Glioblastoma multiforme cell proliferation in vitro. To study the effect of REST on glioblastoma multiforme proliferation, CRISPR-Cas9 was used to generate REST- knockout (REST-KO) homozygous cells from a representative glioblastoma cell line, T98G, containing high REST protein amount. To identify the genes specific to glioblastoma multiforme rather than common REST targets, REST-KO were also generated for non-neural HEK293 cells, which also contain a high REST amount (Figure IE). Accordingly, two single-guide RNA (sgRNA) oligonucleotides targeting two specific human REST genomic regions located in exons 2 (sgRNA RY1) and 3 (sgRNA RG6) were synthesized and cloned into pX330 vector (16). Target cells (T98G or HEK293) were either 1) co-transfected with Cas9-2A-GFP and empty pX330 vector (CRISPR control) or 2) co-transfected with Cas9-2A-GFP and two sgRNA expression vectors for double-nicking recombination (REST-KO). Western blotting confirmed the absence of the protein band corresponding to the observed REST molecular weight, ~ 200 kDa (Figure IE, Figure 7 A- Figure 7B). To identify the DNA sequences of selected REST-KO clones, PCR primers flanking regions of sgRNA-introduced double-stranded breaks were designed and genomic DNA isolated from control or KO cells was amplified (Figure 7C). The resulting PCR products were purified and sequenced. In each case, the REST genomic sequence was repaired so that the resulting protein sequence had a premature stop codon (Table 1).

[0341] Table 1. REST protein sequence(s) for cell lines / clones (stop-codon highlighted in red (-)).

[0342] To evaluate the function of REST in glioblastoma multiforme, the cell proliferation rate in T98G WT and three different REST-KO clones were compared, which showed that REST deficiency resulted in significant cell growth arrest, consistent with higher proliferation doubling time (PDT) (Figure IF). To exclude potential off-target effects of CRISPR-mediated genome editing and furflier verify the specific effect of REST on promoting glioblastoma multiforme proliferation, REST expression was reconstituted in REST knockout cells by transiently transfecting REST (Figure 7D). It was observed that the reconstitution of REST partly restored the cell proliferation rate in the REST-KO CIO clone (Figure 1G). The mean proliferation doubling time of control cells was 24.9 h, whereas REST-KO CIO cells (transfected with empty pLPC) divided on average 1.7-fold slower. Furthermore, the reconstitution of REST rescued proliferation by approximately 56% (Figure 1G). These observations suggest that REST is vital for glioblastoma multiforme cell proliferation.

[0343] Taken together, these data show that REST inhibition significantly slows the proliferation potential of glioblastoma multiforme cells. Thus, targeting REST can have beneficial therapeutic effects in high-REST glioblastoma.

[0344] Covalent inhibition ofSCPl with smaU-moiecule compounds. Though REST can be a therapeutic target for high-REST glioblastoma multiforme due to its effect on proliferation, targeting transcription factors like REST by small molecules is often difficult. However, prior studies have shown that the cellular REST level is primarily regulated by its phosphorylation- triggered degradation. A series of small-molecule covalent inhibitors (T-series) against SCP1 were designed that were capable of reducing REST protein levels in human HEK293 cells (14) (T-65, Figure 2A, left). These compounds have a hydrophobic moiety that recognizes the active site of SCP1 and places the warhead near a cysteine close to the active site allowing for covalent targeting. Despite these initial design efforts, T-65 exhibited limited cytotoxicity towards high- REST glioblastoma multiforme cells (T98G and Al 72), and REST protein amount was not significantly changed after T-65 (4 jiM, 24 h) treatment (Figure 8A-Figure 8C).

[0345] In a newer generation of focused library design, the hydrophobic moiety of the compound was varied to enhance its specific interaction with the SCP1 protein (Figure 2A, right1). When designing covalent inhibitors for SCP1, an aim was to promote the noncovalent interaction of designed compounds with SCP1 to first form a stable complex and second allow tiie slow formation of a covalent bond. Such design reduces the non-specific inhibition of SCP1 by only allowing covalent bond formation within the stably bound SCP1 complex. Based on this rationale, a synthetic scheme was designed (Methods section) and a focused chemical library of ~20 compounds (G-series) was generated.

[0346] The kinetic characterization of GR-28 against the phosphatase activity of SCP 1 is shown in Figure 2B- Figure 2D. To characterize the inhibition of SCP1 phosphatase by the new focused library of covalent inhibitors, para-nitro phenyl phosphate (pNPP) assays were conducted in a time- and concentration-dependent manner (Figure 2B-Figure 2D). The compounds were incubated with pNPP for different durations and then quantified the inhibition. Out of the focused library, compound GR-28 (Figure 2A, right) exhibits the most inhibition, characterized with the following parameters: kimct of 0.00083 (0.00055-0.0015) min"1(shown is best-fit value with 95% CI), Ki of 0.6 (0.13-2.16) μM, and ktnict / Ki was calculated to be 1383 min"1M"1(Figure 2B-Figure 2D). This GR-28 compound exhibits a better Ki than the previous lead (Ki of 4.8 μM for T-65 (14)), suggesting better recognition / binding of the target. The covalent bond formation rate appears to be slower in the GR-28 compound, which can be advantageous to ensure that the covalent bond only forms for stably bound complex, thereby guaranteeing better specificity.

[0347] To test if the mechanism of inhibition is a result of covalent bond formation (14), SCP1 was incubated with the inhibitor and the change in its molecular weight was measured using Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The prolonged incubation of GR-28 (theoretical molecular weight of 476.54 Da) with SCP1 led to a peak shift of 477.21 Da of mass. This shift was consistent with the formation of a covalent bond between the protein and the compound (Figure 2E). Previous studies have identified Cl 81 nearby tire active site as the target for covalent bond formation (14). This was supported by the observation that the SCP1 Cl 81 A mutant shows resistance to GR-28 inhibition (Figure 2F). Furthermore, additional experiments were conducted to identify whether this resistance of the SCP1 C181A variant was attributed to the absence of adduct formation. Despite a prolonged overnight incubation of the SCP1 Cl 81 A variant with GR-28, no discernible covalent adduct peak was observed when analyzed with MALDI-TOF (Figure 2G).

[0348] Molecular docking was further used to model GR-28 inhibition of SCP1 (Figure 2H). The X-ray crystal structure of SCP1 with its selective inhibitor rabeprazole provided a good initial template of the inhibitor binding pocket. GR-28 consists of a hydrophobic moiety, a linker, and a warhead (Figure 2H, schematic). The end phenol ring of the hydrophobic moiety is located in a proximal pocket close to the active site, hydrophobically sandwiched between Y158 and F106. The middle ring of the hydrophobic moiety of the inhibitor does not provide direct interaction with the protein, but its rigidity reduces the entropy cost for compound binding. The amide bond of the hydrophobic moiety is stabilized by extensive interaction with the R178 side chain. The carbon linker is flexible, while the warhead moiety is well anchored to SCP1 when it covalenfly attaches to C181. The warhead forms a cation-z interaction with the benzothiophene ring and a K-U stacking interaction on the other side with Y188. The model also places the amide group of the warhead close to the carbonyl backbone of Al 53 with a potential hydrogm bond. The model suggests a strong interaction network upon tire inhibitor covalmtiy binding to SCP1.

[0349] Transcriptome sequencing reveals distinct gene signatures associated with REST. To understand if the lead compound could reduce the functional activity of REST in vitro, human glioblastoma multiforme cells were exposed to the compound to evaluate its effects on REST- mediated transcriptional silencing. First, to identify REST-controlled genes, Tag-Seq (17) was performed in WT cells and corresponding REST-null clones (Figure 3A-Figure 3B, Figure 9A- Figure 9B) in both T98G and non-neural HEK293 cells. Initially, three "slow” REST-KO T98G clones (CIO, F7, G2) and two REST-KO HEK293 clones (D10, E6) were sequenced.

[0350] Sequencing was performed in duplicate (Figure 9B), and differentially expressed genes (DEGs) were called for every unique clone versus the corresponding control cell line using DESeq2 (log2FC cut-off = 0.58, p-adjusted cut-off = 0.05). An overview of differentially expressed genes can be found in Table 2. Figure 10 shows qPCR validation of transcriptomic changes revealed by Tag-Seq. For instance, REST loss in T98G cells led to a reduction in NEDD9 expression, a marker of glioma invasion potential (18), and an increase in BEX1 expression, a tumor suppressor gene in malignant glioma (19).

[0351] Table 2. Differentially expressed genes.

[0352] A focus was on differentially expressed genes overlapping in several single-cell clones within one cell line (Figure 3B, Figure 11 A). Unlike in non-neural HEK293 cells, downregulated genes common in T98G REST-KO clones formed several significant gene ontology (GO) categories, highlighting tissue-specific functions of REST in glioblastoma (Figure 3C, fewer). These data suggest that targeting REST will inhibit several Glioblastoma multiforme-related pathways (20-23) in contrast with non-neural cells.

[0353] As expected, since REST is a transcriptional silencer, direct REST target genes could be found among upregulated genes that became derepressed after REST knockout. Consistent with known REST functions (21), gene ontology analysis showed that derepressed genes in both T98G and HEK293 were related to neuron-specific activities such as synaptic vesicle cycle, exocytosis, and neurotransmitter secretion (Figure 3C, upper, Figure 1 IB). To identify direct REST targets, derepressed genes were overlapped with a recently published dataset that included genome-wide REST binding sites in human embryonic stem cells (24). As a result, only 40% of derepressed genes in T98G and the absolute majority of derepressed genes in HEK293 (97 / 106) were estimated as direct targets of REST (Figure 3D). REST-target genes common for both cell lines (n = 33, Table 3) were listed as representative REST-controlled genes regardless of cell type. Table 3. Shared REST-controlled genes (T98G, HEK293)

[0354] To identify the genes subject to REST control in Glioblastoma multiforme tumors, a list of representative REST-target genes (n = 6) was prepared using both the top-100 anti-correlated with REST mRNA genes in TCGA-Glioblastoma multiforme dataset and the Tag-Seq data overtyping in T98G and HEK293 cells (Figure 3E). Indeed, all the genes from the list were additionally validated by qPCR assay in two glioblastoma REST-KO clones (Figure 3F). Assessment of functional level of REST in glioblastoma cells is shown in Figure 12A-Figure 12B. A detailed rationale for the inclusion of specific genes in the list of REST-controlled genes is shown in Figure 12B. Interestingly, the majority of included genes were significantly derepressed under REST knockdown in other cancer types, such as endometrial adenocarcinoma, GSE150254 (25), and breast cancer, GSE173857 (26), suggesting they could be further validated as REST-controlled genes in other cancer types with known REST deregulation.

