COMPOSITIONS AND METHODS FOR INHIBITING CARP-1 BINDING TO NEMO

INCORPORATION OF THE SEQUENCE LISTING

The present application contains a sequence listing that is submitted via EFS-Web concurrent with the filing of this application, containing the file name “37759_0257U3_SL” which is 65,252 bytes in size, created on Jan. 27, 2022, and is herein incorporated by reference in its entirety.

BACKGROUND

Diverse pathways of cell survival and apoptosis signaling by the transcription factor NF-κB are yet to be elucidated. CARP-1 (also referred to as CCAR1 or CCAR1/CARP1) is a perinuclear phospho-protein that regulates signaling by chemotherapy and growth factors. Doxorubicin, also known as Adriamycin, is a chemotherapeutic agent used to treat cancer. Doxorubicin works in part by interfering with the function of DNA. Resistance to doxorubicin among cancer cells is considered a barrier to effective treatment. Thus, effective cancer treatments are needed.

SUMMARY

Disclosed herein are methods of treating of cancer, the methods comprising: administering to a subject with cancer a therapeutically effective amount of a CARP-1-NEMO inhibitor.

Disclosed herein are methods of inhibiting cell cycle progression, cell growth or DNA repair, the methods comprising: contacting a cancer cell or malignant tissue or administering to a subject with cancer a therapeutically effective amount of a CARP-1-NEMO inhibitor.

Disclosed herein are methods of enhancing a chemotherapeutic response in a subject, the methods comprising administering to a subject with cancer a therapeutically effective amount of a CARP-1-NEMO inhibitor.

Disclosed herein are methods of reducing chemotherapeutic toxicity in a subject, the methods comprising administering to a subject with cancer a therapeutically effective amount of a CARP-1-NEMO inhibitor and a therapeutically effective amount of chemotherapeutic agent.

Disclosed herein are methods of reducing or preventing chemotherapeutic resistance in a cancer cell, the methods comprising administering to a subject with cancer a therapeutically effective amount of a CARP-1-NEMO inhibitor and a therapeutically effective amount of chemotherapeutic agent.

Disclosed herein are methods of reducing systemic levels of one or more cytokines in a subject, the methods comprising administering to a subject with cancer a therapeutically effective amount of a CARP-1-NEMO inhibitor.

Disclosed herein are methods of inhibiting binding of NEMO to CARP-1, the methods comprising administering to a subject with cancer or contacting a cancer cell with a therapeutically effective amount of a CARP-1-NEMO inhibitor.

Disclosed herein are compositions for treating cancer, wherein the compositions comprise a CARP-1-NEMO inhibitor, a chemotherapeutic agent or a DNA damage-inducing agent, and optionally, a pharmaceutical carrier; wherein the CARP-1-NEMO inhibitor and the chemotherapeutic agent or a DNA damage-inducing agent are present in a therapeutically effective amount.

Disclosed herein are methods of enhancing the efficacy of radiotherapy and/or a chemotherapeutic agent, the methods comprising: administering to a subject with cancer: (a) an effective amount of radiotherapy and/or the chemotherapeutic agent; and (b) a therapeutically effective amount of a CARP-1-NEMO inhibitor, wherein the administration of the CARP-1-NEMO inhibitor enhances the efficacy of the chemotherapeutic agent and/or the radiotherapy in the subject with cancer.

Disclosed herein are methods for screening one or more compounds for pharmacological intervention in cancer, the methods comprising: (a) providing a CARP-1 amino acid fragment capable of binding to a NEMO amino acid fragment or a NEMO amino acid fragment capable of binding to a CARP-1 amino acid fragment; (b) providing a purified or non-purified compound; (c) screening the purified or non-purified compound in an environment that allows for binding of the compound or mixture of compounds in (b) to the CARP-1 amino acid fragment or to the NEMO amino acid fragment; and (d) isolating the compound in (c) that is bound to either the CARP-1 amino acid fragment or the NEMO amino acid fragment.

Disclosed herein are methods for screening one or more compounds for pharmacological intervention in cancer, the methods comprising: (a) providing a CARP-1 amino acid fragment capable of binding to a NEMO amino acid fragment or a NEMO amino acid fragment capable of binding to a CARP-1 amino acid fragment; (b) providing a purified or non-purified mixture of compounds; (c) screening the purified or non-purified mixture of compounds in an environment that allows for binding of a compound or mixture of compounds in (b) to the CARP-1 amino acid fragment or to the NEMO amino acid fragment; and (d) isolating the compound or mixture of compounds in (c) that is bound to either the CARP-1 amino acid fragment or the NEMO amino acid fragment.

Disclosed herein are compounds having a structure represented by a formula:

embedded image

- wherein Z is selected from —S(O)— and —SO₂—;
- wherein each of R^1aand R^1bis independently selected from hydrogen and C1-C4 alkyl,
- or wherein each of R^1aand R^1bare covalently bonded, and, together with the intermediate atoms, comprise a 6-membered heterocycle;
- or wherein each of R^1aand R^1btogether comprise —CH₂—; and
- wherein Ar¹is a structure having a formula selected from:

embedded image

- wherein R², when present, is C1-C4 alkyl;
- wherein each of R^3a, R^3b, R^3c, R^3d, and R^3e, when present, is independently selected from hydrogen, halogen, —CN, —NH₂, —OH, —NO₂, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; and
- wherein each of R^4aand R^4b, when present, is independently selected from hydrogen, halogen, —CN, —NH₂, —OH, —NO₂, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, C1-C4 aminoalkyl, and Ar², provided that at least one of R^4aand R^4b, when present, is not hydrogen; and
- wherein Ar², when present, is selected from C6 aryl and C3-C5 heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, —CN, —NH₂, —OH, —NO₂, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl;
- or wherein each of R^4aand R^4b, when present, are covalently bonded and, together with the intermediate atoms, comprise a 6-membered aryl substituted with 0, 1, 2, or 3 groups independently selected from halogen, —CN, —NH₂, —OH, —NO₂, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl,
- or a pharmaceutically acceptable salt thereof.

Disclosed herein are compounds having a structure selected from:

embedded image

- or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D show CARP-1 binds with NEMO, and CARP-1 amino acids 553-599 (SEQ ID NO: 1) and NEMO amino acids 221-261 (SEQ ID NO: 2) harbor respective epitopes for interaction of CARP-1 and NEMO proteins. FIG. 1A shows protein complexes from the indicated cells were immunoprecipitated (IP.) with noted antibodies followed by the analysis of the immunocomplexes by western blot (W.B.) using anti-NEMO (upper) antibodies. The membrane containing proteins from whole cell lysates was then probed with anti-CARP-1 (middle) or anti-NEMO (lower) antibodies for presence of respective proteins. FIG. 1B shows W.B. analysis of I.P. protein complexes derived from using indicated antibodies from the noted cell lines. The membrane containing I.P proteins was probed with anti-CARP-1 antibodies (upper), while the membrane containing proteins from whole cell lysates was probed with anti-NEMO (middle) or anti-CARP-1 (lower) antibodies for presence of respective proteins. Arrowheads on the left or right side, respectively indicate presence of the proteins and molecular weight markers in panels A, B, of each blot. Schematic of CARP-1 WT and its various mutants (FIG. 1C) and NEMO WT and its mutants (FIG. 1D) that were utilized in co-I.P.-W.B. experiments to elucidate CARP-1 and NEMO interactions and map the respective minimal epitopes. The CARP-1 proteins have myc and 6×His epitopes at their carboxyl termini. The NEMO proteins, with the exception of 2-260 (SEQ ID NO: 3) and 221-261 mutants, harbored 6×myc epitope at their amino termini. NEMO 2-260 and 221-261 mutants had Gst epitope at their amino termini. Positive interactions are indicated by + and loss/absence of interaction is denoted by −.

FIGS. 2A-D show that the interference of CARP-1 binding with NEMO enhances chemotherapy efficacy in part by inhibiting activation of p65/RelA. FIGS. 2A, 2B and 2C show the indicated cell lines were treated with DMSO (Control), or with the noted dose and time of indicated agents. Determination of viable/live cells was carried out by MTT assays The bar chart columns represent means of two independent experiments; bars, SE. For panels A and C, *, @, p≤0.001 relative to respective vector sublines. FIG. 2D shows cells stably expressing myc-His-tagged wild-type CARP-1 or CARP-1 (Δ553-599; SEQ ID NO: 4) mutant were either treated with DMSO (control) or with various agents for indicated doses and time. Cell lysates were then analyzed by Western blot for levels of phosphorylated and total p65/RelA. Arrowheads on the left or right side indicate presence of proteins or molecular weight markers, respectively.

