Non-cleaving gasdermin d agonists

Abstract

Disclosed are Gasdermin D (GSDMD) agonists identified via a high-throughput screen. Quinoxaline 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB) was identified as a direct and selective GSDMD agonist, which activates GSDMD pores and pyroptosis without cleaving GSDMD. Also developed were derivatives of DMB, which showed anti-tumor effects, in vitro and in vivo. In mouse tumor models, pulsed and low level pyroptosis induction by DMB suppressed tumor growth without harming GSDMD-expressing immune cells, in an immune-mediated mechanism. Vaccination with DMB-treated cancer cells protected mice from secondary tumor challenge, indicating immunogenic cell death is induced. DMB treatment also synergized with anti-PD-1. Accordingly, the disclosed GSDMD compounds form the basis for a new strategy for tumor immunotherapy.

Description

[0001] NON-CLEAVING GASDERMIN D AGONISTS

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under Grant Numbers All 39914,

[0004] CA240955 and CA255841, awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0005] FIELD OF THE INVENTION

[0006] The present invention is generally in the field of anti-cancer compounds, and more specifically small molecule compounds.

[0007] BACKGROUND OF THE INVENTION

[0008] Recent research identified a pore forming molecule, gasdermin D (GSDMD), as critically important for inflammasome-mediated cell death and cytokine secretion. Certain gasdermins have been suggested to act as tumor suppressors. Gasdermin-mediated inflammatory cell death (pyroptosis) can activate protective immunity in immunologically cold tumors. Gasdermins are activated by cleavage, which causes the N- terminal domain to form membrane pores that kill the cell.

[0009] Immunity against neoplastic cells is critical for surveillance and control of tumor growth and metastasis. Immune protection is mediated by cytotoxic lymphocyte recognition and elimination of tumor cells as “foreign”, which depends on tumor antigens, costimulation, and a danger signal. These three elements are needed to effectively activate antigen-presenting cells that phagocytose dying tumor cells and induce cytotoxic functions and cytokine secretion by cytotoxic lymphocytes and establish long-lived memory.

[0010] How a tumor cell dies shapes the immune response. Immunogenic cell death of tumor cells promotes effective immunity while apoptosis, which can be induced by hypoxia or nutrient deprivation in the tumor environment or chemotherapy, is an immunologically silent form of cell death and sometimes even induces tolerance of tumor antigens. The success of checkpoint blockade (CPB) and chimeric antigen receptor T cell (CAR-T) therapies has demonstrated that activating T cells to recognize cancer cells can powerfully improve cancer outcome and lead to cures. Yet only a subset of cancers responds to immunotherapy. Most solid tumors are not effectively recognized by immune cells and many tumors are devoid of functional infiltrating immune effector cells. CPB and CAR-T therapies can also cause autoimmune side effects and cytokine release syndrome in some patients. Thus, additional therapies are needed to increase antitumor immunity specifically without activating harmful systemic side effects. Inflammatory cell death is emerging as an important immune mechanism that bridges innate and adaptive immunity to stimulate antitumor immunity and potentiate CPB or CAR-T cell therapy. In addition, the clinical activity of some conventional and targeted antineoplastic agents currently used in humans is attributed to their role in re-establishing immune surveillance. These agents often induce inflammatory or immunogenic cell death, which promotes the antitumor functions of TILs that are associated with more favorable therapeutic responses in patients with cancer. Treatment with antineoplastic agents that do not predictably induce inflammatory cell death or antitumor immunity may be more prone to developing tumor resistance, relapse and metastatic disease.

[0011] Pyroptosis is an immunogenic and inflammatory cell death mediated by the gasdermin family of pore-forming proteins. In myeloid cells and epithelial mucosa, gasdermin D (GSDMD) can be cleaved by inflammatory caspases downstream of pathogen or damage-induced inflammasome activation. The established paradigm is that cleavage releases autoinhibition of the C-terminal (CT) fragment and allows the toxic N-terminal fragment (NT) to form pores in cell membranes, which induce inflammatory cell death to release cytosolic lactate dehydrogenase (LDH) and damage-associated molecules, such as ATP.

[0012] Other signaling pathways also cleave and activate gasdermins. GSDMD is activated by caspase- 8 in the TNF-a receptor pathway and by neutrophil granule enzymes such as the ELANE neutrophil elastase, gasdermin B (GSDMB) by granzyme A (GzmA) from cytotoxic lymphocytes, gasdermin C (GSDMC) by caspase-8, and gasdermin E (GSDME, also known as DFNB59) by granzyme B (GzmB) from cytotoxic lymphocytes and by the apoptotic caspase-3. Gasdermin A (GSDMA) was recently shown to be cleaved by the streptococcal pyrogenic exotoxin B.

[0013] Several lines of evidence suggest that gasdermin activation in cancer cells could in principle boost antitumor immunity. First, the importance of gasdermins in antitumor immunity is highlighted by the tumor suppressor function of GSDME, which is frequently silenced or mutated in cancers. Exogenous GSDME expression in tumors enhances the function of tumorinfiltrating killer cells, likely due to GSDME cleavage and activation by cytotoxic T lymphocyte (CTL) -delivered GzmB, which triggers tumor cell pyroptosis. Second, CTL-delivered GzmA cleaves GSDMB to cause pyroptosis in GSDMB-expressing tumors and promote antitumor immunity. Third, nanoparticle delivery and activation of mouse GSDMA3-NT in cancer cells heightens antitumor immunity.

[0014] Protein expression profiling of gasdermin family members shows that GSDMA, C and E are poorly expressed in most tumors, while GSDMD and GSDMB are often expressed. GSDMB expression is associated with poor prognosis, possibly due to its additional transcriptional activity or expression of dominant negative inhibitory splice variants in tumors.

[0015] While these data support a beneficial role of gasdermin activation in antitumor immunity, it is not known how one can activate an endogenous gasdermin in cancer cells. In particular, the requirement for enzymatic cleavage makes gasdermin activation difficult to achieve without protease delivery.

[0016] It is therefore an object of the present invention to provide compounds that can activate gasdermin in treatment of tumors.

[0017] SUMMARY OF THE INVENTION

[0018] Gasdermin-mediated inflammatory cell death (pyroptosis) can activate protective immunity in immunologically cold tumors. Gasdermins are activated by cleavage, which causes the N-terminal domain to form membrane pores that kill the cell. A high-throughput drug screen was used to identify compounds that could activate gasdermin D (GSDMD), which is expressed widely in some tumors. Quinoxaline 6,7-dichloro-2-methylsulfonyl-3-N-tert- butylaminoquinoxaline (DMB) was identified as a direct and selective GSDMD agonist. Surprisingly, DMB activates GSDMD pores and pyroptosis without cleaving GSDMD. In mouse tumor models, pulsed and low level pyroptosis induction by DMB suppresses tumor growth without harming GSDMD-expressing immune cells. Protection is immune-mediated since it is abrogated in mice lacking lymphocytes. Vaccination with DMB-treated cancer cells protects mice from secondary tumor challenge, indicating immunogenic cell death is induced. DMB treatment also synergizes with anti-PD-1. In animal model studies, DMB treatment does not increase circulating proinflammatory cytokines or leukocyte numbers, or cause weight loss. Accordingly, DMB forms the basis for a new strategy for tumor immunotherapy in which a small molecule-induced low level of tumor cell pyroptosis is sufficient to stimulate antitumor immunity, without causing overt toxicity.

[0019] Structural characterizations of full-length GSDMD suggest that mutations disrupting interactions of the NT and CT can lead to GSDMD constitutive activation without cleavage, by forming membrane-permeabilizing pores. A chemical biology approach was used to identify activators to release the autoinhibition of an endogenous gasdermin in tumors without the need for cleavage. A number of small molecules that activate GSDMD pore formation in vitro and in cells, were identified using a biochemical high-throughput screening assay. These small molecules, some of which are active at nanomolar concentrations in vitro, are useful to treat cancers. The use of these small molecules in cells and a mouse cancer models in inducing pyroptosis and reducing cancer size has been demonstrated. These results provide a means to use gasdermin D as a target for cancer treatment.

[0020] Mass spectrometry analysis revealed that DMB activates full-length GSDMD pore formation without cleavage by selectively and covalently modifying a cysteine residue (Cysl91) of GSDMD.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGs. 1A-1F show high-throughput screening for small-molecule GSDMD agonists. FIG. 1A is a GSDMD-induced liposome leakage assay using time-resolved Terbium (Tb3+) / dipicolinic acid (DPA) fluorescence. FIG. IB shows the percentage activation of liposome leakage by screened compounds, assayed at 25 pg / mL (~50 pM for most compounds). Cutoff was 50% activation relative to detergent. FIG. 1C panels show the 24 screening hits after excluding pan-assay-interference compounds, liposome disruptors, auto-fluorescent compounds and those without saturable EC so curves. ECsos of liposome leakage, GSDMD binding dissociation constants (KD) by microscale thermophoresis (MST) and cell death activity are shown. N.D.: not detected. The selected hit C-185 is labeled in red. FIG. ID is chemical structure of compound C-185, also known as 6,7-dichloro-2-methylsulfonyl-3-N-tert- butylaminoquinoxaline (DMB). FIG. IE is a dose response curve with ECso of DMB in the liposome leakage assay. FIG. IF is the binding curve for Alexa 647-labeled GSDMD with DMB by MST.