[0355] Lead compound, GR-28, reduces the transcriptional activity of REST and is cytotoxic against high-REST Glioblastoma multiforme cells. Since biochemical assays indicated potent in vitro activity of GR-28 against tire SCP1 protein, it was furflier sought to assess the lead compound’s effect on the REST protein level and transcriptional activity of REST in the cellular setting.

[0356] Western blotting showed that adding 4 μM GR-28 resulted in a 2.1 -fold decrease in REST amount in A 172 cells after 24 h of incubation (Figure 4 A). This is a big improvement compared to the parental compound, T-65, which could not reduce REST protein levels in Glioblastoma multiforme cells in the same treatment setting (Figure 8C). Furthermore, GR-28 treatment (4 μM, 18 h) of A172 cells significantly increased the transcriptional activity of 4 genes (RUNDC3A, SCAMPS, AP3B2, CHGB, FC > 1.5, / ? < 0.05) out of 6 genes that were identified as REST-controlled (Figure 4B, leff). As T98G cells were more resistant to GR-28, a longer treatment (48 h) was applied with the compound at a 10 μM dose, which caused a 1.6- fold reduction in REST protein (Figure 4 A). Treatment for 36 hours at 10 μM dose markedly increased expression of the RUNDC3A gene (FC > 1.5, p < 0.05), but expression of other REST- controlled gores did not change significantly (Figure 4B, right).

[0357] Next, the survival rates of high-REST glioblastoma multiforme cells treated with GR-28 at different doses were analyzed and viability normalized to cells treated with DMSO-control was plotted (Figure 4C). The HepG2 control cell line was also treated for a parallel estimation of compound-induced hepatotoxicity (27). Dose-response fitting showed that the LDso of GR-28 was lower than that of the parental compound T-65: 2.9 μM and 10.1 μM in A172 and T98G cells, respectively (Figure 4D, upper. Figure 8B, Table 4). For comparison, the LDso of T-65 was estimated to be 3.9 μM and 12.5 μM in Al 72 and T98G cells, respectively. T98G cells were markedly more resistant to SCP1 inhibitors than Al 72 cells. GR-28 affected control cells with an LDso of approximately 5 μM (Figure 4D, upper). Both small molecules, parental T-65 and optimized GR-28, were predicted to be able to cross the blood-brain-barrier (BBB) using a recently published deep neural network-based model for the prediction of BBB permeability of novel compounds (28).

[0358] These data demonstrate specific and functional REST-inhibitory effects of the top lead, GR-28, on high-REST glioblastoma multiforme cells. However, the compound dosage needed to thwart Glioblastoma multiforme cell growth is still high.

[0359] Table 4. LDso of compounds and compositions.

[0360] REST-null Glioblastoma multiforme cells can rescue their growth through lipid metabolism rewiring. Contrary to the observation of REST-null glioblastoma multiforme cells having growth arrest, it was noticed that some REST-KO Glioblastoma multiforme clones (e.g., T98G REST-KO D4) still divided at tire same rate as control cells even when REST protein was absent (Figure 5 A). Indeed, it has been shown that the integration of mutations into the genome sometimes leads to compensation with little to no effect on cellular phenotype (29). It was speculated that targeting the underlying compensatory pathway in D4 cells might have a synergetic effect with REST inhibition. To identify w'hat kind of adaptation enhanced the growth of these “fast” cells upon REST loss, gene expression in the D4 clone was compared vs. three “slow7” clones (REST-KO CIO, F7, G2) using whole-transcriptome sequencing. First, upregulated differentially expressed genes shared between “slow” clones and upregulated differentially expressed genes identified for the D4 clone were overlapped (Figure 5B) and it was found that the majority of shared genes (73%) were also elevated in D4 cells, indicating underlying universal changes induced by REST-KO regardless of cellular phenotype. However, 761 upregulated genes did not overlap with the common “slow” gene set, were specific to tire D4 clone, and clustered into several gene ontology categories, tire most significant of which was ‘Fatty acid metabolic process’ (g-value = 0.005) (Figure 5C). This category is shown as a gene network based on protein-protein interactions in Figure 5D. As shown in the gene network, the “fast” phenotype was co-occurring with the upregulation of metabolic enzymes such as CPT1C, CROT, and PTGS2 (COX-2), all of which are oncogenic (30-32).

[0361] An important enzyme in the “Fatty acid metabolism” network was ACSL4, acyl-CoA synthetase long-chain family member 4 (Figure 5D). ACSLs (ACSL1, 3, 4, 5, and 6) are a family of CoA synthetases that activate long-chain FAs into acyl-CoA for the synthesis of cellular lipids and are thought to affect cell proliferation, including in nervous system diseases (33). It is currently known that actively proliferating cancer cells often activate de novo fatty add synthesis to provide essential structural components, e.g., structural lipids, for their growth (34). Interestingly, high expression of ACSL4 positively correlated with cell survival and proliferation after REST knockdown, based on results of pan-cancer RNAi screen by the DeμMap project (35) (Table 5). ACSL1 specifically was reported to be associated with a shorter survival time in glioblastoma multiforme patients, and ACSL1 inhibitors could reduce glioblastoma multiforme tumor growth both in vivo and in vitro (36). Based on transcriptome sequencing, expression levels of ACSL1 were downregulated in all “slow” clones (Table 6) and significantly correlated with PDT (r = -0.818, p = 0.0038) across all sequenced cells: T98G control, REST-KO CIO, F7, G2, D4 (Table 7, highlighted). In addition, a qPCR assay confirmed fhatACSLl and ACSLS mRNA levels were lower in “slow” CIO cells compared to control and were increased in “fast” cells compared to CIO (Figure 5E). Table 5. DeμMap predictability data.

[0362] Table 6. Shared differentially expressed genes in "slow” clones.

[0363] Table 7. PDT correlation.

[0364] Thus, it was asked if inhibition of fatty acid metabolic compensation could reverse the

[0365] “fast’7phenotype of T98G D4 cells (Figure 5F-Figure 5G). To this end, the pan ACSL-inhibitor

[0366] Triacsin C (TrC) was used, a pharmacological intervention that has been shown to prevent lipid accumulation, including in glial cells (37). Triacsin C directly inhibits ACSL1, ACSL3, and

[0367] ACSL4 by competing with fatty acids for their catalytic domain (38). Adding as low as 500 nM of the compound to the cell culture media dramatically increased the proliferation doubling time relative to vehicle-treated D4 cells (Figure 5F, right). However, this dose (500 nM) did not result in a profound decrease in D4 cell viability (>90%). Triacsin C reduced the proliferation rate of

[0368] D4 cells to that of “slow” REST-KO clones (Figure 5G). Finally, viability assays showed that

[0369] D4 cells were more resistant to Triacsin C-induced toxicity than T98G WT or “slow” REST-KO cells (Figure 5H). These results establish that glioblastoma cells can compensate for the loss of

[0370] REST required for their growth via upregulation of fatty' acid metabolism, which can be blocked with Triacsin C.

[0371] GR-28 lead exhibits profound synergy with ACSL inhibitor in high-REST

[0372] Glioblastoma multiforme cells. Next, a combination anticancer therapy was investigated to lower effective doses of GR-28 in glioblastoma multiforme cells without severe compound- induced hepatotoxicity. The rationale design of combination drug regimens is a powerful and comprehensive strategy for targeting cancer cells while sparing normal cells (39). First, it was noted that REST knockout led to the activation of genes related to fatty acid metabolism, and then it was observed that the addition of pan- ACSL (long chain fatty acyl-CoA ligase) inhibitor

[0373] Triacsin C was able to suppress this compensatory event in REST-KO glioblastoma cells. Therefore, it was tested whether Triacsin C can sensitize wild-type high-REST Glioblastoma multiforme cells to the top lead, GR-28 (Figure 6A-Figure 6B).

[0374] For combinatorial treatment, three serial 2-fold dilutions were prepared from the maximal dose of the single compounds that corresponded to average viability rates > 40-50% in glioblastoma multiforme cells based on preliminary single-drug treatments (Figure 4C, Figure 13 A). Since it was observed substantial differences in sensitivity to GR-28 and Triacsin C in Al 72 and T98G cells, individual dose ranges were applied. Drug combinations were used to treat Glioblastoma multiforme cells in plates for 72 h, along with vehicle-only controls (media with drugs was also renewed daily). The resulting cell viabilities from 5 x 5 matrices based on this multi-ray design (40) were used as input to build a synergy landscape using a Bliss model in the ‘synergyfinder’ R package (41). Thai, maximal synergy scores were determined for each cell line from at least three independent experiments, and the averages were recorded. Hepatocarcinoma cells (HepG2) were treated al the doses corresponding to the Al 72 glioblastoma cell line, as the one more sensitive to GR-28. It was observed that GR-28 / Triacsin C combination was highly synergetic against Al 72 and T98G cells (Figure 6A-Figure 6B, Figure 13B), but antagonistic in liver cancer cells (Figure 6C, Figure 13B). Low concentrations of Triacsin C (0.625-2.5 μM) markedly sensitized glioblastoma multiforme cells to GR-28, decreasing their LDsos 1.5-1.6-fold on average (Figure 6D, Figure 4D, lower, Table 4). Despite the differences in T98G and A 172 genotypes, the tested drug combination was synergetic against both cell lines. Predictably, adding Triacsin C (2.5 μM) to GR-28 treatment did not further reduce the REST protein amount in Al 72 cells (Figure 4A). Importantly, the GR-28 / Triacsin C combination had significantly lower synergy scores in liver cancer cells (Figure 6E), allowing higher selectivity and a significant therapeutic window between glioblastoma multiforme cell lines and HepG2 (Figure 6F). When treated with a single GR-28 compound, T98G cells were 2.3-fold more resistant to GR-28 than HepG2. Still, adding a low dose of Triacsin C could surprisingly reverse this sensitivity pattern (Figure 6F). These results align with recent suggestions that dysregulated lipid metabolism can contribute to tumor resistance and novel combination therapy strategies can re-sensitize cancer cells to chemotherapy (42). For instance, paired combinations of Triacsin C with classic chemotherapeutic drugs such as cisplatin, doxorubicin, and paclitaxel resulted in remarkable synergistic anti-tumor effects (43).