FIGS. 3A-B shows that interference of CARP-1 binding with NEMO inhibits activation of canonical NF-κB signaling. Indicated cells stably expressing wild-type or mutant CARP-1 protein were treated as in FIG. 2D for 1 h (FIG. 3A) or 6 h (FIG. 3B) durations. Cell lysates were then analyzed by W.B. for levels of CARP-1, phosphorylated and total p65/RelA, NEMO, IKKβ, and JNK1/2 proteins. The Western blot membranes in panels FIG. 3A and FIG. 3B were probed with anti-actin antibodies to assess protein loading. Arrowheads on the left or right side of each blot in panels FIGS. 3A and 3B indicate presence of proteins or molecular weight markers, respectively.

FIGS. 4A-E show the computational analyses of CARP-1 (551-600; SEQ ID NO: 5) binding with NEMO (221-261; SEQ ID NO: 2). FIG. 4A shows the SWISS Model image of CARP-1 (551-600; SEQ ID NO: 5). FIG. 4B shows the PDB ID: 3CL3 image of NEMO (221-261; SEQ ID NO: 2). FIG. 4C, FIG. 4D and FIG. 4E show the three top scoring docked complexes of CARP-1 (551-600; SEQ ID NO: 5) (Grey)/NEMO (221-261; SEQ ID NO: 2) (Green) in descending order, FIG. 4C, FIG. 4D, then FIG. 4E.

FIGS. 5A-F show the kinetics of CARP-1 binding with NEMO, and identification of pharmacologic inhibitors of CARP-1 interaction with NEMO. FIG. 5A depicts a SPR sensogram showing binding of CARP-1 (551-580; SEQ ID NO: 6) and NEMO (221-260; SEQ ID NO: 7) peptides. FIG. 5B shows the solution phase binding of Flag-CARP-1 (546-580; SEQ ID NO: 8) and Biotin-NEMO (221-261; SEQ ID NO: 2) peptides. The histogram shows fluorescence signal following binding of the two peptides over three noted times using AlphaLisa assay format. FIG. 5C and FIG. 5D show the structure and percent inhibition of binding of the Flag-CARP-1 (546-580; SEQ ID NO: 8) and Biotin-NEMO (221-261; SEQ ID NO: 2) peptides by respective compound, chemical name, formula, molecular weight, and abbreviated name of each compound that was identified following HTS. FIG. 5E shows that SNI-1 binds CARP-1. Left panel, His-TAT-HA-CARP-1 (551-580; SEQ ID NO: 6) was affinity-purified and immobilized on Ni-NTA beads, with or without SNI-1, washed 3× with RIPA buffer to remove free compound, and then allowed to bind with affinity-purified Gst-NEMO (221-261; SEQ ID NO: 2). Right panel, Gst-NEMO (221-261; SEQ ID NO: 2) peptide was affinity-purified and immobilized on glutathione sepharose, incubated with or without SNI-1, washed with RIPA buffer to remove free compound, and then allowed to bind with affinity-purified His-TAT-HA-CARP-1 (551-580; SEQ ID NO: 9). The complexes were analyzed by SDS-PAGE followed by WB with noted antibodies in respective top and middle blots. The lower blots in each panel indicate respective input peptides. FIG. 5F shows that SNI-1 does not affect NEMO interaction with RIPK1. HBC cells were untreated (control) or treated with indicated agents for noted dose and time. Protein complexes were immunoprecipitated (IP.) with noted antibodies followed by the analysis of the immunocomplexes by western blot (W.B.) using anti-CARP-1 (upper blot), anti-NEMO (middle blot), and anti-RIPK1 (lower blot) antibodies. Arrowheads on the left or right side of each blot in the left panel indicate presence of proteins or molecular weight markers, respectively. The arrowheads on the left or right side of each blot in right panel indicate presence of molecular weight markers or proteins, respectively.

FIGS. 6A-F show that SNI-1 enhances anti-cancer efficacy of chemotherapy in vitro, and CARP-1 is important for cell growth suppression by SNI-1. FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, and FIG. 6F show that cell viability was determined by MTT assay following treatments of cells with vehicle/DMSO (Control) or indicated time and doses of various agents. The columns in each histogram indicate percent of live/viable cells relative to their DMSO-treated controls and represent means of two-three independent experiments; bars, S.E. *, **, ***, p<0.001 relative to respective cells treated with chemotherapy alone (FIGS. 6A-D) or SNI-1 alone (FIG. 6E); *, **, and ***, p<0.05. 0.01, and 0.001, respectively, relative to corresponding wild-type cells (FIG. 6F).

FIGS. 7A-G show that SNI-1 attenuates chemotherapy-induced phosphorylation/activation of RelA and NEMO, promotes RIPK1 cleavage, and enhances CARP-1 levels and apoptosis. FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F and FIG. 7G show that the indicated cells were either treated with DMSO (control), or treated with noted time and dose of respective agents. In FIG. 7F, cellular proteins were first separated into cyotosolic and nuclear fractions. The cell lysates were analyzed by Western blot for levels of phosphor-p65, p65, CARP-1, PARP, Cleaved Caspase-3, RIPK1, Actin, Lamin B, GAPDH, phospho-NEMO, and NEMO proteins.

FIGS. 8A-H show that SNI-1 attenuates chemotherapy-induced secretion of pro-inflammatory cytokines in vitro. FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, FIG. 8F, FIG. 8G and FIG. 8H show that the indicated cells were either treated with DMSO (control), or treated with noted time and dose of respective agents. The media from the cells were analyzed by ELISA for levels of different pro-inflammatory cytokines.

FIGS. 9A-D show that SNI-1 enhances tumor suppression by Cisplatin in part by attenuating systemic levels of pro-inflammatory cytokines and promoting tumor apoptosis. Histogram columns showing median tumor volume (FIG. 9A) or percent T/C (percent treated (T)/control (C) tumor mass) (FIG. 9B) of the TNBC (4T1) xenograft-bearing mice treated with indicated agents. The xenograft establishment, treatment and analysis procedures were carried out as described herein. FIG. 9C shows the serum levels of noted pro-inflammatory cytokines. The columns in histograms indicate noted systemic cytokine levels in two representative animals from each of the control and treatment groups; bars, standard error (S.E.). FIG. 9D shows the immuno-histochemical staining of the noted proteins in the tumors derived from mouse with median tumor volume from each of the control and treatment groups in FIG. 9A.

FIG. 10 shows the schematic of the mechanism of action of SNI-1.

FIGS. 11A-D show the mapping of minimal epitopes of CARP-1 binding with NEMO. FIG. 11A shows that the noted cells were either untransfected (noted as −) or transfected separately with plasmids expressing indicated, myc-His-tagged CARP-1 mutant proteins in combination with plasmid expressing Gst-tagged NEMO (noted as +). Upper Blot, Western blot analysis of immunoprecipitation protein complexes using indicated antibodies. The membrane containing proteins from whole cell lysates was probed with anti-myc-Tag (middle) or anti-Gst (Lower) antibodies for presence of respective fusion proteins. FIG. 11B shows that the noted cells were either untransfected (noted as −) or transfected separately with plasmids expressing indicated, 6×myc-tagged NEMO mutant proteins in combination with plasmid expressing myc-His-tagged CARP-1 (552-654; SEQ ID NO: 10; noted as +). Upper Blot, Cell lysates were subjected to immunoprecipitation using anti-His tag antibodies to precipitate proteins complexed with His-tagged CARP-1 (552-654; SEQ ID NO: 10). The immunocomplexes were then analyzed by Western blot with anti-myc tag antibodies to detect 6×myc tagged NEMO proteins. The membrane containing proteins from whole cell lysates was probed with anti-His-Tag (Lower) antibodies for presence of CARP-1 (552-654; SEQ ID NO: 10) protein. FIG. 11C shows that the noted cells were first transfected with vector plasmid or plasmids encoding myc-His-tagged WT CARP-1 or its mutants as indicated. The neomycin-resistant, stable sublines were generated and characterized. Upper Blot, Western blot analysis of immunoprecipitation protein complexes using indicated antibodies. The membrane from upper blot was next probed with myc-Tag (middle) or the membrane containing proteins from whole cell lysates was probed with anti-NEMO (lower) antibodies. FIG. 11D shows the Gst-tagged NEMO (2-260) protein, and various His-TAT-HA-tagged CARP-1 peptides were purified following expression in E. coli BL-21 cells. The Gst-NEMO (2-260; SEQ ID NO: 3) protein was immobilized on glutathione sepharose followed by incubation with IgG or indicated CARP-1 peptides. Following stringent washing, the bound proteins were analyzed by Western blot using anti-HA (upper) or anti-Gst (middle) antibodies. The lower blot shows respective HA-tagged CARP-1 peptides used as input. Arrowheads on the left or right side of each blot in panels FIGS. 11A-D indicate presence of proteins or molecular weight markers, respectively.