[0023] FIGs. 2A-2K show that DMB induces pyroptotic cell death in a GSDMD-dependent manner. FIG. 2A is a time course of PI positivity in WT and GSDMD KO THP- 1 cells after treatment with DMSO, DMB (5 and 20 pM) or LPS + nigericin. FIG. 2B shows ECso determination for WT THP- 1 cells using PI positivity at 4 h after treatment with different concentrations of DMB (red) or DMSO (blue). FIG. 2C shows quantification of PI uptake 1, 2 and 4 h after treatment with DMSO or DMB (5 and 20 pM) in GSDMD KO THP-1 cells, GSDMD KO THP-1 cells reconstituted with WT GSDMD or GSDMD KO THP-1 cells reconstituted with D275A uncleavable GSDMD mutant. FIGs. 2D and 2E show time course of LDH release (FIG. 2D) and extracellular ATP measured by a luciferase-based assay (FIG. 2E) after treatment of WT and GSDMD KO THP-1 cells with DMSO, DMB (5 and 20 pM) or LPS + nigericin. Note discontinuous x-axis in (FIG. 2D). FIGs. 2F and 2G show quantification of PI uptake (FIG. 2F) and LDH release (FIG. 2G) after treatment with DMSO, DMB (5 and 20 pM) or LPS + nigericin, with or without pretreatment with DSF in human PBMCs. FIGs. 2H and 21 show quantification of PI uptake (FIG. 2H) and LDH release (FIG. 21) after treatment with DMSO, DMB (5 and 20 pM) or LPS + nigericin in WT and GSDMD KO primary BMDMs. FIGs. 2J and 2K show quantification of PI uptake (FIG. 2J) and LDH release (FIG. 2K) after treatment of WT and caspase- 1 / 11 KO iBMDMs with DMSO or DMB (5 and 20 pM). Error bars represent s.e.m. of 3 independent experiments. Statistics were measured by Student’s t-tests (FIGs. 2C, 2F, 2H, and 2J) or two-way ANOVA (FIGs. 2A, 2D, 2G, 21, and 2K). NS, not significant; ****, p<0.0001. See also FIGs. 8A-8K.

[0024] FIGs. 3A-3J show cleavage-independent and selective activation of GSDMD pore formation by DMB. FIG. 3A shows the chemical structure of biotinylated DMB (left) and biotinylated DMB pulldown of GSDMD in THP-1 whole cell lysates (WCL), which was markedly reduced by excess DMB. The immunoblot used anti-GSDMD antibody NBP2-33422 (Novus Biologicals). Asterisk (*) indicates a non-specific band. Lysate loaded was 5% total and each lane is 15% total. FIG. 3B shows negative staining EM images of liposomes incubated with DMB or GSDMD alone, cleaved GSDMD, or GSDMD plus DMB upon liposome reconstitution and detergent solubilization. Scale bar, 100 nm. Arrowheads point to DMB- induced GSDMD pores. Cleaved GSDMD and DMB+GSDMD bottom left insets show enlarged images of the boxed areas. FIG. 3C presents data from liposome leakage assay showing that DMB activates recombinant GSDMD similarly as cleavage. The NT-CT linker contained an engineered 3C protease cleavage site. FIG. 3D shows results from BRET assay using NT-fused YFP and CT-fused luciferase showing that DMB treatment reduced the intramolecular BRET ratio relative to DMSO treatment, suggesting that DMB increased the distance between the GSDMD NT and CT.FIG. 3E is a nano-LC / MS / MS spectrum of the Cys 191 -containing human GSDMD peptide (aa 184-203; 2057.00 Da) modified on Cysl91 by carbamidomethyl (an increase of 57.0214 Da). A triplet charged precursor ion m / z 705.6812 (mass: 2114.0435 Da) was observed. FIG. 3F shows a mass spectrum of the corresponding GSDMD peptide after GSDMD incubation with DMB, which was modified on Cysl91 by the quinoxaline moiety of DMB (an increase of 267.0330 Da). A triplet charged precursor ion m / z 775.6767 (mass: 2324.0591 Da) was observed. FIG. 3G shows DMB dose-response curves and EC so of liposome leakage after incubation with human GSDMA, GSDMB, GSDMC, GSDMD or GSDME (0.3 pM). FIG. 3H shows PI uptake in HEK293T cells transfected with WT and Cysl91 mutants of GSDMD-NT. C191F and C191R GSDMD-NT induced comparable cell death as WT GSDMD-NT. FIG. 31 shows LDH release in HEK293T cells transfected with full- length GSDMD and treated with DMB (right column) or DMSO (left column). WT, the D275A mutant that cannot be cleaved by inflammatory caspases, and the C268G / D275A double mutant that also cannot be cleaved by ELANE were activated by DMB. By contrast, the C191R and C191F mutants were resistant to DMB activation. FIG. 3J shows leakage of liposomes induced by adding DMB to full-length C191 A, C191R, C191F or WT GSDMD. WT GSDMD was also pretreated with DSF before DMB treatment. Only WT GSDMD that was not pretreated with DSF could be activated by DMB. Data represent mean ± s.e.m. of 3 independent experiments performed in triplicate. Statistics were measured by Student’s t-tests (H). NS, not significant; **, pcO.Ol, ***, pcO.OOl, ****, pcO.OOOl. See also FIGs. 9A-9H and 10A-10E.

[0025] FIGs. 4A-4G show GSDMD-dependent pyroptosis and DAMP release in mouse tumor cells. FIG. 4A shows phase contrast, SYTOX Green staining, and merged images of WT (left) and GSDMD KO clone 10 (right) EMT6 cells treated for 2 h with DMSO, DMB (5, 20 pM) or mitomycin C (MMC), an apoptosis inducer. Squares in the left column of each panel indicate zoomed-in regions. Arrows in left column in each panel point to pyroptotic bubbles. Scale bar: 20 pm. Blobs in the middle and right columns in each panel denote SYTOX green stain. FIG. 4B shows quantification of % of cells that took up SYTOX Green 1, 2 and 4 h after treatment of WT and GSDMD KO clone 10 EMT6 cells with DMSO, DMB (5 and 20 pM) or MMC. FIGs. 4C and 4D show time course of LDH release (FIG. 4C) and ATP release (FIG. 4D) after treatment of WT and GSDMD KO EMT6 clone 10 cells with DMSO, DMB (5 and 20 pM) or MMC. FIG. 4E shows phase contrast, SYTOX Green staining, and merged images of CT26 cells treated for 2 h with DMSO, DMB (5, 20 pM) or mitomycin C (MMC). Squares in the left column of each panel indicate zoomed-in regions. Arrows in left column in each panel point to pyroptotic bubbles. Scale bar: 20 pm. Blobs in the middle and right columns in each panel denote SYTOX green stain. FIG. 4F shows ATP release over time from CT26 cells after treatment with DMSO, MMC, or DMB. Note discontinuous x-axis.

[0026] FIG. 4G shows % SYTOX Green uptake positivity in DMB-treated EMT6 cells that were pretreated or not with the inflammatory caspase inhibitor AC-FLTD-CMK. Error bars represent s.e.m. of 3 independent experiments. Statistics were measured by Student’s t-tests (FIGs. 4B and 4G) or two-way ANOVA (FIG. 4C). NS, not significant; *, p<0.05; **, p<0.01; ***, pc0.001 ; ****, pcO.OOOl. See also FIGs. 11A-11J.

[0027] FIGs. 5A-5J demonstrate that DMB induces antitumor activity that depends on tumor cell GSDMD expression. FIGs. 5A-5D show results for mice bearing orthotopic WT (left), Gsdmd' ' (middle), or Gsdmd' / 'Gsdme / ' (right) EMT6 tumors treated with vehicle (left column) or DMB (right column; 10 mg / kg) every week starting when tumors became palpable and analyzed for tumor volume (FIG. 5A), percentage of CD8+TILs expressing GzmB or PFN (FIG. SB), percentage of CD8+TILs expressing IFN-y or TNF-a after PMA and ionomycin stimulation ex vivo (FIG. SC), and percentage of NK TILs with GzmB and PFN expression (FIG. 5D). n=5 mice / group. FIGs. 5E-5F show results for mice bearing subcutaneous CT26 tumors treated with vehicle (left column) or DMB (right column; 10 mg / kg) and analyzed for tumor volume (FIG. 5E) and percentage of CD8+or NK TILs expressing GzmB or PFN and percentage of CD8+TILs expressing IFN-y or TNF-a after PMA and ionomycin stimulation ex vivo (FIG. 5F). n=6 mice / group. FIGs. 5G-5J show results for subcutaneously implanted KP tumor cells in WT or Gsdmd ^ mice treated with vehicle or DMB and analyzed for tumor volume (FIGs. 5G and 5H), and percentages of CD8+(FIG. 51) and NK TILs (FIG. 5J) expressing GzmB or PFN or expressing IFN-y or TNF-a after PMA and ionomycin stimulation ex vivo. In FIGs. 5F, 51, and 5J data for the vehicle and DMB are shown in the left and right column, respectively. WT mice, vehicle or DMB treatment, n=7 mice / group; Gsdmd^ mice, vehicle treatment, n=5 mice / group or DMB treatment, n=6 mice / group. All data are represented as Mean ± s.e.m. For tumor volume analysis, the area under the tumor growth curves were compared. Two-tailed Student’s t-tests were used to determine differences between two groups. *, p < 0.05; **,p < 0.01; ***, p < 0.001. See also FIGs. 12A-12F.

[0028] FIGs. 6A-6I show that DMB induces immunogenic cell death and its antitumor effect depends on the immune response. FIG. 6A shows an experimental scheme to analyze DMB treatment of EMT6 tumors in WT or NSG mice. FIG. 6B shows that DMB did not affect EMT6 tumor growth in NSG mice. FIG. 6C shows that EMT6 tumor cells (CD45" CD3 ) showed increased PI uptake in DMB-treated WT mice compared to vehicle- treated WT mice (left), but DMB did not significantly change PI uptake in CD45+CD1 lb+F4 / 80+TAMs (right). FIG. 6D shows that DMB treatment did not increase PI uptake of EMT6 tumor cells (CD45- CD3 ) in NSG mice. FIG. 6E is a schematic of a vaccination experiment using MMC- or DMB-treated EMT6 tumor cells as immunogens. BALB / c mice were vaccinated in the left flank with MMC- or DMB-treated EMT6 cells and challenged 8 days later by injecting untreated EMT6 cells in the right mammary fat pad. MMC treatment, n=10 mice / group; DMB treatment, n=12 mice / group. FIG. 6F shows average tumor volumes (left), and individual tumor growth kinetics for MMC (middle) and DMB (right) groups. FIGs. 6G-6I show percentages of CD8+(FIG. 6G), NK (FIG. 6H), and CD4+(FIG. 61) TILs expressing GzmB or PFN and producing IFN-y or TNF-a after PMA and ionomycin activation ex vivo. All data are represented as mean ± s.e.m. For tumor volume analysis, the areas under the tumor growth curves were compared. Two-tailed Student’s t-tests were used to determine differences between two groups. *, p < 0.05; ****, p < 0.0001. See also FIGs. 13A-13G.