[0375] Discussion. In this study, a combination of genetic and cellular approaches were utilized to demonstrate that REST significantly promotes glioblastoma proliferation. A newly designed SCP1 inhibitor GR-28 can reduce REST transcriptional activity by targeting REST for degradation and thus thwart the growth of glioblastoma multiforme cells. Intriguingly, it was noticed that glioblastoma multiforme cells could compensate for the loss of REST via upregulating fatty acid metabolism. Remarkably, the results revealed that combinatorial REST targeting using GR-28 and the fatty acid pathway inhibitor Triacsin C led to synergistic cell death in glioblastoma cells with high basal REST levels (Figure 6G). This drug combination exhibited limited hepatotoxicity, as it induced little adverse effect on human hepatocarcinoma cells. These findings warrant further investigation of the therapeutic potential for drug combinations targeting REST and lipid metabolism in xenograft glioblastoma multiforme models.

[0376] The underlying genetic background of the tumor is crucial to the phenotypic heterogeneity and adaptability of glioblastoma (44). Recent advances in single-cell technologies, particularly single-cell transcriptomics (scRNA-Seq), offer promising avenues for a deeper understanding of intratumor heterogeneity when tailoring personalized cancer therapies (45). Specifically, glioblastomas with a high-REST tumor subpopulation can be potentially diagnosed and then targeted with selective REST inhibitors alone or combined with other chemotherapeutic agents. Since REST serves as a growth promoter in glioblastoma multiforme, targeting REST can multifacetedly impact various signaling pathways for more efficient glioblastoma multiforme eradication (3).

[0377] Given the significant role of phenotypic plasticity in cancer treatment resistance, especially in glioblastoma multiforme tumors, it became evident that targeting plasticity and its regulators is crucial to restrict the adaptive capacities of glioblastoma multiforme (44). While some glioblastoma multiforme cells may already exist in highly resistant states, “persister’* cells can activate various adaptive mechanisms upon treatment, such as entering quiescence or adopting sternness pathways (44). To address the adaptive capacities of tumors, two major strategies can be used to enhance re-sensitization to targeted therapy. First, tumor cells are subject to chemotherapy treatment, and then resulting phenotypic changes are recorded with subsequent identification of adaptive tumor response (39, 42). The second strategy entails using a genetic approach, such as knockdown or knockout, as demonstrated in this study. By targeting tumor adaptive responses, combination therapy regimens can augment the treatment efficacy and curb cancer resistance (46, 47).

[0378] As was observed in this study’, the SCP1 inhibitor GR-28 exhibited limited lethality against glioblastoma multiforme cells when used as a single drug, despite its superior specificity. However, this limitation was effectively overcome by combining GR-28 with a pan ACSL inhibitor, which resulted in a significant resensitization to GR-28 while sparing hepatocarcinoma cells. Targeting key lipid metabolism enzymes (SCD, FADS2, ACLY, and ACSL) to re-sensitize cancer cells to chemotherapy has already been demonstrated as a successful approach to combat glioma and glioblastoma (48-50). Furthermore, in a nude mouse xenograft model, ACSL- inhibitor Triacsin C at a non-toxic dose enhanced the anti-tumor efficacy of low-dose chemotherapy with etoposide, a well-known apoptosis activator (50).

[0379] These results contribute to recent findings regarding the promise of REST inhibition in high REST glioblastoma cells and combination regimens targeting glioblastoma multiforme plasticity. Future studies will determine the optimal lipid metabolism inhibitors to be coupled with REST inhibitors, including an in vivo setting.

[0380] Materials and Methods

[0381] TCGA data mining. TCGA data was analyzed using ‘TCGAbiolinks’ Bioconductor package (51) and GEPIA 2 (Gene Expression Profiling Interactive Analysis) web server (52). REST mRNA expression data (in TPM format, transcripts per million) were compared in TCGA-LGG (low-grade glioma) dataset, TCGA-GBM (glioblastoma) dataset, and matching normal samples from TCGA and GTEx databases via GEPIA 2. Survival analysis was performed using the same tool. For prediction of REST -target genes, RNA-Seq data (FPKM-UQ format) was downloaded from the open-access part of the TCGA database for 169 Glioblastoma multiforme patient samples (Primary Solid Tumor / Recurrent Solid Tumor) on 2 / 13 / 2021 using ‘TCGAbiolinks’. After standard pre-processing and filtering of low-signal mRNAs across all samples, pair-wise correlation coefficients between REST expression and expression of every transcript from the remainder (n = 42,335) were calculated. Then, the coefficients were ranked in the order of increasing magnitude, and top genes negatively correlated with REST were analyzed in follow-up studies.

[0382] Cell culturing and cell treatments. Human embryonic kidney cells (HEK293 / HEK293T), human liver cancer cells (HepG2), human fetal glial cells SVGpl2, and glioblastoma cell lines (Al 72, T98G) were purchased from ATCC (Manassas, VA). U251 glioblastoma cells were purchased from Sigma-Aldrich (St. Louis, MO). HEK293, SVGpl2, and glioblastoma multiforme cells were routinely cultured in minimal essential media (MEM, Sigma-Aldrich) supplemented with 10% Opti-Gold fetal bovine serum, FBS (GenDEPOT, Katy, TX), 1 mM sodium pyruvate (Sigma- Aldrich) and 1% non-essential amino acids (Sigma- Aldrich) at 37 °C in a humidified 5% CO2 atmosphere. HepG2 was cultured in MEM supplemented with 10% FBS. HEK293T cells, CRISPR-edited cell lines, and corresponding CRISPR control cells were cultured in Dulbecco’s modified Eagle’s media (DMEM, Sigma- Aldrich), supplemented with 10% FBS. HyClone penicillin and streptomycin (P / S) mix (Cytiva, Marlborough, MA), at a final concentration of 1%, was added to all media. Prior to the experiments, routinely cultured cell lines were confirmed mycoplasma free by tire Mycoplasma qPCR kit (Minerva Biolabs, Skillman, NJ).

[0383] Small-molecule SCP1 inhibitors (T-65) were synthesized. Triacsin C / TrC was purchased fromTocris (#2472) or Cayman Chemicals (#10007448). Stock solutions of SCP1 inhibitors and Triacsin C were prepared in DMSO and were stored at -80°C, avoiding multiple freeze-thaw cycles.

[0384] For determination of cytotoxicity, cells at a final density of 8,000 / well (100 pL) were seeded in black 96-well plates in their corresponding complete media and treated with compound(s) of interest or solvent-control on the next day. For Western blots or RNA extraction, cells were seeded in complete media in flasks and also treated the next day. Wild- type cell treatments were performed for a specified time duration (18, 24, 36, 48 or 72 h) in MEM supplemented with 5% FBS, 1 mM sodium pyruvate, 1% NEAA, and 1% P / S. CRISPR- edited cells and their corresponding controls were treated in DMEM supplemented with 5% FBS and 1% P / S. Drug mixtures with SCP1 inhibitors were replenished daily when treatment duration exceeded 24 h, based on time-course assessment of REST protein recovery under treatmeit with compounds of this class (14). DMSO concentrations in the incubation mixtures or solvent-control mixtures never exceeded 0.5% (v / v).

[0385] Design and synthesis ofSCPl inhibitors. The syntheses of a large number of analogues with variable targeted covalent functionality has been possible given the design of the route and generality of the reaction used for the syntheses. Amide coupling reactions with the noncovalent amide region were needed for targeting and provided access to improved compounds. Testing of a large number of reactive warheads with structurally diverse, reactively unique, and positional isomers was used to find the optimal reactive group - benzothiophene- 1,1 -dioxide (53). The detailed synthesis followed a route similar to the procedure described by Medellin et al. J. Med. Chem, 2022, 65, 507-519.

[0386] Synthetic Procedures

[0387] To a stirring solution of 2'-methoxy-[l,r-biphenyl]-4-carboxylic acid (200 mg, 0.88 jimol, 1.0 equiv) and tert-butyl (3-aminopropyl)carbamate (198 mg, 1.14 mmol, 1.3 equiv) in DMF (5mL) was added HATU (666 mg, 1.75 mmol, 2 equiv) and DIPEA (340 mg, 2.63 mmol, 3 equiv) and the reaction was stirred at 23°C for 14 hours. The reaction was then diluted with ethyl acetate (25mL) and the organic phase was washed with brine (4 X 25mL). The organics phase was dried over NazSO*, conceitrated, and purified by column chromatography (1:5 EtOAc:Hexanes) to yield tert-butyl (3-(2'-methoxy-[l,r-biphenyl]-4- carboxamido)propyl)carbamate (302 mg, 90%).

[0388] XH NMR (600 MHz, CDCh) 5 7.88 (d, J= 7.9 Hz, 2H), 7.60 (d, J= 8.2 Hz, 2H), 7.34 (dd, J= 17.3, 8.4 Hz, 2H), 7.22 (s, 1H), 7.04 (t, J= 7.4 Hz, 1H), 7.00 (d, J= 8.2 Hz, 1H), 4.98 (s, 1H), 3.81 (s, 3H), 3.53 (dd, J= 12.2, 6.1 Hz, 2H), 3.36 - 3.18 (m, 2H), 1.72 (s, 2H), 1.46 (s, 9H).

[0389] HRMS: m / z: calcd for C22H28N2O4: 385.2122; found 385.2120

[0390] To a stirring solution of tert-butyl (3-(2'-methoxy-[l,T-biphenyl]-4-carboxamido)propyl) carbamate (270 mg, 702 pmol, 1 equiv) in MeOH (3 mL) was added conceitrated aqueous HC1 (15 mL). The solution was stirred at 23°C for 15 minutes. Completion of reaction was monitored by TLC and which was subsequently concentrated in vaccuo to afford 3-(2'-methoxy-[l,r- biphenyl]-4-carboxamido)propan-l-aminium chloride which was use directly for the next reaction. The solid was dissolved in DMF (2 mL) and benzo[b]thiophene-2-carboxylic acid (162mg, 912pmol, 1.3 equiv) was added followed by HATU (533 mg, 1.4 mmol, 2 equiv) and DIPEA (272 mg, 2.1 mmol, 3 equiv). The reaction was stirred at 23°C for 14 hours the diluted with ethyl acetate (15mL). The mixture was washed with brine (4 X 15 mL). The organic extract was dried over NazSCh, concentrated under vacuum, and purified by column chromatography (1:1 EtOAc: Hexanes) to yield N-(3-(2'-methoxy-[l,r-biphenyl]-4-carboxamido)propyl) benzo[b]thiophene-2 -carboxamide (260mg, 82%).Rf= 0.5 (silica gel, 0:1 hexanes: EtOAc) lH NMR (600 MHz, CDCh) 57.93 - 7.89 (m, 3H), 7.85 (t, J= 8.3 Hz, 2H), 7.62 (d, J=

[0391] 8.1 Hz, 2H), 7.59 (t, J= 6.0 Hz, 1H), 7.44 - 7.33 (m, 3H), 7.31 (d, J= 7.4 Hz, 1H), 7.09 (t, J=

[0392] 6.1 Hz, 1H), 7.05 (t, J= 7.4 Hz, 1H), 7.00 (d, J= 8.2 Hz, 1H), 3.81 (s, 3H), 3.62 (dd, J= 11.8,

[0393] 6.2 Hz, 2H), 3.57 (dd, J= 11.7, 6.1 Hz, 2H), 1.88 - 1.82 (m, 2H).