FIGS. 12A-I show that NEMO (221-261; SEQ ID NO: 2) interacts with CARP-1 (551-580; SEQ ID NO: 6). FIGS. 12A, and FIGS. 12D-G show that the noted cells were either untransfected (noted wild-type) or transfected separately with indicated plasmids expressing eGFP, eGFP-tagged CARP-1 (551-580; SEQ ID NO: 6) mutant (FIG. 12A), Gst, Gst-NEMO (wild-type or mutant) proteins (FIGS. 12D-G), and neomycin-resistant, respective stable sublines were generated and characterized. Expression of respective, transfected proteins was analyzed by Western blot (upper blots), and each membrane was probed with anti-actin antibodies to assess protein loading (lower blots). FIGS. 12B-C show the Western blot analysis of immunoprecipitation protein complexes from indicated stable sublines using noted antibodies (upper, middle plots in FIG. 12B, and left blot in FIG. 12C). The membrane containing proteins from whole cell lysates was probed with anti-NEMO antibodies (lower blot in FIG. 12B) or anti-NEMO and anti-CARP-1 antibodies (right side blots in FIG. 12C). FIG. 12H shows that the indicated cells were either untransfected (noted as −) or transfected with Gst-NEMO (221-261; SEQ ID NO: 2) plasmid (noted as +), and Western blot analysis of immunoprecipitation protein complexes was conducted using noted antibodies (upper and lower right blots). The membrane containing proteins from whole cell lysates was probed with anti-CARP-1 (upper left) or anti-Gst (lower left) antibodies. Arrowheads on the left or right side of each blot in FIGS. 12A-G indicate presence of proteins or molecular weight markers, respectively. FIG. 12I shows the alignment of human CARP-1 (550-600; SEQ ID NO: 17) protein with corresponding regions of various vertebrate and invertebrate CARP-1 proteins deduced from GenBank sequences; human, SEQ ID NO: 11; mouse, SEQ ID NO: 12; dog, SEQ ID NO: 13; chimp, SEQ ID NO: 14; xenopus, SEQ ID NO: 15; and apis, SEQ ID NO: 16.

FIGS. 13A-C show that interference of CARP-1 binding with NEMO results in diminished RelA and NEMO phosphorylation. FIG. 13A shows HBC cells stably expressing Gst-NEMO or Gst-NEMO (221-261) were either untreated (Control) or treated with indicated dose and time of each agent, and Western blot analysis of the cell lysates was carried out using anti-phospho-RelA, anti-RelA, and anti-actin antibodies as in FIG. 3. FIGS. 13A-B show that the indicated cells stably expressing wild-type or mutant CARP-1 protein were either untreated (control) or treated with noted dose and time of each agent. Cells were then processed for immunofluorescence staining for CARP-1 (red), DAPI (blue), and phosphorylated NEMO (green). Images were taken using Zeiss LSM 510 Meta NLO. Bar, 2 micrometer.

FIGS. 14A-C show the computational analyses of CARP-1 (551-600; SEQ ID NO: 5) binding with NEMO (221-261; SEQ ID NO: 2). Backbone RMSD and conformation histogram analyses for the three top scoring CARP1/NEMO complexes obtained from docking. FIG. 14A shows the top scoring pose. FIG. 14B shows the second pose. FIG. 14C shows the third pose. The calculations were done over the 24 ns production run.

FIGS. 15A-B show the buffer optimization (FIG. 15A) or DMSO tolerance (FIG. 15B) of the high-throughput screening assay. The Examples provide detail measurements of binding of CARP-1 (551-580; SEQ ID NO: 6) and Biotin-tagged NEMO (221-261; SEQ ID NO: 2), peptides in PBS buffer with or without Tween, BSG, or noted concentrations of DMSO.

FIGS. 16A-H show that 1-(3,4-dihydroxyphenyl)-2-{(1-(4-methylphenyl)-1H-tetrazol-5-yl)thio} ethanone (SNI-1) inhibits cell growth in time (FIG. 16A), dose-dependent matter (FIGS. 16B, C), and enhances efficacy of genotoxic chemotherapy in vitro in drug-resistant and BRCA-mutant TNBC, (FIGS. 16D-E), colon cancer cells (FIG. 16F), while NEMO is required for Adriamycin-dependent transcriptional activation of NF-κB (FIG. 16G) but not for γH2AX (FIG. 16H). FIG. 16A, and FIGS. D-F show that cell viability was determined by MTT assay following treatments of cells with vehicle/DMSO (Control) or indicated time and doses of various agents. The columns in each histogram indicate percent of live/viable cells relative to their DMSO-treated controls and represent means of two-three independent experiments. In FIGS. 16B, C, the columns in each histogram indicate number of live/viable cells; bars, S.E. ** and ***; p=0.005 and 0.001, respectively. FIG. 16G shows that for indicated cells were transfected with NF-κB-TATA-Luc plasmid followed by treatments with time and dose of noted agents. The columns in histogram indicate activities of the NF-κB reporter relative to the DMSO-treated controls and represent two separate experiments; bars, S.E. FIG. 16H shows that HeLa cells (wild-type, CARP-1 ko, and NEMO-ko) were either untreated (control) or treated with Adriamycin for indicated dose and time. Western blot analysis of the cell lysates was carried out using anti-γH2AX, anti-H2AX, and anti-actin antibodies. Arrowheads on the left or right side of each blot in FIG. 16H indicate presence of proteins or molecular weight markers, respectively.

FIGS. 17A-B show Adriamycin activates IKKa/P and caspase-3 independent of NEMO. HeLa cells (wild-type and NEMO-ko) were either untreated (control) or treated with indicated dose and time of noted agents. Western blot analysis of the cell lysates was carried out using anti-phospho RelA, anti-RelA, anti-cleaved caspase-3, anti-phospho-NEMO, anti-NEMO, anti-phospho-IKKα/β, anti-IKKα/β, and anti-actin antibodies. Arrowheads on the left or right side of each blot in FIGS. 17A-B indicate presence of proteins or molecular weight markers, respectively. Vertical bar in FIG. 17B denotes autoradiogram splicing.

FIGS. 18A-D show that 1-(3,4-dihydroxyphenyl)-2-{(1-(4-methylphenyl)-1H-tetrazol-5-yl)thio} ethanone (SNI-1) enhances tumor suppression by chemotherapy in part by attenuating systemic levels of pro-inflammatory cytokines and promoting tumor apoptosis. Histogram columns showing median tumor volume (FIG. 18A) or percent T/C (FIG. 18B) of the TNBC (4T1) xenograft-bearing mice treated with indicated agents. FIG. 17C and FIG. 17D show the serum levels of noted pro-inflammatory cytokines. The columns in histograms indicate noted systemic cytokine levels in two representative animals from each of the control and treatment groups; bars, S.E.

FIGS. 19A-B show that 1-(3,4-dihydroxyphenyl)-2-{(1-(4-methylphenyl)-1H-tetrazol-5-yl)thio} ethanone (SNI-1) administration does not induce apoptosis in various organs of 4T1 tumor-bearing mice. FIG. 19A is a histogram showing quantification of staining for indicated proteins in the tumor tissues in FIG. 9D. FIG. 19B shows immuno-histochemical staining for presence of cleaved caspase-3 was carried out in indicated murine tissues from a representative animal from control or SNI-1 group.

FIGS. 20A-B show that SNI-1 and its water soluble, di-sodium salt have similar activities, in vitro. FIG. 20A shows the chemical Structure of di-Sodium SNI-1. FIG. 20B shows cells that were treated with indicated dose and time of noted agent, and their viabilities determined by MTT assay. Columns represent means of three independent experiments; bars, SE. Please note that HCC1937 are representative of BRCA1-mutant TNBC.

FIG. 21 shows the chemical structures of SNI-1 analogs GL-208-213, GL215 and GL216. GL-214 is SNI-1.

FIGS. 22A-B show SNI-1 analogs enhance Adriamycin inhibition of TNBC cell growth. FIGS. 22A-B show cells treated with indicated dose of noted agent for 24 h, and their viabilities determined by MTT assay. Columns represent means of three independent experiments; bars, SE.

FIGS. 23A-B show that SNI-1 enhances Mam 16C/Adr tumor suppression by Cisplatin. Histogram columns showing median tumor volume (FIG. 23A) and % T/C (FIG. 23B). The subcutaneous tumor bearing mice were treated with indicated agents. (Cisplatin, 3 mg/kg/dose, i.v., day 1, 5, 10, 14; SNI-1, 70 mg/kg/dose, i.p., daily days 1-13) The end-points for assessing anti-tumor activity involved qualitative determination via tumor growth inhibition (% T/C) where T is the median tumor volume of the treated mice and C is the median tumor volume of the Control mice on any given day of measurement. According to the NCI-accepted criteria, a treatment is considered effective if the T/C is <42%. It was found that % T/C remained consistently below 30 for the SNI-1+Cisplatin cohort on any day of the measurement indicated in (FIG. 23B).

FIG. 24 shows an efficacy study using human BRCA1 mutant SUM149 TNBC cell-derived xenografts in SCID mice.