[0029] FIGs. 7A-7H show that DMB inhibits growth of B16 tumors expressing human GSDMD. FIG. 7A shows a design of the human GSDMD-GFP fusion protein ectopically expressed in B16 cells that do not express endogenous GSDMD. Black lines mark the regions of the protein recognized by the anti-GSDMD (ZRB1274, Sigma- Aldrich) and anti-GFP (2956, Cell Signaling Technology) antibodies used in (FIG. 7C). GFP and GSDMD-GFP were expressed in B16 cells to generate B l 6-GFP and B16-GSDMD-GFP cells, respectively. FIG. 7B shows time-lapse images of Bl 6-GFP and B16-GSDMD-GFP cells treated with 10 pM DMB showing morphological changes by brightfield and PI uptake as an indicator of pyroptosis. FIG. 7C shows an immunoblot of Bl 6-GFP or B16-GSDMD-GFP incubated with 10 pM DMB for indicated times. Anti-GAPDH (60004- 1-Ig, Proteintech) is a loading control. GSDMD-GFP was not cleaved by DMB. FIG. 7D shows an experimental scheme for investigating the effect of DMB treatment on Bl 6 tumors expressing human GSDMD-GFP or GFP. B16 clones were implanted subcutaneously (sc) on day 0 and mice were treated ip every 3 days for 6 injections (rarrows) with 10 mg / kg DMB or vehicle beginning 5 days later when all mice had palpable tumors. Fig. 7E shows the growth of B16-GSDMD-GFP tumors (clone 9, left; clone 10, right) after treatment with DMB or vehicle (n = 8 mice / group). Tumor growth curves show mean ± s.e.m. for each timepoint and statistical analysis was performed by two-tailed Student’s t-test comparing the area under the tumor growth curves. FIG. 7F shows the Kaplan-Meier survival curve of mice bearing B16-GSDMD-GFP tumors treated with DMB or vehicle (n = 16 mice / treatment group, combining clone 9 and 10). Survival was analyzed by log-rank test. 5 of the 16 mice survived in the DMB-treated group. FIG. 7G shows an immunofluorescence microscopy analysis of infiltrating CD3+CD8+T cells and MHCII+CDl lc+dendritic cells in B16-GSDMD-GFP tumors after treatment with DMB or vehicle (n = 3 mice). Representative images are shown on the left. Quantifications are shown in the graph, for mean + s.e.m. based on n = 12 images from 3 tumors for each group. In the graph, vehicle: left column; DMB: right column). Anti-CD3, anti-CD8, anti-MHCII and CDl lc antibodies were 100235, 100728, 107619 and 117309 from BioLegend. FIG. 7H shows the response of B 16-GSDMD-GFP tumors according to tumor size at the time of the first DMB treatment, plotted against the tumor size 3 days after the last DMB treatment. Two sets of data were included, mice treated 5 days after implantation shown in (FIGs. 7D, 7E) and mice treated 10 days after implantation when the tumors were larger. Mice in the first and second groups that survived for the duration of the experiment are indicated by red and pink dots, respectively, and those that died or had to be sacrificed are indicated by black dots are indicated by black and gray dots, respectively. Simple linear regression was used to model the relationship between tumor volumes at initiation and after all DMB treatments. A Pearson’s r value was used to assess correlation, and the statistical significance of the respective linear regression slope (Wald test) is shown. See also FIG. 13A- 13G. FIGs. 8A-8K show that DMB induces pyroptotic cell death in a GSDMD-dependent manner (related to FIGs. 2A-2K). FIG. 8A shows microscale thermophoresis (MST) for DMB binding to GSDMD. Curves shown are fluorescence decay (Fhot / Fcoid) over time for a series of DMB concentrations against a low concentration of labeled GSDMD (50 nM) when subjected to a very precise and brief laser-induced temperature change at time 0. Each curve corresponds to a ligand concentration. FIG. 8B shows PI uptake (red) by WT and GSDMD KO THP- 1 cells after treatment with DMSO, DMB (5 and 20 pM) or LPS + nigericin for 2 h. Scale bar: 15 pm. FIG. 8C shows anti-NINJl (AF51O5-SP, R&D Systems) immunoblots of THP- 1 supernatant and cell lysate treated with LPS plus nigericin (N) or DMB on non-reducing SDS-PAGE. G indicates reactions performed in the presence of glycine, which inhibited NINJ1 activation. Asterisk (*) symbols indicate non-specific bands. FIG. 8D shows cell death shown by SYTOX Green uptake (left), inflammasome formation shown by ASC speck formation (middle), and effector activation shown by IL-ip release (right). LPS electroporation (electro), but not LPS priming + DMB treatment, induced ASC speck formation and IL-ip release in THP-1 cells. FIG. 8E shows PI uptake (red) by human PBMCs after treatment with DMSO, DMB (5 and 20 pM) or LPS + nigericin (Nig.) for 2 h, with or without pretreatment by the GSDMD inhibitor disulfiram (DSF). Scale bar: 15 pm. FIG. 8F shows time course of extracellular ATP measured by a luciferase-based assay in PBMCs treated with DMSO, DMB (5 and 20 pM) or LPS + nigericin, with or without pretreatment by DSF. FIG. 8G shows PI uptake (red) by WT and GSDMD KO primary mouse bone marrow derived macrophages (BMDMs) after treatment with DMSO, DMB (5 and 20 pM) or LPS + nigericin (Nig.) for 2 h. Scale bar: 15 pm. FIG. 8H shows the time course of extracellular ATP measured by a luciferase-based assay in WT and GSDMD KO primary mouse BMDMs treated with DMSO, DMB (5 and 20 pM) or LPS + nigericin. FIG. 81 shows PI uptake (red) by WT and Caspase- 1 / 11 KO mouse immortalized bone marrow derived monocytes (iBMDMs) treated with DMSO or DMB (5 and 20 pM) for 2 h. Scale bars: 15 pm. FIG. 8J shows the time course of extracellular ATP measured by luciferase-based assay in WT and Caspase- 1 / 11 KO iBMDMs treated with DMSO or DMB (5 and 20 pM) for 2 h. FIG. 8K shows that DMB (10 pM) does not inhibit caspase- 1 (0.5 pM) activity against its fluorogenic substrate Z-YVAD-AFC (10 pM), shown by time-resolved fluorescence intensity (delay 50 ps, interval 950 ps) of each well measured at 550 nm with an excitation of 400 nm. Reaction buffer contained 40 mM HEPES, pH 7.4, 150 mM NaCl. RFU: relative fluorescence unit. Error bars represent s.e.m. of 3 independent experiments. NS, not significant; ****, p<0.0001.

[0030] FIGs. 9A-9H shows DMB -biotin and modification of Cysl91 (Cysl92) of human (mouse) GSDMD by DMB. FIG. 9A shows GSDMD-mediated liposome leakage assay induced by DMB-biotin, showing an EC50 similar to that of DMB. FIG. 9B shows the molecular structure of another DMB-biotin conjugate (left) and its reduced induction of GSDMD-mediated liposome leakage (right). FIGs. 9C and 9D shows nano-LC-MS / MS spectra for the peptide containing Cysl91 in human GSDMD after DMB treatment, FSLPGATCLQGEGQGHLSQK modified on cysteine by carbamidomethyl (control) (FIG. 9C) and FSLPGATCLQGEGQGHLSQK modified on cysteine by DMB (FIG. 9D). From a calculation based on the abundance values, 64.3% of Cysl91 in human GSDMD was modified by DMB, while only 0.7%, 4.0%, 2.8%, 0.2%, and 0.03% of C56, C268, C309, C445 and C467 residues, respectively, were modified. Modifications of other Cys residues were not observed. FIG. 9E shows a sequence alignment of the NTs of human GSDMD (hGSDMD) and mouse GSDMD (mGSDMD) highlighting the Cys residues (red). FIG. 9F shows liposome leakage assay measuring activity of DMB-treated mouse GSDMD and GSDME (mGSDMD and mGSDME) in comparison to human GSDMD (hGSDMD). The hGSDMD data are the same as in FIG. 3G. FIG. 9G shows a liposome leakage assay of WT C38A and C 191 A GSDMD activated by DMB. FIG. 9H shows PI uptake (red) of HEK293T cells expressing WT and mutant GSDMD-NT. BF, brightfield image. Scale bar: 200 pm.

[0031] FIGs. 10A-10E shows structure-activity relationship analysis of DMB and some DMB derivatives. FIG. 10A shows the structure of DMB with core positions numbered. FIG. 10B shows a schematic of a library design with varied positions labeled as Ri, R2, and R3. FIG. 10C shows non-limiting chemical groups substituted for each library. FIGs. 10D and 10E show GSDMD-induced liposome leakage by DMB derivatives, which were generated by changing the methylsulfonyl moiety (FIG. 10D) or the substitution at carbon positions 6 and 7 (FIG. 10E), along with molecular structure and EC50 (bottom right panels for both FIGs. 10D and 10E.