[0394] HRMS: m / z: calcd for C26H24N2O3S: 445.1580; found 445.1578

[0395] To a vigorously stirred solution of N-(3-(2'-methoxy-[l,l'-biphenyl]-4- carboxamido)propyl) benzo[b]thiophene-2-carboxamide (30 mg, 67 pmol, 1 equiv) in dichloromethane (15mL) was added acetone (5 mL) and aqueous, saturated sodium bicarbonate (lOOmL) solution. To this was added 8 portions of Oxone (total amount 8.00 g, 13.0 mmol, 190 equiv) in 5 minute intervals. Upon completion, by TLC, the reaction is diluted with water (50mL) and dichloromethane (20 mL). The layers were separated and the aqueous layer was extracted with dichloromethane (20 mL). The organics were combined, washed with brine (40 mL), dried with MgSO4, filtered, and concentrated in vaccuo. Crude reaction mixture was purified by column chromatography (1:99 MeOH:DCM) to afford N-(3-(2,-methoxy-[l,r- biphenyl]-4-carboxamido)propyl) cinnamamide 1,1 -dioxide (23 mg, 72%). Rf = 0.3 (silica gel, 9.5:0.5 DCM:MeOH)

[0396] XH NMR (600 MHz, MeOD) 57.93 (s, 1H), 7.87 (d, J= 8.3 Hz, 2H), 7.80 - 7.77 (m, 1H), 7.73 - 7.69 (m, 2H), 7.66 (m, 1H), 7.58 (d, J= 8.2 Hz, 2H), 7.35 (t, J= 7.8 Hz, 1H), 7.30 (d, J= 7.5 Hz, 1H), 7.09 (d, J= 8.3 Hz, 1H), 7.03 (t, J= 7.5 Hz, 1H), 3.81 (s, 3H), 3.50 (m, 4H), 1.93 (m, 2H).

[0397] HRMS: m / z: calcd for C26H24N2O5S: 477.1479; found 477.1483

[0398] Full Synthetic Scheme for the Synthesis of GR-28

[0399] Kinetic characterization of GR-series compounds. To determine the potency of GR- compounds in inhibiting SCP1 phosphatase activity towards the analog substrate pNPP, the protocols and methods described in (54-57) were followed. The enzyme concentration was set at 150 nM for SCP1. To study the inhibitory effect, inhibitors were preincubated for varying time durations ranging from 30 minutes to 23 hours at room temperature (RT), and the concentration of DMSO was normalized to 1% in tire final reaction volume. In tire control group, which did not contain the compound, DMSO was added at a concentration of 1%. The reaction time was set to 3 minutes at 37 °C. The activity of each phosphatase towards pNPP in the presence or absence of tiie inhibitor was measured in an assay buffer (50 mM Tris-acetate pH 7.6, 10 mM MgCh, 0.02% Triton X-100, and 1% DMSO). The amount of released pNP was quantified by measuring the absorbance at 410 nm. The kinact and Ki values were calculated using KaleidaGraph software.

[0400] MALDI-TOF analysis of covalent adducts. SCP1 WT (50 μM) and SCP1 C181A mutant in activity buffer at pH 7.6 (with 5 μM or 500 μM BME) were treated with 500 μM of GR-28 (final 1% DMSO), and control samples were treated with 1% DMSO. The samples were incubated overnight at RT. The pNPP activity was tested the next day by taking a sample from each tube. The samples were then desalted using Ziptip Cl 8 resins (Sigma- Aldrich) following standard protocols. Mass spectrometric analysis of SCP1 treated with GR-28 or DMSO was performed using an Auto-flex Max MALDI-TOF (Broker Corporation, Billerica, MA) with a 1 :1 DHB matrix (ThermoFisher, Waltham, MA).

[0401] Molecular docking. The model of GR28 compound was built using MAESTRO v.

[0402] 13.5.128 from the Schrodinger suite. The compound was positioned manually into the active site of SCP1 in PyMOL v. 2.4.1 (PDB Code: 3PGL). Energy minimization of the protein and the manually positioned GR28 compound was performed in MAESTRO by applying the OPLS_2005 force field. The final model was visualized in PyMOL.

[0403] Establishment of CRISPR / Cas9-REST-KO cell lines. To express REST-KO sgRNAs, two pairs of DNA oligos were synthesized (16):

[0404] REST-RY1F (caccgGTTATGGCCACCCAGGTAAT) (SEQ ID NO: 9) and REST-RY1R (aaacATTACCTGGGTGGCCATAACC) (SEQ ID NO: 10);

[0405] REST-RG6F (caccgGTCTTCTGAGAACTTGAGTA) (SEQ ID NO: 11) and REST-RG6R (aaacTACTCAAGTTCTCAGAAGACC) (SEQ ID NO: 12). Annealed double-stranded sgRNAs were elated into pX330 plasmid (58), and the correct clones were verified by sequencing using U6 promoter primer. Non-neural HEK293 cells and glioblastoma cells T98G were co-transfected with two sgRNA expression vectors (RY1, RG6) and Cas9-2A-GFP plasmid (58) using Fugene11HD (Promega, Madison, WI) transfection reagent according to manufacturer’s guidelines. As a CRISPR-recombination control, cells co- transfected with empty pX330 vector and Cas9-2A-GFP plasmid were used. At 48 h post- transfection, single GFP-expressing cells were sorted using MA900 cell sorter (Sony Biotechnology, San Jose, CA) into 96-well plates (one cell per well) with complete DMEM media for clone expansion. Western blotting was used for screening REST-KO single-cell clones.

[0406] Genotyping of CRISPR / Cas9 repair outcomes. Sanger sequencing was used to identify REST protein sequence in REST-KO clones after double-nicking CRISPR / Cas9 recombination. Briefly, genomic DNA was extracted from target cells using Monarch kit (NEB, Ipswich, MA). Next, primers flanking the regions of sgRNA-guided double-stranded breaks (RY1, RG6) were designed and PCR reactions were performed with subsequent Sanger sequencing of PCR products where applicable (Table 8A-Table 8B). For PCR, GC buffer&Phusion DNA- polymerase (ThermoFisher) and dNTP mixture from NEB were used. PCR reactions were run on MasterCycler nexus (Eppendorf, Enfield, CT), and resulting agarose gels were visualized using GelDoc XR+ imager (Bio-Rad, Hercules, CA). PCR products were purified using PCR / gel purification kits following manufacturer’s instructions (Qiagen, Germany) and submitted for sequencing with original primer sequences (RY1-F, RY1-R, RG6-F, RG6-R). Sequences were translated into protein using Expasy online tool (www.expasy.org).

[0407] Table 8A. Primer sequences.

[0408] Table 8B. PCR protocol

[0409] REST transient overexpression. For the rescue experiments, REST-null glioblastoma cells were seeded at 800,000 cells per t-25 flask. Next day, cells were transiently transfected with 500 ng either REST-WT-expressing pLPC-vector (Addgene, #41903) or empty pLPC vector (Addgene, #12521) with Fugene HD transfection reagent (Promega) following manufacturer’s instructions. At 24 h post-transfection, cells were collected and used for proliferation assay and Western blotting.

[0410] Western blots. Briefly, cells were lysed in RIP A buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% sodium deoxy diolate, 0.1% SDS, 1% Triton x-100, 5 mM EDTA) supplemented with lx Halt protease and phosphatase inhibitor cocktail (ThermoFisher) followed by centrifugation for 15 min at 12,000 rpm. Protein concentration in supernatants was measured with BCA assay. Typically, proteins (50 pg) were separated by Novex 4-12% Tris-Glycine gels (Invitrogen, Waltham, MA), transferred to PVDF membranes (Bio-Rad), followed by membrane blocking at room temperature for 1 h in 1% Tween-20-TBS buffer containing 5% BSA (bovine serum albumin, ThermoFisher) or non-fat milk. Membranes were incubated at 4 °C overnight with primary rabbit antibodies against REST (Proteintech, Rosemont, IL) at 1:500 dilution, or p- tubulin (Abeam, Cambridge, MA) at 1:4,000 dilution. Next day, membranes were washed and incubated with 1:15,000 diluted goat anti-rabbit secondary IRDye 680RD antibody (LI-COR, Lincoln, NE) for 1 h at room temperature. After washing, membranes were visualized on LI- COR Odyssey CLx image reader.

[0411] RNA isolation, library preparation, and Tag-Sequencing. Total RNA was isolated from cells using DirectZol RNA Miniprep kit (Zymo Research, Irvine, CA, product number #R2050). 3’ Tag-Seq was performed by the University of Texas Genomic Sequencing and Analysis Facility, based on the protocols from Lohman BK el al. (17) and Meyer E et al. (59). Libraries were quantified using the Quant-it PicoGreen dsDNA assay (ThermoFisher) and pooled equally for subsequent size selection at 350-550 bp on a 2% gel using the Blue Pippin (Sage Science, Beverly, MA). The final pools were checked for size and quality with the Bioanalyzer High Sensitivity DNA Kit (Agilent, Santa Clara, CA) and their concentrations were measured using the KAPA SYBR Fast qPCR kit (Roche, Basel, Switzerland). Samples were then sequenced on the NovaSeq 6000 (Illumina, San Diego, CA) instrument with single-end, 100-bp reads.