FIGS. 25A-B show that SNI-1 enhances tumor suppression by Cisplatin Renal Cancer Syngeneic Tumor Model. Histogram columns showing median tumor volume (FIG. 25A) and % T/C (FIG. 25B). The subcutaneous tumor bearing mice were treated with indicated agents. (Cisplatin, 2 mg/kg/dose, i.v., day 3, 7, 11, 15; SNI-1, 70 mg/kg/dose, i.p., daily days 3-15) The dose of Cisplatin used was lower (subtherapeutic) than that (3 mg/kg/dose) used in other experiments. The end-points for assessing anti-tumor activity involved qualitative determination via tumor growth inhibition (% T/C) where T is the median tumor volume of the treated mice and C is the median tumor volume of the Control mice on any given day of measurement. According to the NCI-accepted criteria, a treatment is considered effective if the T/C is <42%. It was found that % T/C remained consistently below 40 for the SNI-1+Cisplatin cohort on any day of the measurement indicated in (FIG. 25B). Throughout this experiment, weight loss in animals ranged from 1.9-3.7% in Control group, 0.4-4.0% in SNI-1 group, 0.8-3.2% in Cisplatin group, and 1.6-6.7% in SNI-1+Cisplatin group. The animal weight loss remained below the NCI-accepted criteria od<10%.

DETAILED DESCRIPTION

Many modifications and other embodiments of the present disclosure set forth herein will come to mind to one skilled in the art to which this disclosure pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the present disclosure is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Before the present compositions and methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, example methods and materials are now described.

Moreover, it is to be understood that unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, and the number or type of aspects described in the specification.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosures. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

Definitions

As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of” and “consisting essentially of” “Comprising” can also mean “including but not limited to.”

As used in the specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes mixtures of compounds; reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.

As used herein, the term “sample” is meant a tissue or organ from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g., a polypeptide or nucleic acid), which is assayed as described herein. A sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.

As used herein, the term “subject” refers to the target of administration, e.g., a human. Thus the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). In one aspect, a subject is a mammal. In another aspect, a subject is a human. The term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

As used herein, the term “patient” refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects. In some aspects of the disclosed methods, the “patient” has been diagnosed with a need for treatment for cancer, such as, for example, prior to an administering step.

Ranges can be expressed herein as from “about” or “approximately” one particular value, and/or to “about” or “approximately” another particular value. When such a range is expressed, a further 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,” or “approximately,” it will be understood that the particular value forms a further 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. It is also understood that there are a number of values disclosed herein and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

“Inhibit,” “inhibiting” and “inhibition” mean to diminish or decrease 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 may also include, for example, a 10% inhibition or reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, in an aspect, the inhibition or 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. In an aspect, the inhibition or reduction is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% as compared to native or control levels. In an aspect, the inhibition or reduction is 0-25, 25-50, 50-75, or 75-100% as compared to native or control levels.

“Modulate”, “modulating” and “modulation” as used herein mean a change in activity or function or number. The change may be an increase or a decrease, an enhancement or an inhibition of the activity, function or number.

As used herein, the term “CARP-1” is used interchangeably with “cell cycle and apoptosis regulatory protein 1”. The amino acid sequence of CARP-1 can be found in Table 1.

As used herein, the term “NEMO” is used interchangeably with “NF-kappa-B essential modulator”, “NF-κB essential modulator”, “NF-κB activating kinase IKK subunit γ” and “inhibitor of nuclear factor kappa-B kinase subunit gamma (IKK-7)”. NEMO refers to a protein that in humans is encoded by the IKBKG gene. NEMO is a subunit of the IκB kinase complex that activates NF-κB. The human gene for IKBKG is located on chromosome Xq28. In vivo, NEMO activates NF-κB resulting in activation of genes involved in inflammation, immunity, cell survival, and other pathways. The amino acid sequence of NEMO can be found at Table 1. The Accession number for the nucleic acid sequence of NEMO is #NM_001099857.

As used herein, the term “CARP-1-NEMO inhibitor” is used interchangeably with “cell cycle and apoptosis regulatory protein (CARP)-1-NF-κB activating kinase IKK subunit γ (NEMO) inhibitor”, “cell cycle and apoptosis regulatory protein 1 (CARP-1)-NF-κB activating kinase IKK subunit γ (NEMO) inhibitor” and “cell cycle and apoptosis regulatory protein-1 (CARP-1)-NF-κB activating kinase IKK subunit γ (NEMO) inhibitor”.

As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting or slowing progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. Treatment can also be administered to a subject to ameliorate one more signs of symptoms of a disease, disorder, and/or condition. For example, the disease, disorder, and/or condition can be cancer.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications may be practiced within the scope of the appended claims.

CARP-1 is a ubiquitous, ˜130 kDa peri-nuclear phospho-protein (Rishi, A. K., et al. (2003) J Biol Chem 278, 33422-33435) that has homologs in vertebrates, Apis millifera, and the worm Caenorhabditis elegans. Lst3, the C. elegans ortholog of human CARP-1, is an agonist of Notch signaling that also functions as an inhibitor of the EGFR-MAPK pathway (Yoo et al. (2004) Science 303, 663-666). This EGFR pathway antagonism by Lst3 corroborated prior findings of CARP-1 requirement for EGFR inhibitor-induced apoptosis (Rishi, A. K., et al. (2006) J Biol Chem 281, 13188-13198). Additionally, CARP-1 promoter methylation as well as signaling by protein kinase A (PKA) regulated CARP-1 expression and function, respectively (Rishi, A. K., et al. (2006) J Biol Chem 281, 13188-13198; Jiang, Y., et al. (2010) J Mol Signal 5, 7; and Zhang et al. (2007) Mol Cancer Ther 6, 1661-1672). CARP-1 is a phospho-protein, and although the EGF as well as the ATM kinase signaling target specific serine residues of CARP-1 (Beausoleil, S. A., et al. (2004) Proc Natl Acad Sci USA 101, 12130-12135; Blagoev, B., et al. (2003) Nat Biotechnol 21, 315-318; and Matsuoka et al. (2007) Science 316, 1160-1166), the precise role(s) and kinase(s) of CARP-1 serine phosphorylation remain unclear. CARP-1 binds with the LIM protein Zyxin and regulates apoptosis in response to UV-C irradiation (Hervy et al. (2010) Genes Cancer 1, 506-515), while it also interacts with Necdin to regulate myoblast survival (Francois, et al. (2012) PLoS One 7, e43335). Further, recent studies found CARP-1 as a co-activator of the cell cycle regulatory APC/C E3 ligase (Puliyappadamba et al. (2011) J Biol Chem 286, 38000-38017), the steroid-thyroid family of nuclear receptors (Kim et al., (2008) Mol Cell 31, 510-519), the GR signaling during adipogenesis, β-catenin in colon cancer metastasis, or neurogenin3-mediated pancreatic endocrine differentiation (Ou et al. (2009) J Biol Chem 284, 20629-20637; Ou et al. (2014) J Biol Chem 289, 17078-17086; and Lu et al. (2012) Biochem Biophys Res Commun 418, 307-312). Interestingly, CARP-1 also co-activated tumor suppressor p53 to transduce the DNA-damage-induced transcriptional increase of CDKI p21WAF1 in breast cancer cells (Kim et al., (2008)Mol Cell 31, 510-519).

Chemotherapeutics such as Adriamycin (ADR) induce double-strand breaks (DSBs) while phosphorylation of H2AX at serine139 (γ-H2AX) by ATM/ATR functions to repair DSBs (Pommier et al. (2010) Chem Biol 17, 421-433; Fornari et al. (1994) Mol Pharmacol 45, 649-656; and Podhorecka et al. (2010) J Nucleic Acids 2010). ADR also promotes apoptosis in part by inducing JNK-dependent γH2AX (Picco et al. (2013) Genes Cancer 4, 360-368; and Lu et al. (2006) Mol Cell 23, 121-132). It was shown that ADR induced CARP-1 and γH2AX, and depletion of CARP-1 abrogated γH2AX increase by ADR (Sekhar et al. (2019) Cancers (Basel) 11). CARP-1 binds with H2AX, and abrogation of CARP-1/H2AX binding blocked ADR-induced inhibition of triple negative breast cancer (TNBC) and HeLa cells (Sekhar et al. (2019) Cancers (Basel) 11).