[0032] FIGs. 11A-11J show data for WT, GSDMD KO and GSDMD / GSDME dKO EMT6 lines and the CT26 line. FIGs. 11A and 11B are Western blots of GSDMD (ab209845, abeam) and / or GSDME (ab215191, abeam) expression in Cas9-positive GSDMD KO and GSDMD and GSDME double KO (dKO) clones. Actin (sc-47778, Santa Cruz Biotechnology) served as the loading control. FIG. 11C shows a luciferase-based cell viability assay in WT EMT6 cells after incubation with indicated concentrations of DMB (red) or MMC (blue) for 14 (left), 24 (middle), or 48 (right) h. RLU: relative luminescence unit. FIGs. 11D and HE show quantification of % of cells that took up SYTOX Green 1, 2, and 4 h after DMB (5 and 20 pM) or MMC (30 pM) treatment of WT and GSDMD KO clone 9 (D) and clone 5 (E), MMC-treated cells did not take up SYTOX Green. FIG. HF shows quantification of % of cells that took up SYTOX Green 2 h after DMB (5 and 20 pM) or MMC (30 pM) treatment of WT and GSDMD / GSDME dKO (clones 7, 10, and 12) EMT6 cells. MMC-treated cells did not take up SYTOX Green. FIG. 11G shows LDH release 2 h after treatment of WT and GSDMD / GSDME dKO (clones 7, 10, and 12) EMT6 cells with DMSO, DMB (5 and 20 pM) or MMC (30 pM). FIG. 11H shows quantification of % of CT26 cells that took up SYTOX Green uptake 1, 2, and 4 h after treatment with DMSO, DMB (5 and 20 pM) or MMC (30 pM). FIG. Ill shows LDH release over time from CT26 cells after treatment with DMSO, MMC (30 pM), or DMB (5 and 20 pM). Note discontinuous x-axis. FIG. 11J shows LDH release in DMB-treated WT EMT6 cells that were pretreated or not with the inflammatory caspase inhibitor AC-FLTD-CMK. LDH release was measured 1, 2 and 4 h after adding DMB.

[0033] Error bars represent s.e.m. of 3 independent experiments. Statistics were measured by Student’s t-tests. NS, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0034] FIGs. 12A-12F show that DMB does not significantly alter TIL numbers, blood counts or serum cytokine levels (related to FIGs. 5A-5J and FIGs. 6A-6I). FIG. 12A shows mice bearing orthotopic Gsdmd^ EMT6 tumors treated with vehicle (left column) or DMB (right column, 10 mg / kg) every week starting when tumors became palpable and analyzed for tumor volume. FIG. 12B shows BALB / c mice orthotopically implanted with WT or Gsdmd ' EMT6 tumors were treated with vehicle or DMB. Shown are numbers of CD8+(left), NK (middle), or CD4+(right) TILs. n=5 mice / group. FIG. 12C shows BALB / c mice implanted subcutaneously with CT26 treated with vehicle or DMB. Numbers of CD8+(left), NK (middle), or CD4+(right) TILs were compared. n=6 mice / group. FIG. 12D shows levels of IL- lb, IL- 18, IL-6, or TNF-a in serum were compared in BALB / c mice orthotopically implanted with EMT6 tumors that were treated with vehicle or DMB. n=5 / group. FIG. 12E shows complete blood counts were compared in BALB / c mice orthotopically implanted with EMT6 tumors that were treated with vehicle or DMB. n=5 / group. WBC: white blood cells. FIG. 12F shows WT or Gsdmd ' mice implanted subcutaneously with KP tumors treated with vehicle or DMB. Numbers of CD8+, NK, or CD4+TILs, myeloid-derived suppressor cells (MDSC), and TAMs in tumors were compared. WT mice, vehicle or DMB treatment, n=7 mice / group; Gsdrnd^' mice, vehicle treatment, n=5 mice / group; DMB treatment, n=6 mice / group. For each graph with columns, vehicle = left column; DMB = right column. Error bars represent s.e.m. of 3 independent experiments. Statistics were measured by Student’s t-tests. NS, not significant; *, p<0.05; **, p<0.01 ; ***, pcO.001; ****, p<0.0001.

[0035] FIGs. 13A-13G shows results for in vivo treatment with DMB in B16 tumors (related to FIGs. 7A-7H). FIG. 13A shows DMB effect on tumor volumes in WT mice implanted with EMT6 WT tumors. FIG. 13B shows Gsdmd mRNA in mouse cancer cell lines, assessed by qRT-PCR, relative to Gapdh. FIG. 13C shows GFP expression of untransfected B16 and B16 transfected to ectopically express GFP or human GSDMD-GFP, analyzed by flow cytometry for GFP mean fluorescence intensity (MFI). FIG. 13D shows in vitro PI uptake measured 2 h after adding indicated concentrations of DMB to B16-GFP or B16-GSDMD-GFP cells (pools or indicated clones). DMB EC50 curves were fit based on the percentage of cells that took up PI. FIGs. 13E and 13F show tumor growth (FIG. 13E) and survival (FIG. 13F) curves of B16-GFP tumors treated with DMB (10 mg / kg, n = 4) or vehicle (n = 4). B16-GFP cells were implanted sc on day 0 and mice were treated ip every 3 days for 6 injections with 10 mg / kg DMB or vehicle, starting 5 days post-implantation (red arrow) when all mice had palpable tumors. Growth curves show mean ± s.e.m. at each time point and are representative of two independent experiments. The area under the tumor growth curves was compared by 2-tailed Student’s t-test. Kaplan- Meier survival curve was analyzed by log-rank test (n = 8 / group). FIG. 13G show mouse weight before and after DMB (black) or vehicle (red) treatment (begun on day 5 (arrow). There was no significant difference in weight between mice treated with DMB or vehicle. NS, not significant; *, p<0.05; **, p<0.01 ; ***, p<0.001 ; ****, p<0.0001.

[0036] FIGs. 14A-14F show that DMB used at low dose synergizes with anti-PD-1 to exert potent antitumor activity in 4T1E tumor model (FIGs. 7A-7H). FIG. 14A show orthotopically implanted 4T1E tumors in BALB / c mice treated with vehicle, anti-PD-1 every two days, DMB (1 mg / kg) every week, or anti-PD-1 combined with DMB (1 mg / kg) starting when tumors became palpable and analyzed for tumor volume. n=8 mice / group. FIG. 14B show expression of co-inhibitory molecules PD-1 and CTLA-4 on antigen-experienced CD44+CD8+TILs in each group of tumors on day 21 (end of the study). MFI: mean fluorescence intensity. FIG. 14C show percentages of CD8+TILs expressing GzmB or PFN (left), or IFN-y or TNF-a by PMA and ionomycin stimulation ex vivo (middle), and percentages of NK+TILs expressing GzmB or PFN (right). FIG. 14D show numbers of CD8+, NK, or CD4+TILs in tumors compared between vehicle, anti-PD-1, DMB, or DMB + anti-PD-1 treatments. FIG. 14E shows numbers of myeloid-derived suppressor cells (MDSC), and tumor-associated macrophages (TAMs) in tumors compared between vehicle, anti-PD-1, DMB, or DMB + anti-PD-1 treatments. FIG. 14F shows animal body weight at different time points before and after vehicle, anti-PD-1, DMB, and anti-PD-1 +DMB treatments. All data are represented as mean ± s.e.m. For tumor volume analysis, the area under the tumor growth curves were compared by a two-tailed Student’s t-test. For expression analyses, one- or two-way ANOVA was used to calculate differences among the different groups. * for p < 0.05, ** for p < 0.01, *** for p < 0.001 and **** for p < 0.0001. FIG. 15 shows relative fluorescence unit (RFU) for DMB and derivatives with modification at position 3. Also shown are IC50 values.

[0037] FIGs. 16-20 show microscale thermophoresis (MST) results for the different DMB derivatives portrayed in FIG. 15. Also shown are IC50 (p M) values and MST KD (pM) for each DMB derivative.

[0038] FIG. 21 shows in vivo time vs tumor volume results of DMB also labeled as C-185. The tumor model is orthotopic breast cancer mouse model, and the drug is injected intraperitoneally for systemic administration. The drug works well and does not show side effects on mice. Tumor injection at 0 day, and drug (DMB or C-285) is administered on the first, third, and fifth days.

[0039] FIGs. 22A-22C show in vivo results of a DMB derivative (C185A24) in terms of time vs body weight (FIG. 22A), tumor volume (FIG. 22B), and survival (FIG. 22C).

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] I. DEFINITION

[0042] “Electron withdrawing group,” as used herein refers to a functional group or atom within a molecule that has a tendency to withdraw electron density from the rest of the molecule typically due to its electronegative nature or its ability to attract electrons through resonance or inductive effects. Electron withdrawal can occur through different mechanisms, such as: (1) Electronegativity; (2) Resonance Effects; (3) Inductive Effects. Non-limiting exemplary functional groups include -S(O)2R; -S(O)R; -C(O)OR; -C(O)R’; -C(O)NRIDR2D; hydroxyl; nitrile; an amino group (primary, secondary, or tertiary); nitro (-NO2); haloalkyl; and halogens (e.g. , -F, -Cl, -Br, -I); where R, R’, RID, and R D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted Ce-Cn aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2- C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl.

[0043] “Nanoparticle” refers to any particle having a diameter greater than 1 nm and less than 1000 nm. “Microparticle” refers to any particle having a diameter of at least 1 pm and less than 1000 pm. Nanoparticles and microparticles having a spherical shape are generally referred to as “nanospheres” and “microspheres,” respectively. The term “pharmaceutically acceptable salts” refers to the modification of the original compound by making the acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines and alkali or organic salts of acidic residues such as carboxylic acids. For original compounds containing a basic residue, pharmaceutically acceptable salts can be prepared by treating the compounds with an appropriate amount of a non-toxic inorganic or organic acid. Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acids; suitable organic acids include acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxy maleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, naphthalenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic acids. For original compounds containing an acidic residue, pharmaceutically acceptable salts can be prepared by treating the compounds with an appropriate amount of a non-toxic base. Suitable non-toxic bases include ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine , ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, and histidine. Generally, pharmaceutically acceptable salts can be prepared by reacting the free acid or base form of the original compounds with a stoichiometric amount of the appropriate base or acid, respectively, in water or in an organic solvent, or in a mixture thereof. Non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, acetonitrile, or combinations thereof can be used. Lists of suitable pharmaceutically acceptable salts can be found in Remington’s Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704; Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VCH, Weinheim, 2002, and Kumar, et al., Pharmaceutical Technology-03-02-2008, Volume 32, Issue 3.