[0412] Tag-Seq data analysis. Quality of raw reads was assessed using FastQC read quality reports (https: / / usegalaxy.org (60)). Adaptor trimming, deduplicating, and quality filtering were performed using a published pipeline (https: / / gitfiub.com / zOon / tag-based_RNAseq). Next, trimmed reads were aligned to human reference genome, GRCh38 version, using H1SAT2 fast aligner v.2.2.1 (61) with default parameters, except Forward (F) -ma-strandedness. Gencode v38 gtf file was used as annotation gtf. Lastly, mapped fragments were quantified by featureCounts v.2.0.1 (62) in Galaxy. Differential expression was analyzed using DESeq2 v.1.30.1 (63) in R; genes with adjusted p-value < 0.05 and FC cut-off of 1.5 were considered as differentially expressed. Tag-Seq data was deposited in Gene Expression Omnibus / GEO under the accession number GSE234912. Gene ontology-enrichment analysis of gene clusters was performed using Bioconductor R package ‘clusterProfiler’ v.3.18.1 (64) and STRING v.il.5 (65). For every gene network based on protein-protein interactions (PPI), protein-protein interaction enrichment p-value or FDR was recorded. For correlation with PDT (proliferation doubling time), raw counts were converted to FPKM in R. qPCR. Total RNA was isolated from cells using either DirectZol RNA Miniprep kit (Zymo Research) or Trizol reagent with subsequent isopropanol precipitation. 0.5 pg of total RNA was used for reverse transcription using the AzuraQuant™ cDNA Synthesis Kit, #AZ- 1995 (Azura, Raynham, MA, USA) using manufacturer’s guidelines. Relative gene expression was measured using AzuraQuant™ Green Fast qPCR Mix, Lo-Rox (Azura) and normalized toACTB gene expression. Amplification was performed using the ViiA 7 Real-Time PCR System (Applied Biosystems, Waltham, MA). Specificity of amplification was controlled with melting curves / primer efficiency calculation. Analysis of qPCR data was performed using the AACt method. Primer sequences (designed to span exon-exon junctions or to be separated by a relatively large intron) and qPCR conditions are shown in Table 8B.

[0413] Cytotoxicity assays (resazurin reduction) and drug combination landscapes. After treatment (72 h), 20 pl 0.15 mg / ml resazurin solution (ThermoFisher) was added to each well of a 96-well plate. After 3 h incubation, fluorescence was recorded using a 560 nm excitation / 590 nm emission filter set on an Infinite F200 microplale reader (Tecan, Switzerland) (66). Cell viability was normalized to that of control wells after background subtraction. LD50s of selected compounds were fitted based on cell survival data using the ‘drc’ (dose-response curves) R package (67). To assess synergetic effects of GR-28 with Triacsin C, 5x5 drug combination landscapes were built using the Bioconductor package ‘synergyfinder’ and its Bliss model (41). For every landscape, cells were seeded in plates and treated the next day with serial 2-fold dilutions of drug(s), and tire maximal dose of single drugs corresponded to 40-50% or higher mean viability. HepG2 cells were treated at the doses corresponding to the A 172 cell line which was more sensitive to SCP1 inhibitors. Maximal synergy coefficients were extracted from each landscape and recorded.

[0414] Cell proliferation assay. For the proliferation assay, cells were seeded at a density of 50,000 cells per well in complete media in 24-well plates. Then, cells were counted every 24 h for four subsequent days using Trypan Blue exclusion assay (0.4%) on automated Luna-II automated cell counter (Logos Biosystems, Annandale, VA). Population doubling time (PDT) was estimated with the following formula, PDT = (72 h x ln2) / ln(N4 / Ni), where Ni and N4 are cell counts in every well on I*1and 4thdays, respectively.

[0415] Statistical analyses. Statistical analyses were performed using RStudio v.4.0.5 and GraphPad Prism v.9.5. One-tailed or two-tailed, unpaired or paired (where applicable) t-test was used for comparing two groups. ANOVA was used when comparing several groups vs control. p < 0.05 values were considered as significant. Correlations were assessed using two-tailed Pearson r coefficients. Protein bands were quantified and compared using Image! software. Illustrations were created using BioRender software. The statistical details of experiments can be found in the figure legends.

[0416] References

[0417] 1. Poon MTC et al. Sci Rep. 2020;10(l): 11622.

[0418] 2. Zhou W et al. Cancers (Basel). 2019; 11(9).

[0419] 3. Pearson JRD et al. Signal Transduct Target Then 2017;2: 17040.

[0420] 4. Chong JA et al. Cell. 1995;80(6):949-57.

[0421] 5. Negrini S et al. Trends Cell Biol. 2013;23(6):289-95.

[0422] 6. Zhang Y et al. Front Med (Lausanne). 2021;8:739624.

[0423] 7. Zhang P et al. Trends Neurosci. 2009;32(l l):559-65.

[0424] 8. Kamal MM et al. Stem Cells. 2012;30(3):405-14.

[0425] 9. Ballas N et al. Curr Opin Neurobiol. 2005;15(5):500-6.

[0426] 10. Koehler AN. Curr Opin Chem Biol. 2010;14(3):331-40.

[0427] 11. Guardavaccaro D et al. Nature. 2008;452(7185):365-9.

[0428] 12. Nesti E et al. Proc Natl Acad Sci U S A. 2014:11 l(37):E3929-36.

[0429] 13. Burkholder NT et al. J Biol Chem. 2018;293(43): 16851-61.

[0430] 14. Medellin B et al. J Med Chem 2022;65(l):507-19.

[0431] 15. Jones FS et al. Bioessays. 1999;21(5):372-6.

[0432] 16. ZhouZ et al. Cancers (Basel). 2020; 12(6).

[0433] 17. Lohman BK et al. Mol Ecol Resour. 2016;16(6):1315-21.

[0434] 18. SperanzaMC et al. Oncotarget. 2012;3(7):723-34.

[0435] 19. Foltz G et al. Cancer Res. 2006;66(13):6665-74.

[0436] 20. Marisetty AL et al. Neuro Oncol. 2017;19(4):514-23.

[0437] 21. Marisetty AL et al. Neuro Oncol. 2019;21(6):775-85.

[0438] 22. Conti L et al. PLoS One. 2012;7(6):e38486.

[0439] 23. Zhang D et al. Int J Mol Sci. 2016;17(5).

[0440] 24. Rockowitz S et al. Nucleic Acids Res. 2015;43(12):5730-43.

[0441] 25. Tang Y et al. Nucleic Adds Res. 2021:49(8):4506-21.

[0442] 26. Cloud AS et al. BMC Cancer. 2022;22(l):180.

[0443] 27. Van Summeren A et al. Toxicol Sci. 201 l;120(l): 109-22.

[0444] 28. Kumar R et al. Front Neurosci. 2022;16:858126.

[0445] 29. Rossi A et al. Nature. 2015;524(7564):230-3.

[0446] 30. Zaugg K et al. Genes Dev. 201 l;25(10):1041-51.

[0447] 31. Lasheras-Otero I et al. J Invest Dermatol. 2023;143(2):305-16.e5.

[0448] 32. LiuB et al. Cancer Cell Int. 2015;15:106. 33. Wu Z et al. Front Neurosci. 2022; 16: 1030512.

[0449] 34. ROhrig F et al. Nat Rev Cancer. 2016;16(l l):732-49.

[0450] 35. Tshemiak A et al. Cell. 2017;170(3):564-76.el6.

[0451] 36. Xu C et al. Front Oncol. 2022; 12: 888922.

[0452] 37. Victor MB et al. Cell Stem Cell. 2022;29(8): 1197-212.e8.

[0453] 38. Rossi Sebastiano M et al. Int J Mol Sci. 2019;20(15).

[0454] 39. Pei S et al. J Biol Chem. 2016;291(42):21984-2000.

[0455] 40. Foucquier J et al. Pharmacol Res Perspect. 2015;3(3):e00149.

[0456] 41. lanevski A et al. Bioinformatics. 2017;33(15):2413-5.

[0457] 42. Zhang T et al. EBioMedicine. 2022;77: 103872.

[0458] 43. Orlando UD et al. Biochem Pharmacol. 2019;159:52-63.

[0459] 44. Yabo YA et al. Neuro Oncol. 2022;24(5): 669-82.

[0460] 45. Nalh A et al. Trends Cancer. 2021;7(4):359-72.

[0461] 46. Bayat Mokhtari R et al. Oncotarget. 2017;8(23):38022-43.

[0462] 47. Shi ZD et al. Signal Transduct Target Ther. 2023;8(l):l 13.

[0463] 48. Parik S et al. Front Oncol. 2022;12:988872.

[0464] 49. Beckner ME et al. Int J Cancer. 2010;126(10):2282-95.

[0465] 50. Mashima T et al. Cancer Sci. 2009; 100(8): 1556-62.

[0466] 51. Colaprico A et al. Nucleic Acids Res. 2016;44(8):e71.

[0467] 52. Tang Z et al. Nucleic Acids Res. 2019;47(Wl):W556-W60.

[0468] 53. Gehringer M et al. J Med Chem. 2019;62(12):5673-724.

[0469] 54. Zhang Y et al. Mol Cell. 2006;24(5):759-70.

[0470] 55. Mons E et al. Curr Protoc. 2022;2(6):e419.

[0471] 56. Pettinger J et al. J Med Chem. 2019;62(24): 11383-98.

[0472] 57. Strelow JM. SLAS Discov. 2017;22(l):3-20.

[0473] 58. Ran FA et al. Nat Protoc. 2013;8(ll):2281-308.

[0474] 59. Meyer E et al. Mol Ecol. 2011;20(17):3599-616.

[0475] 60. Afgan E et al. Nucleic Acids Res. 2018;46(Wl):W537-W44.

[0476] 61. Kim D et al. Nat Methods. 2015;12(4):357-60.

[0477] 62. Liao Y et al. Bioinformatics. 2014;30(7):923-30.

[0478] 63. Love MI et al. Genome Biol. 2014;15(12):550.

[0479] 64. Yu G et al. OMICS. 2012;16(5):284-7.

[0480] 65. Szklarczyk D et al. Nucleic Acids Res. 2019;47(Dl):D607-D13.

[0481] 66. Riss T et al. Assay Guidance Manual2016. 67. Ritz C et al. PLoS One. 2015;10(12):e0146021.

[0482] Example 2 -4 method of combinatorial targeting of REST and fatty add metabolic pathway for eradicating REST-dependent human glioblastoma cells

[0483] According to American Cancer Society (cancer.org), more than 24,000 new cases of brain cancer (all the subtypes) will be diagnosed in the US in 2023. From these, about 19,000 people will die. Glioblastoma is the deadliest type of primary brain cancer and accounts for 50.1 percent of all primary malignant brain tumors.

[0484] Glioblastoma multiforme (GBM) is an aggressive type of brain cancer that is associated with poor prognosis, intrinsic heterogeneity and plasticity, as well as stubborn therapy resistance. In some GBMs, tumorigenidty is fueled by the activity of a transcriptional regulator REST (repressor element- 1 silencing transcription factor).