NF-κB is a pro-inflammatory transcription factor that is a regulator of the immune system, and is responsive to a large number of stimuli that engage signaling pathways to activate this transcription factor and effect distinct cellular responses (Graef et al., (2001) Proc Natl Acad Sci USA 98, 5740-5745). With the exception of C. elegans, the NF-κB signaling components exist in most multicellular organisms (Zhang et al. (2017) Cell 168, 37-57). In mammalian cells, five members of the NF-κB family include RelA (p65), RelB, c-Rel, p50/p105 (NF-κB1), and p52/p100 (NF-κB2) that function by forming homo- and hetero-dimers. A family of inhibitory proteins called IκBs sequester the NF-κB complexes in the cytoplasm. IκBs are phosphorylated by IκB kinase (IKK), which leads to IκB degradation by ubiquitin-proteasome pathway, followed by release of NF-κB for its translocation to the nucleus where it functions as transcription factor (Zhang et al. (2017) Cell 168, 37-57). The TKK complex contains two kinase subunits, IKKα and IKKβ, and an associated regulatory subunit called NEMO (IKKγ). NF-κB regulates cellular homeostasis as well as tumor cell proliferation, survival, metastasis, inflammation, invasion, and angiogenesis, and often contributes to a resistant phenotype and poor prognosis (Liu et al. (2006) Mol Cell 21, 467-480). Although, a pro-apoptotic function for NF-κB has also been suggested (Shou et al. (2002) J Neurochem 81, 842-852; Martin et al. (2009) Aging (Albany NY) 1, 335-349; and Ryan et al. (2000) Nature 404, 892-897), and possibly involves NF-κB-regulation of transducers of receptor-mediated apoptosis, a full characterization of the complex molecular details of the apoptotic functions of NF-κB remain to be accomplished. However, therapy-induced DNA damage that causes ATM/ATR activation to promote H2AX-dependent DSB repair, also stimulates phosphorylation of NEMO in the nucleus by ATM. The phosphorylated NEMO is mono-ubiquitinated, which triggers its nuclear export and IKK activation in the cytoplasm (Wu et al. (2006) Science 311, 1141-1146). This therapy-induced activation of canonical NF-κB promotes production of pro-inflammatory cytokines, cell growth and survival signaling, and contributes to therapy resistance.

Since, CARP-1 is a regulator of cell growth and survival signaling and a component of the NF-κB proteome, and CARP-1 depletion inhibited transcriptional activation of NF-κB by ADR, TNFα, or an experimental CARP-1 Functional Mimetic (CFM) compound, the molecular mechanism of CARP-1-dependent regulation of NF-κBf signaling was investigated as described herein. It was determined that CARP-1 directly binds with NEMO, and blockage of this interaction interferes with ADR-induced activation of canonical NF-κBf. Pharmacological inhibition of NEMO-CARP-1 binding enhances Cisplatin efficacy in part by impacting levels of circulating pro-inflammatory cytokines in immuno-competent mice bearing subcutaneous tumors of murine breast cancer cells.

Table 1 provides sequences of the various molecules described herein.

TABLE 1

Sequences.