[0044] The terms “effective amount” or “therapeutically effective amount” means a dosage sufficient to alleviate one or more symptoms of a disorder, disease, or condition being treated, or to otherwise provide a desired pharmacologic and / or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder being treated, as well as the route of administration and the pharmacokinetics of the agent being administered. The term “prevention” or “preventing” means to administer a composition to a subject or a system at risk for or having a predisposition for one or more symptoms caused by a disease or disorder to cause cessation of a particular symptom of the disease or disorder, a reduction or prevention of one or more symptoms of the disease or disorder, a reduction in the severity of the disease or disorder, the complete ablation of the disease or disorder, stabilization or delay of the development or progression of the disease or disorder.

[0045] “Small molecule” refers to an organic molecule that is less than about 2500 g / mol in molecular weight, less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. The molecular weight is between about 50 g / mol and about 2500 g / mol, between about 50 g / mol and about 2000 g / mol, between about 50 g / mol and about 1500 g / mol, between about 50 g / mol and about 1000 g / mol, between about 50 g / mol and about 800 g / mol, between about 50 g / mol and about 500 g / mol. In some forms, small molecules are non-polymeric and / or non-oligomeric.

[0046] The term “treat” or “treating” refers to administering a composition to a subject having one or more symptoms caused by a disease or disorder to inhibit the disease or disorder, e.g., impeding its progress; and relieving the disease or disorder, e.g., causing regression of the disease or disorder. Treating the disease or disorder includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating the tumor of a subject by administration of a gasdermin D agonist.

[0047] IL GSDMD Agonists

[0048] A small molecule agonist, DMB, that activates cleavage- independent GSDMD pore formation, is a GSDMD agonist that can trigger pyroptotic death of tumor cells and activate antitumor immunity in multiple mouse tumor models, without causing measurable toxicity. GSDMD expression in tumor cells, rather than host cells, is required for this activity, and agonist-treated tumor cells can act as an effective vaccine against secondary tumor challenge. When used at a lower dose (lOx less), DMB synergizes with anti-PD-1 to mount effective antitumor immunity in a tumor model in which DMB or anti-PD- 1 alone was ineffective. These data indicate that small molecule-mediated activation of endogenous GSDMD can act as a switch to mount effective antitumor immunity for treating GSDMD-positive cancers without inducing unnecessary inflammation or other toxicity.

[0049] A high-throughput drug screen to identify compounds that could activate gasdermin D (GSDMD), which is expressed widely in some tumors, and identified quinoxaline 6,7-dichloro- 2-methylsulfonyl-3-N- tert-butylaminoquinoxaline (DMB) as a direct and selective GSDMD agonist. Surprisingly, DMB activates GSDMD pores and pyroptosis without cleaving GSDMD. In mouse tumor models, pulsed and low level pyroptosis induction by DMB suppresses tumor growth without harming GSDMD-expressing immune cells. Protection is immune-mediated since it is abrogated in mice lacking lymphocytes. Vaccination with DMB-treated cancer cells protects mice from secondary tumor challenge, indicating immunogenic cell death is induced. DMB treatment also synergizes with anti-PD- 1.

[0050] DMB treatment does not increase circulating proinflammatory cytokines or leukocyte numbers, or cause weight loss. Thus, the studies reveal a new strategy for tumor immunotherapy in which a small molecule-induced low level of tumor cell pyroptosis is sufficient to stimulate antitumor immunity, and raise the possibility of exploiting pyroptosis without causing overt toxicity. a. Chemical structure

[0051] The compounds described herein have a structure: wherein:

[0052] A and B are independently absent, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of A and B is present, wherein when B is absent, Ra and Rb are bonded to A, or wherein when A is absent, Rc and Rd are bonded to B; preferably both A and B are present; at least one Ra and at least one Rb are independently a first electron withdrawing group and a second electron withdrawing group, respectively, independently selected from -S(O)2R, - S(O)R, -C(O)OR, -C(O)R’, -C(0)NRIDR2D, -OR, nitrile, -NRIDR2D (primary, secondary, or tertiary), nitro, haloalkyl, and halogen (e.g., -F, -Cl, -Br, -I), wherein R, R’, RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2- C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3- C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of Ra and Rb is -S(O)2R;

[0053] Rc and Rd are independently hydrogen, halogen, azide, -S(O)2R, -S(O)R, -C(O)OR, - C(O)R’, -C(0)NRIDR2D, -OR, -SR, hydroxyl, nitrile, -NRIDR2D (primary, secondary, or tertiary), nitro, haloalkyl, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2- C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein R, R’, R ID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of Rc and Rd is a halogen; and m, n, o, and p are independently integers from 1 to 4, such as 1, 2, 3, and 4.

[0054] In some forms, the compounds are as described above, except that A and B are independently substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2- C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl.

[0055] In some forms, the compounds are as described above, except that A and B are independently substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group.

[0056] In some forms, the compounds are as described above, except that A and B are independently substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, wherein one of A and B is substituted C6-C12 aryl group or unsubstituted C6-C12 aryl group, and the other of A and B is substituted C2- C12 heteroaryl group or unsubstituted C2-C12 heteroaryl group. In some forms, the compound is as described above, except that the compound has a structure: wherein: xl, x2, x3, and x4 are independently carbon or nitrogen;

[0057] Ri, R2, R3, and R4 are independently absent, -S(O)rR, -S(O)R, -C(O)OR, -C(O)R’, - C(0)NRIDR2D, -OR, nitrile, -NRIDR2D (primary, secondary, or tertiary), nitro, haloalkyl, and halogen (e.g., -F, -Cl, -Br, -I), wherein R, R’, RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2- C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of Ri, R2, R3, and R4 is -S(O)2R, and the other of Ri, R2, R3, and R4 is -NRIDR2D, wherein Ri, R2, R3, and R4 are present or absent according to valency;

[0058] R5, Re, R7, and Rs are independently hydrogen, halogen, azide, -S(O)2R, -S(O)R, - C(O)OR, -C(O)R’, -C(0)NRIDR2D, -OR, -SR, hydroxyl, nitrile, -NRIDR2D (primary, secondary, or tertiary), nitro, haloalkyl, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein R, R’, RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of R5, Re, R7, and Rs is a halogen.

[0059] In some forms, the compound is as described above for Formula II, except that R, R’, RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of RID, and R2D are independently selected from substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl.

[0060] In some forms, the compound is as described above for Formula II, except that R, R’, RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3- C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of RID, and R2D are independently selected from substituted C1-C5 alkyl group or unsubstituted C1-C5 alkyl group.

[0061] In some forms, the compound is as described above for Formula II, except that R is substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2- C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl. In some forms, the compound is as described above for Formula II, except that RID and RZD are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2- C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2- C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of RID, and R2D are independently selected from substituted C1-C5 alkyl group or unsubstituted C1-C5 alkyl group.

[0062] In some forms, the compound is as described above for Formula II, except that RID is hydrogen and R2D is independently substituted C1-C5 alkyl group or unsubstituted C1-C5 alkyl group.

[0063] In some forms, the compound is as described above for Formula II, except that two of xl, x2, x3, and x4 are nitrogen, and the other of xl, x2, x3, and x4 are carbon. In some forms, the compound is as described above for Formula II, except that x 1 and x4 are nitrogen, and x2 and x3 are carbon.

[0064] In some forms, the compound is as described above for Formula II, except that the compound has a structure:

[0065] Formula III

[0066] In some forms, the compound is as described above for Formula II or Formula III, except that at least one of Re and R7 are a halogen. In some forms, the compound is as described above for Formula II or Formula III, except that Re is a halogen and R7 is hydrogen. In some forms, the compound is as described above for Formula II or Formula Ill, except that Re is hydrogen and R7 is a halogen. In some forms, the compound is as described above for Formula II or Formula III, except that Re and R7 are both halogens that may be the same or different.

[0067] In some forms, the compound is as described above for Formula II or Formula III, except that at least one of R5 and Rs are independently hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, or unsubstituted C2-C5 alkynyl group. In some forms, the compound is as described above for Formula II or Formula III, except that at least one of R5 and Rs are independently hydrogen, substituted C1-C5 alkyl group, or unsubstituted C1-C5 alkyl group. In some forms, the compound is as described above for Formula II or Formula III, except that at least one of R5 and Rs are hydrogen.

[0068] In some forms, the compound is as described above for Formula II or Formula II, except that the compound has a structure:

[0069] Formula IV

[0070] In some forms, the compound is as described above for Formula II, Formula III, or Formula IV, except that at least one of Re and R7 is a halogen.

[0071] In some forms, the compound is as described above for Formula II, Formula III, or Formula IV, except that Re is a halogen and R7 is hydrogen. In some forms, the compound is as described above for Formula II, Formula III, or Formula IV, except that Re is hydrogen and R7 is a halogen. In some forms, the compound is as described above for Formula II, Formula Ill, or Formula IV, except that Re and R7 are both halogens that may be the same or different.

[0072] In some forms, the compound is not:

[0073] In some forms, the compound is as described above for Formula I, Formula II, Formula

[0074] Ill, or Formula IV, except that the compound has a structure:

[0075]

[0076] In some forms, the compound of Formula I, Formula II, Formula III, Formula IV, or the above-represented non-limiting can exist as its pharmaceutically acceptable salt.