[0485] Described herein is anew generation of small-molecule compounds (GR-series) that reduced protein level and transcriptional activity of REST via covalent targeting its stabilizer SCP1 (C-terminal domain small phosphatase 1) in glioblastoma cells. Top lead from the compound series, GR-28, was cytotoxic against high-REST GBM cell lines (LD50 2.9 μM in A172 cells and 10.1 μM in T98G cells).

[0486] CRISPR / Cas9 genetic approach showed that REST-null cells could acquire resistance to REST loss via rewiring of fatty acid metabolism. Blocking this compensatory' adaptation simultaneously with REST inhibition using Triacsin C (pan-ACSL inhibitor) and GR-28, respectively, exhibited substantial synergetic potential in GBM cell lines (max synergy score 39.8 ± 9.6 in Al 72, 53.8 ± 15.3 in T98G), but not in liver cancer cells, HepG2 (max synergy score 6.0 ± 5.0). Low concentrations of Triacsin C (0.625-2.5 μM) markedly sensitized GBM cells to GR-28 decreasing their LD50s 1.5-1.6-fold on average: from 2.9 μM to 1.9 μM in A172 cells and from 10.1 μM to 6.4 μM in T98G cells. Cytotoxicity assays showed that GR- 28 / Triacsin C drug pair eradicated GBM cells at doses lower than needed to induce hepatotoxicity, e.g. treatment with 2.5 μM GR-28 / 1.25 μM Triacsin C for 72 h resulted in mean 45-48% viability of GBM cells and 81% viability of liver carcinoma cells (HepG2).

[0487] Described herein are small-molecule rationally-designed compounds (GR-series with GR-28 top lead), that were designed and chemically tailored to target REST regulator SCP1. Also described herein is the identification of specific metabolic adaptation upon REST inhibition in GBM cells via CRISPR / Cas9 knockout approach.

[0488] Also described herein is tire synergetic killing of glioblastoma cells dependent on REST by REST inhibitor coupled with fatty acid metabolism inhibitor. This drug combination is an effective and selective therapeutic strategy to combat REST-dependent GBM cells in vitro. The combination overcomes metabolic adaptation acquired by glioblastoma cells in response to REST inhibition.

[0489] Advantages include, but are not limited to, therapeutic efficacy in eradicating glioblastoma cells in vitro; and limited hepatotoxicity in vitro.

[0490] Potential in vitro toxicity against normal glial cells can be overcome by testing other lipid / fatty" acid metabolism inhibitors, such as TVB-2640 (FASN inhibitor, currently in clinical trials in cancer patients), avasimibe (SOAT inhibitor), perhexiline (CPT inhibitor), etc.

[0491] This treatment approach may be advantageous to combat other cancer types, associated with upregulated REST level, e.g. low-grade glioma (LGG), neuroblastoma, or medulloblastoma.

[0492] Example 3 - A method of combinatorial targeting of REST and fatty acid metabolic pathway for eradicating REST-dependent human glioblastoma cells

[0493] According to American Cancer Society, more than 24,000 new cases of brain cancer will be diagnosed in the US in 2023. From these, about 19,000 people will die. About 3 out of 10 of all brain tumors are gliomas, and half of these are glioblastomas (grade IV), the most common malignant brain tumors in adults.

[0494] Current therapy of glioblastoma (GBM) includes surgery followed by adjuvant chemotherapy and radiation therapy. According to National Cancer Institute information, there are many challenges in GBM therapy including limited use of radiotherapy due to damage to the nearby normal tissue and a necessity for chemotherapy to be able to cross blood-brain-barrier. Consistently, there is an urgent need to improve the treatment of adult GBM by developing novel effective agents that can cross blood-brain barrier and testing them eventually in clinical trials.

[0495] One of deregulated genes in GBM is a repressor element- 1 silencing transcription factor (REST), a transcriptional repressor, which has been recently noted as an oncogenic protein in several brain tumor types including neuroblastoma, medulloblastoma, and glioblastoma [PMID 23414932], High REST expression was significantly associated with worse overall survival in glioma patients [PMID 34859007],

[0496] Using rational chemical and drug combination design, described herein is a synergistic drug combination simultaneously targeting REST transcription factor and fatty acid metabolism for eradication of glioblastoma cells. In vitro cytotoxicity estimation shows that the drug cocktail allows for significant therapeutic window between target GBM cells and liver carcinoma cells, e.g. drug combination is characterized by limited hepatotoxicity.

[0497] An example of the drug combination is comprised of 1) small-molecule inhibitor of REST (e.g., GR-28) which specifically binds to REST regulator SCP1; and 2) pan-ACSL inhibitor Triacsin C.

[0498] Benefits include, but are not limited to:

[0499] • With REST serving as an oncogene, targeting REST can have a multifaceted impact on various signaling pathways for more efficient GBM eradication;

[0500] • By targeting tumor adaptive response (fatty acid metabolism activation), drug combination can augment the treatment efficacy and curb GBM resistance;

[0501] • Synergetic effect of combination allows decrease in single drug effective doses, therefore chance of side effects and systemic toxicity is decreased.

[0502] Example 4 - A method of combinatorial targeting of REST and fatty acid metabolic pathway for eradicating REST-dependent human glioblastoma cells

[0503] According to American Cancer Society, more than 24,000 new cases of brain cancer (all subtypes) will be diagnosed in the US in 2023. It’s estimated that about 19,000 people will die. About 3 out of 10 of all primary malignant brain tumors are gliomas, and 50.1% of these are glioblastomas (grade IV), the most common malignant brain tumors in adults.

[0504] There is an urgent need to improve the treatment of adult glioblastoma multiforme (GBM).

[0505] One of deregulated genes in some GBMs is a transcriptional repressor, repressor element- 1 silencing transcription factor (REST), which has been recently noted as an oncogenic protein in several brain tumor types including neuroblastoma, medulloblastoma, and glioblastoma

[0506] CRISPR / Cas9 knockout approach showed that REST-null cells could acquire resistance to REST loss via rewiring of fatty acid metabolism.

[0507] Described herein is a synergistic drug combination simultaneously targeting REST transcription factor and fatty acid metabolism for eradication of glioblastoma cells. An example of the drug combination is comprised of 1) novel small-molecule inhibitor of REST compounds (~20 GR-series with GR-28 as the top lead) which specifically binds to the REST regulator SCP1; and 2) pan-ACSL inhibitor Triacsin C.

[0508] In vitro studies indicated synergetic results in two different GBM cell lines (Al 72 and T98G) but not in liver cancer cells (HepG2).

[0509] This treatment approach may be advantageous to combat other cancer types, associated with upregulated REST level, e.g. low-grade glioma (LGG), neuroblastoma, or medulloblastoma.

[0510] Potential in vitro toxicity against normal glial cells can be overcome by testing other lipid / fatty acid metabolism inhibitors, such as TVB-2640 (FASN inhibitor, currently in clinical trials in cancer patients), avasimibe (SOAT inhibitor), perhexiline (CPT inhibitor), etc.

[0511] EXEMPLARY ASPECTS

[0512] In view of the described compositions, devices, systems, and methods, herein below are described certain more particularly described aspects of the inventions. The particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language and formulas literally used therein.

[0513] Example 1: A composition comprising: a REST (repressor element- 1 silencing transcription factor) inhibitor; and a fatty acid metabolism inhibitor.

[0514] Example 2: The composition of any examples herein, particularly example 1, wherein the composition is synergistic (e.g. LDso of each active ingredient is lower than when used alone).

[0515] Example 3: The composition of any examples herein, particularly example 1 or example 2, wherein the composition exhibits limited hepatotoxicity.

[0516] Example 4: The composition of any examples herein, particularly examples 1-3, wherein the fatty acid metabolism inhibitor comprises Triacsin C, TVB-2640 / Denifanstat, avasimible, 2- fluoropalmitic add, their homologues, or a combination thereof.

[0517] Example 5: The composition of any examples herein, particularly examples 1-4, wherein the fatty acid metabolism inhibitor comprises a pan-ACSL inhibitor.

[0518] Example 6: The composition of any examples herein, particularly examples 1-5, wherein the fatty acid metabolism inhibitor comprises Triacsin C.

[0519] Example 7: The composition of any examples herein, particularly examples 1-6, wherein the REST inhibitor targets SCP1.

[0520] Example 8: The composition of any examples herein, particularly examples 1-7, wherein the REST inhibitor covalently interacts with SCP1.

[0521] Example 9: The composition of any examples herein, particularly examples 1-8, wherein the REST inhibitor comprises a compound described by Medellin et al. J Med. Chem, 2022, 65, 507-519, or a pharmaceutically acceptable salt thereof.

[0522] Example 10: The composition of any examples herein, particularly examples 1-9, wherein the REST inhibitor comprises a compound defined by Formula I, or a pharmaceutically acceptable salt thereof: wherein

[0523] R1, R2, R3, and R4are each independently H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 alkoxy, NRxRy, or wherein, as valence permits, R1and R2, R2and R3, or R3and R4, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms;

[0524] Rxand Ryare independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl;

[0525] R5is H, OH, halogen, or substituted or unsubstituted C1-C20 alkyl;

[0526] R6is substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, or substituted or unsubstituted C1-C20 alkoxy;

[0527] Q is C(O) or S(O)2; and

[0528] A is a cyclic hydrophobic group, such as a substituted or unsubstituted aromatic group.

[0529] Example 11: The composition of any examples herein, particularly example 10, wherein R6is substituted or unsubstituted C1-C20 alkyl, or substituted or unsubstituted C3-C20 aryl.

[0530] Example 12: The composition of any examples herein, particularly example 10 or example 11, wherein R6is substituted or unsubstituted Ci-Ce alkyl or substituted or unsubstituted benzyl.

[0531] Example 13: The composition of any examples herein, particularly examples 10-12, wherein R6is substituted or unsubstituted Ci-Ce alkyl. Example 14: The composition of any examples herein, particularly examples 10-13, wherein the REST inhibitor is defined by Formula II, or a pharmaceutically acceptable salt thereof:

[0532] 5 wherein n is an integer from 0 to 2.

[0533] Example 15: The composition of any examples herein, particularly examples 10-14, wherein Q is C(O).

[0534] Example 16: The composition of any examples herein, particularly examples 10-15, wherein the REST inhibitor is defined by Formula III: or a pharmaceutically acceptable salt thereof.

[0535] Example 17: The composition of any examples herein, particularly examples 10-16, wherein n is 0.