SEQ

ID NO:
NAME
SEQUENCE

1
CARP-1:
PEETHKGRTVPAHVETVVLFFPDVWHCLPTRSEWETL

553-599
SRGYKQQLVE

2
NEMO:
SEEKRKLAQLQVAYHQLFQEYDNHIKSSVVGSERKRG

221-261
MQLE

3
NEMO:
NRHLWKSQLCEMVQPSGGPAADQDVLGEESPLGKPA

2-260
MLHLPSEQGAPETLQRCLEENQELRDAIRQSNQILRER

CEELLHFQASQREEKEFLMCKFQEARKLVERLGLEKL

DLKRQKEQALREVEHLKRCQQQMAEDKASVKAQVT

SLLGELQESQSRLEAATKECQALEGRARAASEQARQL

ESEREALQQQHSVQVDQLRMQGQSVEAALRMERQA

ASEEKRKLAQLQVAYHQLFQEYDNHIKSSVVGSERK

RGMQL

4
CARP-1:
PEETHKGRTVPAHVETVVLFFPDVWHCLPTRSEWETL

553-599
SRGYKQQLVE

5
CARP-1:
YHRPEETHKGRTVPAHVETVVLFFPDVWHCLPTRSEW

550-600
ETLSRGYKQQLVEK

6
CARP-1:
HRPEETHKGRTVPAHVETVVLFFPDVWHCL

551-580

7
NEMO:
EEKRKLAQLQVAYHQLFQEYDNHIKSSVVGSERKRGM

221-260
QLE

8
CARP-1:
AEIRYHRPEETHKGRTVPAHVETVVLFFPDVWHCL

546-580

9
His-
MHHHHHHKLYGRKKRRQRRRGSYPYDVPDYAGSPEE

Tat-HA-
THKGRTVPAHVETVVLFFPDVWHCLPTRSEWETLSRG

CARP-1:
YKQQLVE

553-599

10
CARP-1:
RPEETHKGRTVPAHVETVVLFFPDVWHCLPTRSEWETL

552-654
SRGYKQQLVEKLQGERKEADGEQDEEEKDDGEAKEIS

TPTHWSKLDPKTMKVNDLRKELESRALS

11
CARP-1:
MAQFGGQKNPPWATQFTATAVSQPAALGVQQPSLLGA

human
SPTIYTQQTALAAAGLTTQTPANYQLTQTAALQQQAAA

AAAALQQQYSQPQQALYSVQQQLQQPQQTLLTQPAVAL

PTSLSLSTPQPTAQITVSYPTPRSSQQQTQPQKQRVFTGVV

TKLHDTFGFVDEDVFFQLSAVKGKTPQVGDRVLVEATY

NPNMPFKWNAQRIQTLPNQNQSQTQPLLKTPPAVLQPIA

PQTTFGVQTQPQPQSLLQAQISAASITPLLQTQPQPLLQQ

PQQKAGLLQPPVRIVSQPQPARRLDPPSRFSGRNDRGDQ

VPNRKDDRSRERERERRRSRERSPQRKRSRERSPRRERE

RSPRRVRRVVPRYTVQFSKFSLDCPSCDMMELRRRYQN

LYIPSDFFDAQFTWVDAFPLSRPFQLGNYCNFYVMHRE

VESLEKNMAILDPPDADHLYSAKVMLMASPSMEDLYH

KSCALAEDPQELRDGFQHPARLVKFLVGMKGKDEAMA

IGGHWSPSLDGPDPEKDPSVLIKTAIRCCKALTGIDLSVC

TQWYRFAEIRYHRPEETHKGRTVPAHVETVVLFFPDVW

HCLPTRSEWETLSRGYKQQLVEKLQGERKEADGEQDEE

EKDDGEAKEISTPTHWSKLDPKTMKVNDLRKELESRALS

SKGLKSQLIARLTKQLKVEEQKEEQKELEKSEKEEDEDDD

RKSEDDKEEEERKRQEEIERQRRERRYILPDEPAIIVHPNW

AAKSGKFDCSIMSLSVLLDYRLEDNKEHSFEVSLFAELFNE

MLQRDFGVRIYKSLLSLPEKEDKKEKDKKSKKDERKDKKE

ERDDETDEPKPKRRKSGDDKDKKEDRDERKKEDKRKGDSK

DDDETEEDNNQDEYDPMEAEEAEDEEDDRDEEEMTKRDDK

RDINRYCKERPSKDKEKEKTQMITINRDLLMAFVYFDQSHC

GYLLEKDLEEILYTLGLHLSRAQVKKLLNKVVLRESCFYRK

LTDTSKDEENHEESESLQEDMLGNRLLLPTPTVKQESKDVE

ENVGLIVYNGAMVDVGSLLQKLEKSEKVRAEVEQKLQLLE

EKTDEDEKTILNLENSNKSLSGELREVKKDLSQLQENLKISE

NMSLQFENQMNKTIRNLSTVMDEIHTVLKKDNVKNEDKDQK

SKENGASV

12
CARP-1:
MAQFGGQKNPPWATQFTATAVSQPAALGVQQPSLLGASPTIY

mouse
TQQTALAAAGLTTQTPANYQLTQTAALQQQAAAVLQQQYSQ

PQQALYSVQQQLQQPQQTILTQPAVALPTSLSLSTPQPAAQITV

SYPTPRSSQQQTQPQKQRVFTGVVTKLHDTFGFVDEDVFFQLG

AVKGKTPQVGDRVLVEATYNPNMPFKWNAQRIQTLPNQNQSQ

TQPLLKTPTAVIQPIVPQTTFGVQAQPQPQSLLQAQISAASITPLL

QTQPQPLLQQPQQKAGLLQPPVRIVSQPQPARRLDPPSRFSGRN

DRGDQVPNRKDDRSRERDRERRRSRERSPQRKRSRERSPRRER

ERSPRRVRRVVPRYTVQFSKFSLDCPSCDMMELRRRYQNLYIP

SDFFDAQFTWVDAFPLSRPFQLGNYCNFYVMHREVESLEKNM

AVLDPPDADHLYSAKVMLMASPSMEDLYHKSCALAEDPQDLR

DGFQHPARLVKFLVGMKGKDEAMAIGGHWSPSLDGPNPEKDP

SVLIKTAIRCCKALTGIDLSVCTQWYRFAEIRYHRPEETHKGRT

VPAHVETVVLFFPDVWHCLPTRSEWETLSRGYKQQLVEKLQG

ERKKADGEQDEEEKDDGEVKEIATPTHWSKLDPKAMKVNDLR

KELESRALSSKGLKSQLIARLTKQLKIEEQKEEQKELEKSEKEEE

DEDDKKSEDDKEEEERKRQEEVERQRQERRYILPDEPAIIVHPN

WAAKSGKFDCSIMSLSVLLDYRLEDNKEHSFEVSLFAELFNEM

LQRDFGVRIYKSLLSLPEKEDKKDKEKKSKKEERKDKKEERED

DIDEPKPKRRKSGDDKDKKEDRDERKKEEKRKDDSKDDDETE

EDNNQDEYDPMEAEEAEDEDDDREEEEVKRDDKRDVSRYCK

DRPAKDKEKEKPQMVTVNRDLLMAFVYFDQSHCGYLLEKDL

EEILYTLGLHLSRAQVKKLLNKVVLRESCFYRKLTDTSKDDEN

HEESEALQEDMLGNRLLLPTPTIKQESKDGEENVGLIVYNGAM

VDVGSLLQKLEKSEKVRAEVEQKLQLLEEKTDEDGKTILNLEN

SNKSLSGELREVKKDLGQLQENLEVSENMNLQFENQLNKTLRN

LSTVMDDIHTVLKKDNVKSEDRDEKSKENGSGV

13
CARP-1:
MFFAAYQDVRRCYRRQTSEDFYPPFIMAQFGGQKNPPWATQFT

dog
ATAVSQPAALGVQQPSLLGASPTIYTQQTALAAAGLTTQTPANY

QLTQTAALQQQAAAAAAALQQLQQPQQTLLTQPAVALPTSLSL

STPQPAAQITVSYPTPRSSQQQTQPQKQRVFTGVVTKLHDTFGF

VDEDVFFQLSAVKGKTPQVGDRVLVEATYNPNMPFKWNAQRI

QTLPNQNQSQTQPLLKTPPAVLQPIAPQTTFGVQAQPQPQSLLQ

AQISAASITPLLQTQPQPLLQQPQQKAGLLQPPVRIVSQPQPARR

LDPPSRFSGRNDRGDQVPNRKDDRSRERERERRRSRERSPQRKR

SRERSPRRERERSPRRVRRVVPRYTVQFSKFSLDCPSCDMMELR

RRYQNLYIPSDFFDAQFTWVDAFPLSRPFQLGNYCNFYVMHRE

VESLEKNMAILDPPDADHLYSAKVMLMASPSMEDLYHKSCAL

AEDPQELRDGFQHPARLVKFLVGMKGKDEAMAIGGHWSPSLD

GPDPEKDPSVLIKTAIRCCKALTGIDLSVCTQWYRFAEIRYHRPE

ETHKGRTVPAHVETVVLFFPDVWHCLPTRSEWETLSRGYKQQL

VEKLQGERKEADGEQALNANPFFYFRFSQDEEEKDDGEAKEIST

PTHWSKLDPKTMKVNDLRKELESRALSSKGLKSQLIARLTKQLK

VEEQKEEQKELEKSEKEEEEEDDRKSEDDKEEEERKRQEEMERQ

RRERRYILPDEPAIIVHPNWAAKSGKFDCSIMSLSVLLDYRLEDN

KEHSFEVSLFAELFNEMLQRDFGVRIYKSLLSLPEKEDKKEKEKK

SKKDERKDKKEDRDDETDEPKPKRRKSGDDKDKKEDRDERKKE

DKRKEDSKDDDETEEDNNQDEYDPMEAEEAEDEEDDRDEEEINK

RDDKRDINRYCKERPSKDKEKEKTQMITINRDLLMAFVYFDQSHC

GYLLEKDLEEILYTLGLHLSRAQVKKLLNKVVLRESCFYRKLTDT

SKDEENHEESEALQEDMLGNRLLLPTPTVKQESKDVEENVGLIVY

NGAMVDVGSLLQKLEKSEKVRAEVEQKLQLLEEKTDEDEKTILNL

ENSNKSLSGELREVKKDFSQLQENLKISENMNLQFENQLNKTIRNL

STVMDEIHTVLKKDNVKNEDKDQKSKENGASV

14
CARP-1:
MWRRGAAWRKRGKLAHAPKADGFEMASMLAGTRLRPGAASPTP

chimp
TARLFRCPQRPSASAWLRCSPPPHCSRAAAVLPSWPPGPGHRGCSR

RRGSWGIGAFSVRGKRAQGSRDPSSVVGRWVPPSVAGGRHGAGTG

GRWTAELWPLRVAAAEEGVRGRRIFAFSAALGVQQPSLLGASPTIY

TQQTALAAAGLTTQTPANYQLTQTAALQQQAAAAAAALQQQYSQ

PQQALYSVQQQLQQPQQTLLTQPAVALPTSLSLSTPQPTAQITVSYP

TPRSSQQQTQPQKQRVFTGVVTKLHDTFGFVDEDVFFQLSAVKGK

TPQVGDRVLVEATYNPNMPFKWNAQRIQTLPNQNQSQTQPLLKTP

PAVLQPIAPQTTFGVQTQPQPQSLLQAQISAASITPLLQTQPQPLLQQ

PQQKAGLLQPPVRIVSQPQPARRLDPPSRFSGRNDRGDQVPNRKDD

RSRERERERRRSRERSPQRKRSRERSPRRERERSPRRVRRVVPRYTV

QFSKFSLDCPSCDMMELRRRYQNLYIPSDFFDAQFTWVDAFPLSRPF

QLGNYCNFYVMHREVESLEKNMAILDPPDADHLYSAKVMLMASPS

MEDLYHKSCALAEDPQELRDGFQHPARLVKFLVGMKGKDEAMAIG

GHWSPSLDGPDPEKDPSVLIKTAIRCCKALTGIDLSVCTQWYRFAEIR

YHRPEETHKGRTVPAHVETVVLFFPDVWHCLPTRSEWETLSRGYKQ

QLVEKLQGERKEADGEQALNANPFFYFRFSQAQEHSSSHGYLKLDN

HKSERFEISGYVATSLDEEEKDDGEAKEISTPTHWSKLDPKTMKVND

LRKELESRALSSKGLKSQLIARLTKQLKVEEQKEEQKELEKSEKEEDE

DDDRKSEDDKEEEERKRQEEIERQRRERRYILPDEPAIIVHPNWAAKS

GKFDCSIMSLSVLLDYRLEDNKEHSFEKEDKRKDDSKDDDETEEDNN

QDEYDPMEAEEAEDEEDDEDEKTILNLENSNKSLSGELREVKKDLSQL

QENLKISENMNLQFENQLNKTIRNLSTVMDEIHTVLKKYLRPWGTDVE

GYSSTSTNHQAPKLYVGSERPCNGPYCIASETSWSLVSISTGCSSWLLT

WNGPKARSKASLPALGTPGAAVRTADGRSQALQEAAGSPRTWKSPRA

RPWGKGSSGPRGGWKSRASPGGRVGLGCGERSRTLGSGISSTALRRPK

HGCPTPGPPGAVGPAPWSSVPPAASAADPRAVGPSSRRASGVVAAALA