[0077] In some forms, the compounds have an IC50 towards GSDMD of less than or equal to 15 pM or a KD values of less than 15 pM as measured by a microscale thermophoresis (MST) assay, performed using about 30% LED power and 40% MST power. b. Methods of manufacture

[0078] The general syntheses of quinoxaline compounds are well-known to those of skill in the art, and have been described in the literature. Some of these methods include condensation reactions between aromatic- 1 ,2-diamines (e.g. , phenyl- 1 ,2-diamines) and dicarbonyl compounds (e.g., vicinal dicarbonyl compounds) (Khatoon and Abdulmalek, Molecules 2021, 26, 1055), aromatic- 1,2-diamines e.g., phenyl- 1 ,2-diamines) and vicinal diol compounds (Adhikari, et al., J. Org. Chem. 2020, 85, 14971-14979), aromatic- 1 ,2-diamines (e.g., phenyl- 1,2-diamines) and 2- arylethylamine compounds (Luo, el al., Org. Lett. 2017, 19, 5629-5632), one-pot synthesis between aromatic- 1 ,2-diamines (e.g. , phenyl- 1 ,2-diamines) and benzyl ketones (Chen, et al. , Synthesis 2011, 387-396) or aldehydes (Cheon, et al., J. Org. Chem. 2014, 79, 901-907); and aromatic- 1,2-diamines (e.g., phenyl- 1,2-diamines) and ynones via Michael addition, dehydration condensation, and base-promoted C-a-CI h-extrusion under metal-free conditions (Cui, et al., Org. Lett. 2016, 18, 1378-1381). The contents of which are herein incorporated by reference. In some forms, the compounds can be synthesized from further modifications of substituted or unsubstituted quinoxalin-2(lH)-ones and substituted or unsubstituted quinoxalines (e.g., 2,3, 6,7-tetrachloro quinoxalines) as described in Knudsen, et al. , Proc. Natl. Acad. Sci. (USA) 2007, 104 (3), 937-942, the contents of which are herein incorporated by reference.

[0079] III. Formulations

[0080] As used herein, the term “carrier” or “excipient” refers to an organic or inorganic ingredient, natural or synthetic inactive ingredient in a formulation, with which one or more active ingredients are combined.

[0081] As used herein, the term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.

[0082] Pharmaceutical compositions can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), enteral, transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, pulmonary, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0083] The compositions are most typically administered systemically or intra-tumorally.

[0084] Drugs can be formulated for immediate release, extended release, or modified release. A delayed release dosage form is one that releases a drug (or drugs) at a time other than promptly after administration. An extended release dosage form is one that allows at least a twofold reduction in dosing frequency as compared to that drug presented as a conventional dosage form (e.g. as a solution or prompt drug-releasing, conventional solid dosage form). A modified release dosage form is one for which the drug release characteristics of time course and / or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as solutions, ointments, or promptly dissolving dosage forms. Delayed release and extended release dosage forms and their combinations are types of modified release dosage forms.

[0085] Formulations are prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The “carrier” is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. The term “carrier” includes but is not limited to diluents, binders, lubricants, desintegrators, fillers, and coating compositions.

[0086] “Carrier” also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. The delayed release dosage formulations may be prepared as described in references such as “Pharmaceutical dosage form tablets”, eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington - The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6thEdition, Ansel et.al., (Media, PA: Williams and Wilkins, 1995) which provides information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

[0087] The effective amount of the compounds or a pharmaceutically acceptable salt thereof, can be ascertained from assays investigating the (i) killing cancer cells and / or inhibition cancer cell growth, (ii) membrane pore formation, (iii) cleavage-independent activation of GSDMD, (iv) immune-mediated killing of cancer cells and / or inhibition of cancer cell growth, or a combination thereof, compared to a control that does not contain the compounds or a pharmaceutically acceptable salt thereof. In some forms, the compound or a pharmaceutically acceptable salt thereof, has a half-maximal inhibitory concentration (IC50) of GSDMD activation of less than 15 pM, less than 10 pM, less than 5 pM, less than 2.5 pM, less than 1 pM, less than 0.1 pM, less than 0.01 pM, or less than 0.001 pM; for example, 0.001 pM - 15 pM, 0.001 pM - 10 pM, 0.001 pM - 5 pM, 0.01 pM - 15 pM, 0.01 pM - 10 pM, 0.01 pM - 5 pM, 0.1 pM - 15 pM, 0.1 pM - 10 pM, 0.1 pM - 5 pM, 1 pM - 15 pM, 1 pM - 10 pM, 1 pM - 5 pM, or any subrange or specific number therebetween. In some forms, the concentration of the compound or a pharmaceutically acceptable salt thereof in the formulation is less than 30 mM, less than 25 mM, less than 20 mM, less than 15 mM, less than 10 mM, less than 5 mM, or less than 1 mM; for example, 0.001 mM - 30 mM, 0.01 mM - 30 mM, 0.1 mM - 25 mM, 0.1 mM - 30 mM, 1.0 mM - 20 mM, 5 mM - 20 mM, 10 mM - 20 mM, or any subrange or specific number therebetween. In some forms, the dose of the compound or a pharmaceutically acceptable salt thereof in the formulation can be expressed in terms of mass of drug / mass of a subject in need thereof. Suitable doses include less than 50 mg / kg; less than 45 mg / kg; less than 40 mg / kg; less than 35 mg / kg; less than 30 mg / kg; less than 25 mg / kg; less than 20 mg / kg; less than 15 mg / kg; less than 10 mg / kg; or less than 5 mg / kg, such as between 0.5 mg / kg and 50 mg / kg; between 1.25 mg / kg and 45 mg / kg; between 1.25 mg / kg and 40 mg / kg; between 1.25 mg / kg and 35 mg / kg; between 1.25 mg / kg and 30 mg / kg; between 1.25 mg / kg and 25 mg / kg; between 1.25 mg / kg and 20 mg / kg; between 1.25 mg / kg and 15 mg / kg; between 1.25 mg / kg and 10 mg / kg; between 1.25 mg / kg and 5 mg / kg; about 10 mg / kg, about 5 mg / kg, about 2.5 mg / kg, about 1.25 mg / kg, or about 1 mg / kg. The compound can be administered to a subject with or without the aid of a delivery vehicle. Appropriate delivery vehicles for the compounds are known in the art and can be selected to suit the particular active agent. For example, in some forms, the active agent(s) is incorporated into or encapsulated by a nanoparticle, microparticle, micelle, synthetic lipoprotein particle, or carbon nanotube. For example, the compositions can be incorporated into a vehicle such as polymeric microparticles which provide controlled release of the active agent(s). In some forms, release of the drug(s) is controlled by diffusion of the active agent(s) out of the microparticles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation.

[0088] In some forms, the compounds described herein can be formulated as conjugates, such as antibody-compound conjugates (such as antibody-drug conjugates), polymer-compound conjugates (such as polymer-drug conjugates), small molecule-compound conjugates (such as small molecule-drug conjugates), etc. The antibody moiety may be a monoclonal antibody, an antigen-binding antibody fragment, a bispecific or other multivalent antibody, or other antibodybased molecule. The antibody can be of various isotypes, preferably human IgGl, IgG2, IgG3 or IgG4, such as human IgGl hinge and constant region sequences. The antibody or fragment thereof can be a chimeric, a humanized, or a human antibody, as well as variations thereof, such as half-IgG4 antibodies (referred to as “unibodies”). Suitable antibodies or fragments thereof include, but are not limited to, anti-PD-1 antibody, anti-Trop-2 antibody, HER2 antibody, etc. Suitable polymers include, but are not limited to, polyesters (such as polyhydroxy acids), polyanhydrides, poly(ortho)esters, poly(p-dioxanone), poly(polyurethane), polycarbonate, polyphosphate, polyphosphonate, polyalkylene glycols (e.g., poly(ethylene glycols), and a combination thereof.

[0089] Combination Therapies

[0090] The formulations or compounds described herein can also be administered alone or in combination with one or more therapies, such as cancer therapy. In some forms, the one or more therapies include administration of one or more of the formulations or compounds in combination with one or more additional active agents. The term “combination” or “combined” is used to refer to either concomitant, simultaneous, or sequential administration of two or more agents. Therefore, the combinations can be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second). The additional therapeutic agents can be administered locally or systemically to the subject, or coated or incorporated onto or into a sustained release delivery system. Therefore, in some forms, the formulation includes two, three, or more active agents. The additional active agent(s) can have the same, or different mechanisms of action. In some forms, the combination results in a synergistic effect or an additive effect on the treatment of the cancer. Preferably, the combination results in a synergistic effect on the treatment of the cancer.

[0091] In some forms, the one or more therapies includes administration of immune check inhibitors, such as PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, etc.

[0092] IV. Methods of Use

[0093] The studies provide a new paradigm in immunotherapy by inducing a low level of pyroptosis to stimulate antitumor immunity. DMB is a small molecule agonist capable of inducing pyroptotic inflammatory cell death by directly activating a gasdermin family protein without cleaving it. DMB works by targeting Cysl91 selectively on human and mouse GSDMD to trigger oligomerization and pore formation, by overcoming autoinhibition. It induces GSDMD-dependent pyroptotic cell death in cancer lines within hours at pM potency, which results in plasma membrane rupture to release LDH and DAMPs, such as extracellular ATP. DMB reduces tumor burden in mice challenged with breast, colorectal and lung tumors and melanoma and increases the cytotoxicity and cytokine secretion of tumor-infiltrating CD8+and NK cells.

[0094] Tumor reduction depends on GSDMD expression in the tumor but not the host, supporting direct killing of cancer cells and tumor-autonomous activation of antitumor immunity. Importantly, DMB induces immunogenic cell death since vaccination with DMB- treated cells protected mice against subsequent tumor challenge. In addition, low dose DMB synergized with anti-PD-1 to suppress growth of a CPB-resistant tumor. Importantly, DMB recognized human GSDMD and showed anti-tumor activity in vivo against a tumor line expressing human GSDMD. Direct induction of pyroptosis by GSDMD agonists may have several advantages over secondary pyroptosis induction by chemo- and radiotherapy. First, tumor cell-killing by chemotherapy and radiotherapy is mostly by apoptosis unless the tumor cell expresses GSDME. Apoptosis can be converted to pyroptosis if a tumor expresses GSDME, but GSDME expression in tumors is typically repressed by DNA hypermethylation. Thus, pyroptosis induction by these therapies can be useful but does not occur predictably. Second, induction of pyroptosis in a small number of tumor cells can stimulate widespread antitumor immune control. A low degree of pyroptosis induced directly by DMB is implied by the data, since tumors implanted in NSG mice showed no significant increase in pyroptosis after treatment with DMB compared to vehicle. The strong vaccination effects from DMB- treated pyroptotic tumor cells suggest that the combined release of tumor antigens and DAMPs from pyroptotic tumor cells may be potent immunogens. Third, while radiotherapy and chemotherapy induce somatic mutations in tumor and healthy cells that can lead to drug resistance or secondary malignancies, pyroptotic agonists might more precisely trigger immunogenic cell death. Fourth, knowing the direct target of the GSDMD agonists could enable patient stratification depending on GSDMD expression. Fifth, the synergy of pyroptosis with anti-PD- 1 or other CPB inhibitors could expand the range of immunotherapy responsive tumors. Finally, the effectiveness of GSDMD agonists against smaller tumors suggests its combination with therapies that reduce tumor burden such as surgery, chemo- and radiotherapy for advanced disease.