[0536] Example 18: The composition of any examples herein, particularly examples 10-17, wherein the REST inhibitor is defined by Formula IV : IV or a pharmaceutically acceptable salt thereof.

[0537] Example 19: The composition of any examples herein, particularly examples 10-18, wherein A is a substituted or unsubstituted heteroaiyl group or a substituted or unsubstituted non-heteroaiyl group.

[0538] Example 20: The composition of any examples herein, particularly examples 10-19, wherein A is a substituted or unsubstituted monocyclic group or a substituted or unsubstituted bicyclic group.

[0539] Example 21: The composition of any examples herein, particularly examples 10-20, wherein A is a substituted or unsubstituted: benzene, furan, indole, benzothiophene, thiophene, quinoline, naphthalene, pyrazole, pyridine, pyrazine, tretrazole, or a combination thereof.

[0540] Example 22: The composition of any examples herein, particularly examples 10-21, wherein A is a substituted or unsubstituted benzyl group.

[0541] Example 23: The composition of any examples herein, particularly examples 10-22, wherein the REST inhibitor is defined by Formula V, or a pharmaceutically acceptable salt thereof: wherein

[0542] R7, R8, R9, R10, and R11are each independently H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaiyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 alkoxy, or NRxRy, or wherein, as valence permits, R7and R8, R8and R9, R9and R10, or R10and R11, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms; and

[0543] Rxand Ryare independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl.

[0544] Example 24: The composition of any examples herein, particularly example 23, wherein R7, R8, R9, R10, and R11are each independently H, OH, nitro, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Cs-Cs aryl, substituted or unsubstituted C4-C10 alkylaryl, substituted or unsubstituted C3-C10 heteroaryl, substituted or unsubstituted Ci-Ce acyl, substituted or unsubstituted Ci-Ce alkoxy, or wherein, as valence permits, R7and R8, R8and R9, R9and R10, or R10and R11, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms.

[0545] Example 25: The composition of any examples herein, particularly example 23 or example 24, wherein R7, R8, R9, R10, and R11are each independently H, NO2, CH3, OCH3, substituted or unsubstituted tetrazole, substituted or unsubstituted benzyl, or wherein, as valence permits, R9and R10, together with the atoms to which they are attached, form a substituted or unsubstituted benzyl.

[0546] Example 26: The composition of any examples herein, particularly examples 23-25, wherein R7, R8, R10, R11, or a combination thereof is hydrogen.

[0547] Example 27: The composition of any examples herein, particularly examples 23-26, wherein R7, R8, R10, and R11are all hydrogen.

[0548] Example 28: The composition of any examples herein, particularly examples 23-27, wherein R9is a substituted or unsubstituted aryl group.

[0549] Example 29: The composition of any examples herein, particularly examples 23-28, wherein R9is a substituted or unsubstituted benzyl group.

[0550] Example 30: The composition of any examples herein, particularly examples 10-29, wherein the REST inhibitor is defined by Formula VI, or a pharmaceutically acceptable salt thereof: wherein

[0551] R12, R13, R14, R15, and R16are each independently H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C3-C20 aryloxy, substituted or unsubstituted C1-C20 ester, substituted or unsubstituted C1-C20 carbonate ester, substituted or unsubstituted C1-C20 sulfonate ester, substituted or unsubstituted C1-C20 sulfamate, or NR'Rb, or wherein, as valence permits, R12and R13, R13and R14, R14and R15, or R15and R16, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms; and

[0552] R* and Rbare independently selected from substituted or unsubstituted C1-C20 allyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl.

[0553] Example 31: The composition of any examples herein, particularly example 30, wherein R7, R8, R10, and R11are each independently H, OH, nitro, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Cs-Cs aryl, substituted or unsubstituted C4-C10 alkylaryl, substituted or unsubstituted C3-C10 heteroaryl, substituted or unsubstituted Ci-Ce acyl, substituted or unsubstituted Ci-Ce alkoxy, or wherein, as valence permits, R7and R8, or R10and R11, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms.

[0554] Example 32: The composition of any examples herein, particularly example 30 or example 31, wherein R7, R8, R10, and R11are each independently H, NO2, CH3, OCH3, substituted or unsubstituted tetrazole, substituted or unsubstituted benzyl.

[0555] Example 33: The composition of any examples herein, particularly examples 30-32, wherein R7, R8, R10, R11, or a combination thereof is hydrogen.

[0556] Example 34: The composition of any examples herein, particularly examples 30-33, wherein R7, R8, R10, and R11are all hydrogen.

[0557] Example 35: The composition of any examples herein, particularly examples 10-34, wherein the REST inhibitor is defined by Formula VI- A: or a pharmaceutically acceptable salt thereof.

[0558] Example 36: The composition of any examples herein, particularly examples 30-35, wherein R12, R13, R14, R15, and R16are each independently H, OH, nitro, substituted or unsubstituted Ci-Cs alkyl, substituted or unsubstituted Cs-Cs aryl, substituted or unsubstituted C4-C10 alkylaryl, substituted or unsubstituted C3-C10 heteroaryl, substituted or unsubstituted Ci- Cs acyl, substituted or unsubstituted Ci-Cs alkoxy, substituted or unsubstituted Cs-Cs aryloxy, substituted or unsubstituted Ci-Cs ester, substituted or unsubstituted C1-C10 carbonate ester, substituted or unsubstituted C1-C10 sulfonate ester, or substituted or unsubstituted C1-C4 sulfamate.

[0559] Example 37: The composition of any examples herein, particularly examples 30-36, wherein R12, R13, R14, R15, and R16are each independently H, halogen, nitro, CH3, tert-butyl, - OCH3, -CH(O), -C(O)OCH3, -OC(O)CH3, -OC(O)CH2CH3, -OCH2C(O)CH3, -OC(O)OCH3, - OC(O)OCH2CH3, -O(CH2)2O(CH2)2OCH3, -O(CH2)2O(CH2)2O(CH2)2OCH3, o or

[0560] Example 38: The composition of any examples herein, particularly examples 30-37, wherein R13, R15, or a combination thereof is hydrogen.

[0561] Example 39: The composition of any examples herein, particularly examples 30-38, wherein R13and R15are both hydrogen.

[0562] Example 40: The composition of any examples herein, particularly examples 30-39, wherein R12, R16, or a combination thereof is hydrogen.

[0563] Example 41: The composition of any examples herein, particularly examples 30-40, wherein R12and R16are both hydrogen.

[0564] Example 42: The composition of any examples herein, particularly examples 30-41, wherein the REST inhibitor is defined by Formula VII: or a pharmaceutically acceptable salt thereof.

[0565] Example 43: The composition of any examples herein, particularly examples 30-39, wherein R14is hydrogen.

[0566] Example 44: The composition of any examples herein, particularly examples 30-39 or 43, wherein the REST inhibitor is defined by Formula VIII: or a pharmaceutically acceptable salt thereof.

[0567] Example 45: The composition of any examples herein, particularly examples 230-39 or 43-44, wherein R12is hydrogen.

[0568] Example 46: The composition of any examples herein, particularly examples 30-39 or 43-45, wherein the REST inhibitor is defined by Formula IX: or a pharmaceutically acceptable salt thereof.

[0569] Example 47: The composition of any examples herein, particularly examples 10-46, wherein R5is hydrogen or halogen.

[0570] Example 48: The composition of any examples herein, particularly examples 10-47, wherein R5is hydrogen.

[0571] Example 49: The composition of any examples herein, particularly examples 10-48, wherein R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted Ci- C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms.

[0572] Example 50: The composition of any examples herein, particularly examples 10-49, wherein R1, R2, R3, and R4are each independently H, halogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, or wherein, as valence permits, R2and R3, together with the atoms to which they are attached, form a 6 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms.

[0573] Example 51: The composition of any examples herein, particularly examples 10-50, wherein R*-R4are each independently H, halogen, OCHs, CH3, or wherein R2and R3, together with the atoms to which they are attached form a benzyl group.

[0574] Example 52: The composition of any examples herein, particularly examples 10-51, wherein at least one of R^R4is not hydrogen.

[0575] Example 53: The composition of any examples herein, particularly examples 10-51, wherein Rx-R4are all hydrogen.

[0576] Example 54: The composition of any examples herein, particularly examples 10-53, wherein the REST inhibitor is selected from the group consisting of:

[0577] <n

[0578] , pharmaceutically acceptable salts thereof, and combinations thereof.

[0579] Example 55: The composition of any examples herein, particularly examples 10-54, wherein the REST inhibitor is selected from the group consisting of: pharmaceutically acceptable salts thereof, and combinations thereof. Example 56: The composition of any examples herein, particularly examples 10-55, wherein the REST inhibitor comprises: or a pharmaceutically acceptable salt thereof.

[0580] Example 57: A pharmaceutical composition comprising the composition of any examples herein, particularly examples 10-56.

[0581] Example 58: The pharmaceutical composition of any examples herein, particularly example 57, further comprising a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, or a combination thereof.

[0582] Example 59: A method of making the composition of any examples herein, particularly examples 1-58.

[0583] Example 60: A method of use of the composition of any examples herein, particularly examples 1-58.

[0584] Example 61 : A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any examples herein, particularly examples 1-58.

[0585] Example 62: The method of any examples herein, particularly example 61, wherein the disease comprises cancer.

[0586] Example 63: A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any examples herein, particularly examples 1-58.

[0587] Example 64: The method of any examples herein, particularly example 62 or example 63, further comprising co-administering an anticancer agent to the subject.

[0588] Example 65: The method of any examples herein, particularly examples 62-64, wherein the cancer has an upregulated REST level.

[0589] Example 66: The method of any examples herein, particularly examples 62-65, wherein the cancer comprises glioblastoma multiforme, low-grade glioma, neuroblastoma, medullablastoma, or a combination thereof.

[0590] Example 67: The method of any examples herein, particularly examples 62-66, wherein the cancer comprises glioblastoma.

[0591] Example 68: A method of suppressing tumor growth in a subject in need thereof, the method comprising contacting at least a portion of the tumor with a therapeutically effective amount of the composition of any examples herein, particularly examples 1-58.

[0592] Other advantages which are obvious and which are inherent to the invention will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

[0593] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative method steps disclosed herein are specifically described, other combinations of the method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitiy mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitiy stated.