EALRCGLPAAGESMARPVQLAPGSLALVLCRLEAQKAAGAAEEPGGRA

VFRAFRRANARCFWNARLARAASRLAFQGWLRRWVLLVRAPPACLQIC

SGRHSGFHVLQCGGLGSGPSSFGVVNFLGKTSDVFPVQMNPITQSQFVPL

GEVLCCAISDMNTAQIVVTQESLLERLMKHYPGIAIPSEDILYTTLGTLIK

ERKIYHTGEGYFIVTPQTYFITNTTTQENKRMLPSDESRLMPASMTYLDT

ESGI

15
CARP-1:
MAQFGGQKNPPPWATQFTATAVSQPGPLAVQQSSLLGASPTIYTQQS

xenopus
ALAAAGLASPSPANYQLSQTAALQQQAAAAAAAAAAALQQQYTQP

QQTIYSVQQQLQPPPQAILTQPAVALPTSLALSTPQQAAQITVSYPTPR

SNQQQTQPQKQRVFTGVVTKLHETFGFVDEDVFFQLTAVKGKSPQA

GDRVLVEATYNPNMPFKWNAQRIQTLPNQNPASAQSLIKNPAAVMQ

PVAQPTAYAVQTQPPPQAQTLLQAQISAATLTPLLQTQTSPLLQQPQQ

KAGLLQTPVRIVSQPQPVRRIEPPSRFSVRNDRGDSILSRKDDRNRERE

RERRRSRDRSPQRKRSRERSPRRERERSPRRPRRVVPRYTVQISKFCLD

CPGCDTMELRRRYQNLYIPSDFFDAQFTWVDAFPISRPFQLGNYSNFY

IMHKEVDPLEKNTAIVDPPDADHTYSAKVMLLASPSLEELYHKSCAL

AEDPIEVREGFQHPARLIKFLVGMKGKDEAMAIGGHWSPSLDGPNPD

KDPSVLIRTAVRCCKALTGIELSLCTQWYRFAEIRYHRPEETHKGRTV

PAHVETVVLFFPDVWHCLPTRSEWENLCHGYKQQLVDKLQGDRKE

ADGEQEEEDKEDGDAKEISTPTHWSKLDPKIMKVNDLRKELESRTLS

SKGLKSQLIARLTKQLRIEEQKEEQKELEKCEKEEEEEEERKSEDDKE

EEERKRQEELERQRREKRYMLPDEPAIIVHPNWSAKNGKFDCSIMSL

SVLLDYRIEDNKEHSFEVSLFAELFNEMLQRDFGVRIYRELLALPEKE

EKKDKEKKCKKEDKRERKEDKDDDDEPKPKRRKSSDDKIKLEEKEE

RKRDDRRKEDYREEDDPDYENQDDYEPIAAEEDDGDYDDREDDDD

DSSSKDKREDKRDGNRYSKERQSKDKEKDKKQMVTVNRDLLMAFV

YFDQSHCGYLLEKDLEEILYTLGLHLSRAQVKKLFTKILLKESLLYRK

LTDTATEDGSHEETDPLHNDILGNCSLLPSKAVRTGLSTVEDKGGLIV

YKGAMVDVGSLLQKLEKSEKTRTELEHRLQTLESKTEEDEKTISQLE

ASNRNLSEELKQTKDDVGHLKDSLKAAEDTRSLYEDQLTNTIKNLSA

AMGEIQVVLNKNPSTTEDQKSKENGSS

16
CARP-1:
MSNLSPFGGGKNPPWVRNAGQGIQNIQQQMLGQAMGSIGGQPMVQ

apis
YQQQTQQVYQQSLGLQQPNITMASMATLGSNLPSGIAGQLYPQVAT

VSYPPPRALNTNAFQPSVAGVPQQVQQNVPSSSTKQRVFTGTVTQV

YDNFGFVDEDVFFQTNACVKGSNPVVGDRVLVEASYNPSMPFKWS

ATRIQVLPMGTNTQQNNQNTRQQQQQSQPQQNRTSGTYNA

VPPPAENANNRFTTSATNANTASNRNKVGRVRERSPRERKNEEEEIE

RKRRREERIREREKKEERSPSRTRRSKSPRPRRRTRVVPRYMVQIPKI

ALDLPEADVLEIRRRYQNMYIPSDFFSTGFRWVDAFPPHMPFALNKP

YVDPCSENTAVLEPSDADYLFSAKVMLISMPAMEEIYKRCCGVSEDR

DPDRDYVHPTRLINFLVGLRGKNETMAIGGPWSPSLDGPNPEKDPSV

LIRTAVRTCKALTGIDLSSCTQWYRFLELYYRRAETTHKSGRVVPSR

VETVILFLPDVWSCVPIKLEWDGLQLSYKKQLERKLLRAASSPDDLD

AANDTDEAAVADQKALPTSSHITFTFLLHYIIVQLFPITKLNFQYRLY

LLIDPIADDPVPEKKDPTHYSELDPKSMNVTELRQELAARNLNCKGL

KSQLLARLMKAITSEQAKEEGRQDDIEENDKDISPPPKEEEDKKFKD

IKDHDEDRRKLCERERAALEKRYTLPESSHIIVHPSRMAKSGKFDCT

VMSLSVLLDYRPEDTKDDDSIKDGRRDREKDGRKRKIKLYTHDPYL

LLSFVYFDQTHCGYIFDKDIEELIYTLGLKLSRAQVRKLVQKVVTRD

SLHYRKLTDRSKEDDLKDEKKDEKEIDKTDSIKIENEEEILRSLALGN

KKLLPVFVGSGPPSKRVHREDAIIEQSDESIVSDGFVIYKGSLLDVEKL

VSQLKRSEKARLDTEERLMELQHELCIVNEKSTKQTNNIKALSEDLK

VYKDKLRNTDEKLKKVSSECHTYLTAVKNMYHIAAKMMQSDTKK

VEVVEIQDEKVSEVNGSEIETKFKMDSRWGDNKVPIKKEFTETDKDK

KCDNKVSIKKEIIETDKEKK

17
CARP-1:
YHRPEETHKGRTVPAHVETVVLFFPDVWHCLPTRSEWETLSRGYKQ

550-600
QLVEK

18
CARP-1:
MASPSMEDLYHKSCALAEDPQELRDGFQHPARLVKFLVGMKGKDE

452-654
AMAIGGHWSPSLDGPDPEKDPSVLIKTAIRCCKALTGIDLSVCTQWY

RFAEIRYHRPEETHKGRTVPAHVETVVLFFPDVWHCLPTRSEWETLS

RGYKQQLVEKLQGERKEADGEQDEEEKDDGEAKEISTPTHWSKLDP

KTMKVNDLRKELESRALS

19
NEMO:
SEEKRKLAQLQVAYHQLFQEYDNHIKSSVVGSERKRGMQLEDLKQQ

221-405
LQQAEEALVAKQEVIDKLKEEAEQHKIVMETVPVLKAQADIYKADF

QAERQAREKLAEKKELLQEQLEQLQREYSKLKASCQESARIEDMRK

RHVEVSQAPLPPAPAYLSSPLALPSQRRSPPEEPPDFCCPKCQYQAP

20
CARP-1:
IKTAIRCCKALTGIDLSVCTQWYRFAEIRYHRPEETHKGRTVPAHV

521-566

21
CARP-1:
KLQGERKEADGEQDEEEKDDGEAKEISTPTHWSKLDPKTMKVND

600-650
LRKELE

22
CARP-1:
RPEETHKGRTVPAHVETVVLFFPDVWHCL

552-580

23
NEMO:
SEEKRKLAQLQVAYHQLFQEYDNHIKSSVVGSERKRGM

221-258

24
CARP-1:
HRPEETHKGRTVPAHVETVVLFFPDVWHCLPTRSEWETLSRGYK

551-599
QQLVE

25
NEMO:
RKRG

254-257

26
NEMO:
RKRH

357-360

27
CARP-1:
MAQFGGQKNPPWATQFTATAVSQPAALGVQQPSLLGASPTIYTQQ

1-198
TALAAAGLTTQTPANYQLTQTAALQQQAAAAAAALQQQYSQPQQ

ALYSVQQQLQQPQQTLLTQPAVALPTSLSLSTPQPTAQITVSYPTPR

SSQQQTQPQKQRVFTGVVTKLHDTFGFVDEDVFFQLSAVKGKTPQ

VGDRVLVEATYNPNMPF

28
CARP-1:
PFKWNAQRIQTLPNQNQSQTQPLLKTPPAVLQPIAPQTTFGVQTQPQ

197-454
PQSLLQAQISAASITPLLQTQPQPLLQQPQQKAGLLQPPVRIVSQPQP

ARRLDPPSRFSGRNDRGDQVPNRKDDRSRERERERRRSRERSPQRK

RSRERSPRRERERSPRRVRRVVPRYTVQFSKFSLDCPSCDMMELRR

RYQNLYIPSDFFDAQFTWVDAFPLSRPFQLGNYCNFYVMHREVES

LEKNMAILDPPDADHLYSAKVMLMAS

29
CARP-1:
GERKEADGEQDEEEKDDGEAKEISTPTHWSKLDPKTMKVNDLRK

603-898
ELESRALSSKGLKSQLIARLTKQLKVEEQKEEQKELEKSEKEEDED

DDRKSEDDKEEEERKRQEEIERQRRERRYILPDEPAIIVHPNWAAK

SGKFDCSIMSLSVLLDYRLEDNKEHSFEVSLFAELFNEMLQRDFG

VRIYKSLLSLPEKEDKKEKDKKSKKDERKDKKEERDDETDEPKP

KRRKSGDDKDKKEDRDERKKEDKRKGDSKDDDETEEDNNQDE

YDPMEAEEAEDEEDDRDEEEMTKRDDKRD

30
CARP-1:
KRDINRYCKERPSKDKEKEKTQMITINRDLLMAFVYFDQSHCGYL

896-1150
LEKDLEEILYTLGLHLSRAQVKKLLNKVVLRESCFYRKLTDTSKD

EENHEESESLQEDMLGNRLLLPTPTVKQESKDVEENVGLIVYNGA

MVDVGSLLQKLEKSEKVRAEVEQKLQLLEEKTDEDEKTILNLENS

NKSLSGELREVKKDLSQLQENLKISENMSLQFENQMNKTIRNLST

VMDEIHTVLKKDNVKNEDKDQKSKENGASV

31
CARP-1:
MASPSMEDLYHKSCALAEDPQELRDGFQHPARLVKFLVGMKGK

452-625
DEAMAIGGHWSPSLDGPDPEKDPSVLIKTAIRCCKALTGIDLSVC

TQWYRFAEIRYHRPEETHKGRTVPAHVETVVLFFPDVWHCLPT

RSEWETLSRGYKQQLVEKLQGERKEADGEQDEEEKDDGEAKEI

32
CARP-1:
MASPSMEDLYHKSCALAEDPQELRDGFQHPARLVKFLVGMKGK

452-610
DEAMAIGGHWSPSLDGPDPEKDPSVLIKTAIRCCKALTGIDLSVC

TQWYRFAEIRYHRPEETHKGRTVPAHVETVVLFFPDVWHCLPTR

SEWETLSRGYKQQLVEKLQGERKEADG

33
CARP-1:
MASPSMEDLYHKSCALAEDPQELRDGFQHPARLVKFLVGMK

452-552
GKDEAMAIGGHWSPSLDGPDPEKDPSVLIKTAIRCCKALTGID

LSVCTQWYRFAEIRYHR

34
CARP-1:
RPEETHKGRTVPAHVETVVLFFPDVWHCLPTRSEWETLSRGY

552-640
KQQLVEKLQGERKEADGEQDEEEKDDGEAKEISTPTHWSKL

DPKTMK

35
CARP-1:
RPEETHKGRTVPAHVETVVLFFPDVWHCLPTRSEWETLSRGY

552-625
KQQLVEKLQGERKEADGEQDEEEKDDGEAKEI

36
CARP-1:
RPEETHKGRTVPAHVETVVLFFPDVWHCLPTRSEWETLSRGYK

552-610
QQLVEKLQGERKEADG

37
CARP-1:
LFFPDVWHCLPTRSEWETLSRGYKQQLVEK

571-600

38
CARP-1:
RGYKQQLVEKLQGERKEADGEQDEEEKDDG

591-620

39
NEMO:
SEEKRKLAQLQVAYHQLFQEYDNHIKSSVVGSERKRGMQLED

221-317
LKQQLQQAEEALVAKQEVIDKLKEEAEQHKIVMETVPVLKAQ

ADIYKADFQAERQ

40
NEMO:
ETVPVLKAQADIYKADFQAERQAREKLAEKKELLQEQLEQLQ

296-419
REYSKLKASCQESARIEDMRKRHVEVSQAPLPPAPAYLSSPLAL

PSQRRSPPEEPPDFCCPKCQYQAPDMDTLQIHVMECIE

41
CARP-1:
EQDEEEKDDGEAKEISTPTHWSKLDPKTMK

611-640

42
CARP-1:
WSKLDPKTMKVNDLRKELESRALSSKGLKS

631-660

43
NEMO:
MNRHLWKSQLCEMVQPSGGPAADQDVLGEESPLGKPAMLHLPSE