[0095] One concern over any immunotherapy is that they might theoretically cause systemic inflammation and toxicity. However, no evidence of DMB cytotoxicity to host immune cells, such as TAMs, which express GSDMD constitutively, or to other host cells, was observed, since treated mice showed no abnormalities in peripheral leukocyte counts, plasma inflammatory cytokines, or body weight. The minimal toxicity of DMB could be because administration of DMB only leads to direct killing of a small number of cells, tumor or host, which is adequate to boost specific antitumor immunity but not high enough to lead to systemic inflammation. This hypothesis of amplification of tumor cell pyroptosis by killer cells is consistent with our in vivo pyroptosis data that DMB caused more pyroptosis in GSDMD-expressing tumor cells than in host cells. IN these studies, no evidence was found that host GSDMD contributes to DMB- induced antitumor immunity since Gsdmd1' mice retained sensitivity to the antitumor effect of DMB. Thus, the GSDMD-agonistic tool compound DMB and the mechanism of its action reveal a new strategy of immunotherapy that is triggered by a low level pyroptosis induction.

[0096] The compositions are administered to an individual with a tumor, systemically, locally or intratumorally, in an amount that elicits an anti-tumor response, for a period of time to reduce or eliminate the tumor.

[0097] The effective dose can be determined by one skilled in the art, based on pharmacokinetic studies, EC50 and IC50.

[0098] Examples

[0099] The present invention will be further understood by reference to the following nonlimiting examples. Example 1: Identifying GSDMD agonists, characterizing binding mode, and demonstrating antitumor activity Materials and Methods

[0100] TABLE 1: Table of key resources, source, and identifier

[0101]

[0102]

[0103] Experimental Model and Subject Details

[0104] Cell lines

[0105] THP-1 and CT26 cells were grown in Roswell Park Memorial Institute RPMI, Gibco) medium with 10% fetal bovine serum (FBS, EMD Sciences), supplemented with 100 U / ml penicillin G, 100 pg / ml streptomycin sulfate (Pen-strep, Gibco). EMT6 cells were grown in Waymouth’s media (Gibco) supplemented with 15% FBS and Pen-strep. HEK293T, B16, KP, and 4T1E cells were grown in Dulbeccos Modified Eagle Medium (DMEM) with 10% FBS and Pen-Strep. B16 cell line is a gift from Gordon J. Freeman. KP cell line is a gift from Tyler Jacks. 4T1E was generated by sorting 4T1 cells (provided by Fred Miller) for high E-cadherin expression. WT and caspase-1 / 11 KO iBMDMs, and WT and GSDMD KO primary BMDMs are gifts from Jonathan C. Kagan. Human Peripheral Blood Mononuclear Cells (PBMCs) were purchased from BPS Bioscience. All other lines were obtained from American Type Culture Collection (ATCC) and were maintained at 37 °C under 10% CO2.

[0106] Mice

[0107] Animal studies were conducted in compliance with the ethical regulations and were approved by the Harvard Medical School Institutional Animal Care and Use Committee. 6-8- week-old female BALB / c, C57BL / 6, or NOD. C -Prkdcsc,dU2rgtml^lISz] (NSG) mice were purchased from The Jackson Laboratory and maintained at the SPF facility at Harvard Medical School. Gsdmd '' mice in the C57BL / 6 background were bred on site. All mouse experiments were conducted using protocols approved by the Animal Care and Use Committees of Boston Children's Hospital and Harvard Medical School.

[0108] Chemical reagents

[0109] 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB), terbium(III) chloride (TbCL), P-mercaptoethanol (2ME), dithiothreitol (DTT), dipicolinic acid (DPA), phorbol 12-myristate 13 -acetate (PM A), DMSO and the cOmplete protease inhibitor cocktail were from Sigma- Aldrich. Nigericin was from InvivoGen.

[0110] Protein expression and purification

[0111] Full-length human GSDMD sequence was cloned into the pDB.His.MBP vector with a tobacco etch virus (TEV)-cleavable N-terminal Hise-MBP tag using Ndel and Xhol restriction sites. For expression of full-length GSDMD, E. coli BL21 (DE3) cells harboring the indicated plasmids were induced with 0.5 mM isopropyl-0-D-thiogalactopyranoside (IPTG) at ODeoo of 0.8, and grown at 18 °C overnight in LB medium supplemented with 50 pg ml-1kanamycin. Cells were sonicated in lysis buffer containing 40 mM HEPES at pH 7.0, 150 mM NaCl, and 5 mM imidazole. The lysate was clarified by centrifugation at 40,000 x g at 4 °C for 1 h. The supernatant containing the target protein was incubated with Ni-NTA resin (Qiagen) for 30 min at 4 °C. After incubation, the resin-supernatant mixture was poured into a column and the resin was washed with lysis buffer. The protein was eluted using the lysis buffer supplemented with 300 mM imidazole. The Hise-MBP tag was removed by overnight TEV protease digestion at 16 °C. The cleaved protein was purified using HiTrap Q ion-exchange and Superdex 200 gelfiltration columns (GE Healthcare Life Sciences).

[0112] Liposome preparation

[0113] PC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 25 mg / mL in chloroform; 80 pL), PE (l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine, 25 mg / mL in chloroform; 128 pL) and CL [l’,3'-bis(l,2-dioleoyl-5 / i-glycero-3-phospho)-i77-glycerol (sodium salt), 25 mg / mL in chloroform; 64 pL] were mixed and the solvent was evaporated under a stream of N2 gas. The lipid mixture was suspended in 1 mL Buffer A (20 mM HEPES, 150 mM NaCl, 50 mM sodium citrate, and 15 mM TbCh) for 3 min. The suspension was pushed through an 100 nm Whatman® Nuclepore™ Track-Etched Membrane 30 times to obtain homogeneous liposomes. The filtered suspension was purified by size exclusion column (Superose 6, 10 / 300 GL) in Buffer B (20 mM HEPES, 150 mM NaCl) to remove TbCh outside liposomes. Void fractions were pooled to produce a stock of PC / PE / CL liposomes (1.6 mM). The liposomes were diluted to 50 pM with Buffer C (20 mM HEPES, 150 mM NaCl and 50 pM DPA) for use in the high- throughput screening.

[0114] TRF high-throughput screen for GSDMD agonists

[0115] Liposome leakage was detected by an increase in fluorescence when Tb3+bound to DPA in Buffer C. Human GSDMD (0.3 pM) dispensed into 384-well plates (Corning 3820) was incubated with compounds from the ICCB-Longwood Screening Facility collection for 1 h before addition of PC / PE / CL liposomes (50 pM liposome lipids) to each well. The time-resolved fluorescence intensity (delay 50 ps, interval 950 ps) of each well was measured at 545 nm with an excitation of 276 nm 1 h after addition of liposomes using a Perkin Elmer EnVision plate reader. The final percent activation was calculated as [(fluorescencetest compound - fluorescencenegative controi) / (fluorescencePositive control - fluorescencenegative control)] X 100, where wells with GSDMD without agonists was used as negative control and with 0.1% SDS as positive control. 50% inhibition was arbitrarily chosen as a threshold. The hits were evaluated in concentration-response experiments in a dose range of 0.023-50 pM to determine EC50.

[0116] Fluorescent protein labelling and microscale thermophoresis binding assay

[0117] Human GSDMD was labelled with AlexaFluor-647 using the Molecular Probes protein labelling kit. Ligand binding to GSDMD was evaluated using microscale thermophoresis (MST). Ligands (0.023-50 pM) were incubated with purified AlexaFluor-647 -labeled protein (50 nM) for 30 min in assay buffer (20 mM HEPES at pH 7.4, 150 mM NaCl, 0.05% Tween 20). The sample was loaded into NanoTemper Monolith NT.l 15 glass capillaries and MST was carried out using 30% LED power and 40% MST power. Kn values were calculated using the mass action equation and NanoTemper software.

[0118] Mass spectrometry and sample preparation

[0119] Recombinant GSDMD (1 pM) was incubated with DMB (20 pM) or not for 1 h at room temperature and run on a non-reducing SDS-PAGE. The gel was stained with Coomassie blue, and the GSDMD bands were excised and placed into separate 1.5 mL polypropylene tubes. 100 pL of 50% acetonitrile in 50 mM ammonium bicarbonate buffer were added to each tube and the samples were then incubated at room temperature for 20 min. This step was repeated if necessary to destain the gel. Then, the gel slice was incubated with 55 mM iodoacetamide (in 50 mM ammonium bicarbonate) for 45 min in the dark at room temperature, before the gel was washed sequentially with 50 mM ammonium bicarbonate, water and acetonitrile. Samples were then dried in a Speedvac for 20 min. Trypsin or chymotrypsin (10 ng / pL in 25 mM ammonium bicarbonate, pH 8.0) was added to each sample tube to just cover the gel, and samples were then incubated at 37 °C for 6 h or overnight.

[0120] After digestion, samples were acidified with 0.1% formic acid (FA) and 3 pL of tryptic peptide solution was injected. Nano-LC / MS / MS was performed on a Thermo Scientific Orbitrap Fusion system, coupled with a Dionex Ultimat 3000 nano HPLC and auto sampler with 40 well standard trays. Samples were injected onto a trap column (300 pm i.d. x 5mm, C18 PepMap 100) and then onto a Cl 8 reversed-phase nano LC column (Acclaim PepMap 100 75 pm X 25 cm), heated to 50 °C. Flow rate was set to 400 nL / min with 60 min LC gradient, using mobile phases A (99.9% water, 0.1% FA) and B (99.9% acetonitrile, 0.1% FA). Eluted peptides were sprayed through a charged emitter tip (PicoTip Emitter, New Objective, 10 + / - 1 pm) into the mass spectrometer. Parameters were: tip voltage, +2.2 kV; Fourier Transform Mass Spectrometry (FTMS) mode for MS acquisition of precursor ions (resolution 120,000); Ion Trap Mass Spectrometry (ITMS) mode for subsequent MS / MS via higher-energy collisional dissociation (HCD) on top speed in 3 s. Proteome Discoverer 1.4 was used for protein identification and modification analysis.