[0594] SEQUENCES

[0595] SEQ ID NO: 1 : Wild Type REST (CRISPR Control) MATQVMGQSSGGGGLFTSSGNIGMA

[0596] SEQ ID NO: 2: HEK293 REST-KO DIO (1) and HEK293 REST-KO E6 MATQVVRTKFTFIWAFSCKISII

[0597] SEQ ID NO: 3: HEK293 REST-KO DIO (2) MATQVQVLRRLI

[0598] SEQ ID NO: 4: T98G REST-KO CIO (1) MATQVWGSLLEEEGCLPAVATLEWPCLTTCMTCMTFPKLNWPHLSLLCWQMWP

[0599] SEQ ID NO: 5: T98G REST-KO CIO (2) MATQVELPRSLTRCARSFGCGERYQLTQRR

[0600] SEQ ID NO: 6: T98G REST-KO F7 (1) MASSQKTHLTRHMRTHSGW

[0601] SEQ ID NO: 7: T98G REST-KO F7 (2) MGVLLQNFNNIRVFKCHEIAKYNSH

[0602] SEQ ID NO: 8: T98G REST-KO D4 MHLRKFF

[0603] SEQ ID NO: 9: REST-RY1F caccgGTTATGGCCACCCAGGTAAT

[0604] SEQ ID NO: 10: REST-RY1R aaacATTACCTGGGTGGCCATAACC

[0605] SEQ ID NO: 11 : REST-RG6F caccgGTCTTCTGAGAACTTGAGTA SEQ ID NO: 12: REST-RG6R aaacTACTCAAGTTCTCAGAAGACC

[0606] SEQ ID NO: 13: ACTB Forward (5’-3’) Primer TCAAGATCATTGCTCCTCCTG

[0607] SEQ ID NO: 14: ACTB Reverse (5’-3’) Primer ACTCGTCATACTCCTGCTTG

[0608] SEQ ID NO: 15: REST Forward (5’-3’) Primer ACTCATTCAGGTGAGAAGCC

[0609] SEQ ID NO: 16: REST Reverse (5 ’-3’) Primer GGCGGGTTACTTCATGTTGA

[0610] SEQ ID NO: 17: NEDD9 Forward (5’-3’) Primer CAGCAGTGATGGTTCTGAGA

[0611] SEQ ID NO: 18: NEDD9 Reverse (5 ’-3 ’) Primer TCTCCACGGGCTTTGTAATC

[0612] SEQ ID NO: 19: ID3 Forward (5’-3’) Primer AAGGAGCTTTTGCCACTGAC

[0613] SEQ ID NO: 20: ID3 Reverse (5’-3‘) Primer TGAGCACCAGGTTTAGTCTC

[0614] SEQ ID NO: 21: BEX1 Forward (5’-3’) Primer CCAGGAGTAATGGAGTCCAA

[0615] SEQ ID NO: 22: BEX1 Reverse (5’-3’) Primer ATTCACCAGCATCCAAAGGG

[0616] SEQ ID NO: 23: L1CAM Forward (5’-3’) Primer CGGATACAATGTGACGTACTG SEQ ID NO: 24: L1CAM Reverse (5 ’-3’) Primer CAAGCCACTGAGGATGACA

[0617] SEQ ID NO: 25: CELSR3 Forward (S’-3’) Primer ACATATTCCACGGGTGAATCC

[0618] SEQ ID NO: 26: CELSR3 Reverse (5*-3 ’) Primer TCTCAGATGCACTGTATGCC

[0619] SEQ ID NO: 27: RUNDC3A Forward (S’ -3’) Primer TTCACGCAGAGCTACGACTA

[0620] SEQ ID NO: 28: RUNDC3A Reverse (5’-3’) Primer TTGTGCCACTTGCTGTACCA

[0621] SEQ ID NO: 29: SCAMPS Forward (5 ’-3’) Primer TCGCCTTTCTCTGGCTCAT

[0622] SEQ ID NO: 30: SCAMPS Reverse (5’-3’) Primer GGATGATGCTGATGACCAAC

[0623] SEQ ID NO: 31: CPLX1 Forward (5’-3’) Primer GAAGGAAGAACACTCGCTC

[0624] SEQ ID NO: 32: CPLX1 Reverse (5 ’-3’) Primer GAGCCTGCTTCATCACAAAC

[0625] SEQ ID NO: 33: AP3B2 Forward (5’-3’) Primer TTTCTGTGACTCAACCCAGG

[0626] SEQ ID NO: 34: AP3B2 Reverse (5’-3’) Primer TGTCTGGCAGCATGAGTTTC

[0627] SEQ ID NO: 35: CHGB Forward (S’-3’) Primer TGAAGGAATGGTGACTCGCT SEQ ID NO: 36: CHGB Reverse (5 ’-3’) Primer CGTCTTTTCTACTCGTCTTCAGG

[0628] SEQ ID NO: 37: SYP Forward (5’-3’) Primer GACATGGACGTGGTGAATC

[0629] SEQ ID NO: 38: SYP Reverse (5’-3’) Primer TCGGTCTTGTTGGCACAATC

[0630] SEQ ID NO: 39: ACSL1 Forward (5’-3’) Primer GGTTACCAAATGGCACCTTGA

[0631] SEQ ID NO: 40: ACSL1 Reverse (5’-3’) Primer TTCTCCGTGGACAAACACCT

[0632] SEQ ID NO: 41: ACSL3 Forward (5’-3’) Primer AACTGGGAGGAAGGTGGATA

[0633] SEQ ID NO: 42: ACSL3 Reverse (5’-3’) Primer CCATCGGGTTCAAACTCTCC

[0634] SEQ ID NO: 43: RY1 Forward (5’-3’) Primer GCTTGTGGATTTCTGGGCTTTC

[0635] SEQ ID NO: 44: RY1 Reverse (5’-3’) Primer CAGCAGACTCTTCAAGTCCTTCTC

[0636] SEQ ID NO: 45: RG6 Forward (5’-3’) Primer ATGTGCTGAGCGCTGAACATTG

[0637] SEQ ID NO: 46: RG6 Reverse (5 ’-3’) Primer CTGGAGTGGACGAATCTCTCATGT

Claims

CLAIMSWhat is claimed is:

1. A composition comprising: a REST (repressor element- 1 silencing transcription factor) inhibitor; and a fatty acid metabolism inhibitor.

2. The composition of claim 1, wherein the composition is synergistic (e.g. LDso of each active ingredient is lower than when used alone) and / or wherein the composition exhibits limited hepatotoxicity.

3. The composition of any one of claims 1-2, wherein the fatty acid metabolism inhibitor comprises Triacsin C, TVB-2640 / Denifanstat, avasimible, 2-fluoropalmitic acid, their homologues, or a combination thereof.

4. The composition of any one of claims 1-3, wherein the fatty acid metabolism inhibitor comprises a pan-ACSL inhibitor.

5. The composition of any one of claims 1-4, wherein die REST inhibitor covalentiy interacts with SCP1.

6. The composition of any one of claims 1-5, wherein die REST inhibitor comprises a compound described by Medellin et al. J Med. Chem, 2022, 65, 507-519, or a pharmaceutically acceptable salt thereof.

7. The composition of any one of claims 1-6, wherein die REST inhibitor comprises a compound defined by Formula I, or a pharmaceutically acceptable salt thereof:whereinR1, R2, R3, and R4are each independentiy H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted orunsubstituted C4-C20 alkylaiyl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 alkoxy, NRxRy, or wherein, as valence permits, R1and R2, R2and R3, or R3and R4, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms;Rxand Ryare independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaiyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl;R5is H, OH, halogen, or substituted or unsubstituted C1-C20 alkyl;R6is substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaiyl, substituted or unsubstituted C3-C20 heteroaiyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, or substituted or unsubstituted C1-C20 alkoxy;Q is C(O) or S(O)2; andA is a cyclic hydrophobic group, such as a substituted or unsubstituted aromatic group.

8. The composition of claim 7, wherein the REST inhibitor is defined by Formula II, or a pharmaceutically acceptable salt thereof:wherein n is an integer from 0 to 2.

9. The composition of any one of claims 7-8, wherein the REST inhibitor is defined by Formula III:or a pharmaceutically acceptable salt thereof.

10. The composition of any one of claims 7-9, wherein the REST inhibitor is defined by Formula IV:or a pharmaceutically acceptable salt thereof.

11. The composition of any one of claims 7-10, wherein the REST inhibitor is defined by Formula V, or a pharmaceutically acceptable salt thereof:whereinR7, R8, R9, R10, and R11are each independently H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alky nyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 alkoxy, or NRxRy, or wherein, as valence permits, R7and R8, R8and R9, R9and R10, or R10and R11, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms; andRxand Ryare independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl.

12. The composition of any one of claims 7-11, wherein the REST inhibitor is defined by Formula VI, or a pharmaceutically acceptable salt thereof:whereinR12, R13, R14, R15, and R16are each independently H, OH, halogen, nitro, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted orunsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 heteroaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C3-C20 aryloxy, substituted or unsubstituted C1-C20 ester, substituted or unsubstituted C1-C20 carbonate ester, substituted or unsubstituted C1-C20 sulfonate ester, substituted or unsubstituted C1-C20 sulfamate, or NR*Rb, or wherein, as valence permits, R12and R13, R13and R14, R14and R15, or R15and R16, together with the atoms to which they are attached, form a 3-10 membered substituted or unsubstituted cyclic moiety optionally including from 1 to 3 heteroatoms; andR* and Rbare independently selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C3-C20 aryl, substituted or unsubstituted C4-C20 alkylaryl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heteroaryl, or substituted or unsubstituted C1-C20 acyl.

13. The composition of any one of claims 7-12, wherein the REST inhibitor is defined by Formula VI-A:or a pharmaceutically acceptable salt thereof.

14. The composition of any one of claims 12-13, wherein the REST inhibitor is defined by Formula VII:or a pharmaceutically acceptable salt thereof.

15. The composition of any one of claims 12-13, wherein the REST inhibitor is defined by Formula VIII:or a pharmaceutically acceptable salt thereof.

16. The composition of any one of claims 7-15, wherein the REST inhibitor is selected from the group consisting of:, pharmaceutically acceptable salts thereof, andcombinations thereof.

17. The composition of any one of claims 7-16, wherein the REST inhibitor is selected from the group consisting of:pharmaceutically acceptable salts thereof, and combinations thereof.

18. The composition of any one of claims 7-17, wherein the REST inhibitor comprises:or a pharmaceutically acceptable salt thereof.

19. A pharmaceutical composition comprising the composition of any one of claims 7-18.

20. A method of treating cancer or suppressing tumor growth in a subject in need thereof, die method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1-18.

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