human
QGAPETLQRCLEENQELRDAIRQSNQILRERCEELLHFQASQREEKE

FLMCKFQEARKLVERLGLEKLDLKRQKEQALREVEHLKRCQQQM

AEDKASVKAQVTSLLGELQESQSRLEAATKECQALEGRARAASEQ

ARQLESEREALQQQHSVQVDQLRMQGQSVEAALRMERQAASEEK

RKLAQLQVAYHQLFQEYDNHIKSSVVGSERKRGMQLEDLKQQLQ

QAEEALVAKQEVIDKLKEEAEQHKIVMETVPVLKAQADIYKADFQ

AERQAREKLAEKKELLQEQLEQLQREYSKLKASCQESARIEDMRK

RHVEVSQAPLPPAPAYLSSPLALPSQRRSPPEEPPDFCCPKCQYQAP

DMDTLQIHVMECIE

Compounds

In some aspect, the compounds disclosed herein can be CARP-1-NEMO inhibitors. In some aspects, the invention relates to compounds useful in treating disorders associated with CARP-1 signaling including, but not limited to, cancer. In some aspects, the compounds described herein are useful in inhibiting cell cycle progression, cell growth, DNA repair, enhancing a chemotherapeutic response in a subject, reducing chemotherapeutic toxicity in a subject, reducing or preventing chemotherapeutic resistance in a cancer cell, inhibiting binding of NF-κBf activating kinase TKK subunit γ (NEMO) to cell cycle and apoptosis regulatory protein (CARP)-1, reducing systemic levels of one or more cytokines in a subject, and enhancing the efficacy of radiotherapy and/or a chemotherapeutic agent.

Disclosed herein are compounds for administering to a subject. Disclosed herein are compounds for treating a subject with a cancer. Disclosed herein are also compounds that can be useful for inhibiting cell cycle progression, cell growth or DNA repair. The compounds disclosed herein can also be useful for enhancing a chemotherapeutic response in a subject. Further, the compounds disclosed herein can be useful for reducing chemotherapeutic toxicity in a subject. The compounds disclosed herein can also be useful reducing or preventing chemotherapeutic resistance in a cancer cell. The compounds disclosed herein can be useful for inhibiting binding of NF-κB activating kinase TKK subunit γ (NEMO) to cell cycle and apoptosis regulatory protein (CARP)-1. The compounds disclosed herein can be useful for reducing systemic levels of one or more cytokines in a subject. Further, the compounds disclosed herein can be useful for enhancing the efficacy of radiotherapy and/or a chemotherapeutic agent.

In some aspects, the disclosed compounds exhibit chemotherapeutic activity.

In some aspects, the compounds of the invention are useful in inhibiting CARP-1 NEMO in a mammal. In some aspects, the compounds of the invention are useful in inhibiting CARP-1 NEMO in at least one cell.

In some aspects, the compounds of the invention are useful in the treatment of cancer, as further described herein.

It is contemplated that each disclosed derivative can be optionally further substituted.

It is also contemplated that any one or more derivative can be optionally omitted from the invention. It is understood that a disclosed compound can be provided by the disclosed methods. It is also understood that the disclosed compounds can be employed in the disclosed methods of using.

Structure. In some aspects, disclosed are compounds having a structure represented by a formula:

embedded image

wherein Z is selected from —S(O)— and —SO₂—; wherein each of R^1aand R^1bis independently selected from hydrogen and C1-C4 alkyl, or wherein each of R^1aand R^1bare covalently bonded, and, together with the intermediate atoms, comprise a 6-membered heterocycle; or wherein each of R^1aand R^1btogether comprise —CH₂—; and wherein Ar¹is a structure having a formula selected from:

embedded image

wherein R², when present, is C1-C4 alkyl; wherein each of R^3a, R^3b, R^3c, R^3d, and R^3e, when present, is independently selected from hydrogen, halogen, —CN, —NH₂, —OH, —NO₂, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; and wherein each of R^4aand R^4b, when present, is independently selected from hydrogen, halogen, —CN, —NH₂, —OH, —NO₂, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, C1-C4 aminoalkyl, and Ar², provided that at least one of R^4aand R^4b, when present, is not hydrogen; and wherein Ar², when present, is selected from C6 aryl and C3-C5 heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, —CN, —NH₂, —OH, —NO₂, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; or wherein each of R^4aand R^4b, when present, are covalently bonded and, together with the intermediate atoms, comprise a 6-membered aryl substituted with 0, 1, 2, or 3 groups independently selected from halogen, —CN, —NH₂, —OH, —NO₂, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, or a pharmaceutically acceptable salt thereof.

In some aspects, disclosed are compounds having a structure selected from:

embedded image

or a pharmaceutically acceptable salt thereof.

In some aspects, disclosed are compounds having a structure represented by a formula:

embedded image

wherein Z is selected from —S—, —S(O)—, and —SO₂—; wherein each of R^1aand R^1bis independently selected from hydrogen and C1-C4 alkyl, or wherein each of R^1aand R^1bare covalently bonded, and, together with the intermediate atoms, comprise a 6-membered heterocycle; or wherein each of R^1aand R^1btogether comprise —CH₂—; and wherein Ar¹is a 5- to 10-membered heteroaryl substituted with 0, 1, 2, or 3 substituents independently selected from hydrogen, halogen, —CN, —NH₂, —OH, —NO₂, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, C1-C4 aminoalkyl, and Ar²; and wherein Ar², when present, is selected from C6 aryl and C3-C5 heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, —CN, —NH₂, —OH, —NO₂, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, provided that when Ar¹is thiazole, then Ar¹is substituted by at least 1 group, provided that when Ar¹is thiazole and Z is —S—, then each of R^1aand R^1bis hydrogen, provided that when Ar¹is benzo[d]thiazole and Z is —S—, then each of R^1aand R^1bis hydrogen, and provided that when Ar¹is tetrazole, then Ar¹has a structure represented by a formula:

embedded image

wherein R²is C1-C4 alkyl, and Z is —S(O)— or —SO₂—, or a pharmaceutically acceptable salt thereof.