[0121] Caspase- 1 activity assay

[0122] To assess the effect of DMB on caspase- 1 activity, a fluorogenic assay was performed using recombinant caspase- 1. Caspase- 1 (0.5 pM) was pre-incubated with DMB (10 pM) or not for 2 h at room temperature in a buffer containing 40 mM Hepes, pH 7.4 and 150 mM NaCl. Samples were then incubated with the fluorogenic substrate Z-YVAD-AFC (10 pM) and immediately analyzed for 2 h at 30-second intervals using a Perkin Elmer EnVision plate reader. The excitation and emission wavelengths were 400 nm and 505 nm, respectively, measured by time-resolved fluorescence (delay 50 ps, interval 950 ps).

[0123] Biotin-DMB pulldown of THP-1 lysates

[0124] To investigate the binding of GSDMD to DMB in cells, a biotin pull-down assay was performed using biotin-DMB and the THP-1 cell line. For each sample, 2 million THP-1 cells were resuspended and lysed in PBS with 1% NP-40 for 20 minutes. The sample was clarified through pre-incubation with streptavidin resin for 30 minutes. The resin was removed by centrifugation at 500 x g for 2 minutes. Each sample was then incubated with the indicated concentration of DMB for 30 min. Subsequently, biotinylated-DMB was added to the samples and incubated for an additional 30 min. 50 pL of streptavidin resin was added to each sample and incubated for 1 h. The resin was recovered via centrifugation at 500 x g for 2 min and washed several times, including twice with 0.1% SDS in PBS, twice with PBS, once with 3 M urea in PBS, twice with PBS, and twice with water. All the steps were performed at room temperature. Finally, the samples were analyzed by western blot to determine the binding of GSDMD to DMB in THP-1 cells.

[0125] GSDMD pore reconstitution and negative-staining electron microscopy

[0126] To reconstitute GSDMD pores on liposomes, liposomes containing phosphatidic acid (PA), an acidic lipid, and phosphatidylcholine (PC), and an engineered human GSDMD construct previously designed for structural determination were used. Dried lipid films consisting of 20% PA and 80% PC were resuspended in Buffer D (40 mM HEPES at pH 7.0, 150 mM NaCl), extruded the liposomes through a 100-nm filter, and the liposomes purified using a SUPEROSE® 6 10 / 300 GL size-exclusion column equilibrated in Buffer D. The GSDMD construct contains an N-terminal TEV-removable MBP tag and a 3C protease cleavage site in place of the GSDMD inter-domain linker (L259 - D275). The MBP fusion protein was expressed in E. coli BL21 (DE3) and purified according to previous protocols.61The MBP tag was removed by TEV cleavage prior to pore reconstitution experiments. Purified GSDMD was incubated with the 3C protease or DMB in the presence of liposomes on ice overnight. Afterwards, the liposomes were pelleted in an ultracentrifuge at 40,000 rpm for 1 h, and the supernatant was removed. The pellet was washed with Buffer D, and then solubilized using Buffer D supplemented with 1% C12E8 (Anatrace). Undissolved aggregates were removed by centrifugation at 15,000 rpm for 10 min at 4 °C. Clarified supernatants containing detergent- solubilized GSDMD pores (5 uL) were applied to glow-discharged Formvar-coated copper grids (Electron Microscopy Sciences), washed twice with 30 mL Buffer D supplemented with 1% C12E8, stained with 1% uranyl formate, and then blotted dry with filter paper. Imaging of the copper grids was performed using a Tecnai G2Spirit BioTWIN electron microscope (FEI) at the Electron Microscopy Facility at Harvard Medical School.

[0127] Cell viability and microscopy-based cytotoxicity assays

[0128] Lactate dehydrogenase and ATP release were measured using LDH-Glo Cytotoxicity Assay kit (Promega) and RealTime-Glo Extracellular ATP assay (Promega), respectively, according to the manufacturer’s instructions. Cell viability was assessed by measuring ATP levels using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7570) according to the manufacturer’s instructions. Luminescence was measured on a BioTek Synergy 2 plate reader. For microscopy-based cytotoxicity analysis, cells were seeded in 24-well plates or CELL view Cell Culture Dishes with four compartments) (USA Scientific, 5662-7870) in culture media with 1 pg / ml propidium iodide (PI, BD Bioscience, 556463) or 0.5 pM Sytox Green (Thermo Fisher Scientific, S7020) followed by calculating PI positivity or Sytox Green positivity on either an Inverted Nikon Ti2 fluorescence microscope with a 40X objective (N.A. = 1.2) with a stage top incubator to maintain 37 °C and 5% CO2 or on a Leica TCS SP8 Laser Scanning Confocal (Leica) fluorescence microscope with a heated stage to maintain 37 °C and 5% CO .

[0129] Plasmid and LPS electroporation

[0130] 0.24 x 106THP-1 cells were electroporated with WT or D275A GSDMD constructs (1 pg) using the Neon™ Transfection System (Invitrogen) at the following setting: 1,400 volt, 20 ms pulse width and 2 pulses. After electroporation, cells were plated in an appropriate tissue culture plate. 20 hours post-electroporation, cells were treated with indicated chemicals and assayed. 1 x 106THP-1 cells were electroporated with LPS (1 pg) using the Neon™ Transfection System (Invitrogen). After electroporation, cells were plated in an appropriate tissue culture plate, and treated or not with indicated chemicals and assayed.

[0131] HEK293T transfection

[0132] HEK293T cells were plated in 24-well plates at 5 x 104cells per well. After 20 h, WT or mutant GSDMD-NT or GSDMD-FL constructs were transfected into HEK293T cells by using Lipofectamine 3000 (Invitrogen, Cat# L3OOOOO8). 20 hours post-transfection, cells were used for assays.

[0133] Immunoblot

[0134] Cell extracts were prepared using Lysis buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40 supplemented with Halt protease inhibitor cocktail (Invitrogen)]. Samples were subjected to SDS-PAGE on 4-12% Tris-Glycine gels (Bio Rad) and transferred to a 0.2 pm nitrocellulose membrane using the iBlot system (Invitrogen) followed by blocking in 5% milk in Tris-buffered saline containing 0.1% Tween- 20 (TBST) followed by incubation with primary antibody, extensive washing, and incubation with secondary antibodies: 1 : 1000 HRP-goat antiRabbit IgG (BD Biosciences) or 1: 1000 HRP-Goat anti-mouse IgG (ab97040, abeam). Immunoblots were probed with the following primary antibodies: 1 : 1000 Rabbit anti-mouse GSDMD (ab209845, abeam), 1:1000 Rabbit anti-mouse GSDME (ab219151, abeam), 1: 1000 Rabbit anti-human GSDMD (NBP-33422, Novus Biologicals), 1:1000 mouse anti- Actin (C4, Santa Cruz Biotechnology) and visualized using a SuperSignal West Pico chemiluminescence ECL kit (Pierce). NINJ1 oligomerization assay

[0135] 2X106THP-1 cells were differentiated into macrophages by 50 nM PMA treatment for 48 hours. The indicated PMA-differentiated THP-1 cells were primed with LPS (1 pg / mL) for 4 hours. For inflammasome activation, the indicated cells were treated with nigericin (20 pM) for 1 hour. DMB-treatment was administered at 20 pM for 2 or 4 hours. To prevent Ninjurin-1 oligomerization, cells in certain conditions were pretreated with 20 mM glycine 1 hour before nigericin or DMB treatment. Cell media were collected and filtered through 0.45 pM filters and spun for 1500 rpm for 5 minutes. Supernatants were collected. Cell lysates were also obtained using RIPA buffer. Samples were subjected to SDS-PAGE electrophoresis under either reducing or non-reducing conditions. Gels were transferred to PVDF membranes using standard Towbin buffer at constant 200 mA for 1 hour and immunoblotted with following antibodies: Ninjurin-1 (sc-136295) 1 :1000 dilution, Vinculin (SAB4200729) 1: 1000 dilution, Actin (sc-47778) 1: 1000 dilution.

[0136] Generation of CRISPR / Cas9 knockout EMT6 lines

[0137] GSDMD and GSMDE CRISPR / Cas9 knockout cell lines were generated using the pGuide-it-ZsGreenl system (Takara, Tokyo, Japan). Briefly, the selection of sgRNA target sites was performed using CHOPCHOP (https: / / chopchop.cbu.uib.no / ). The pGuide-it- ZsGreenl / GSDMD and pGuide-it-ZsGreenl / GSDME plasmids were constructed following the manufacturer’s protocol (Takara, Cat. No. 632601) using the specific oligonucleotides listed in the Key Resources Table. The constructed plasmid was transfected into EMT6 cells using Lipofectamine® 2000 Reagent (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. After 24 h, Cas9-positive cells (marked by GFP) were sorted using a FACS Aria II flow cytometer (BD Bioscience). Clones were selected by limiting dilution and by immunoblot for lack of detectable GSDMD and / or GSDME.

[0138] Generation of B16-GFP and B16-GSDMD-GFP cell lines

[0139] Human GSDMD was cloned or not into the pLenti-CMV-eGFP-SV40p-BlasR plasmid for ectopic expression of GFP or GSDMD-GFP fusion protein. Lentiviruses were generated by transfecting HEK293T cells with 10 pg pLenti-CMV-eGFP-SV40p-BlasR or pLenti-CMV- GSDMD-eGFP-SV40p-BlasR, 7.5 pg pSPAX2 and 2.5 pg pCMV-VSV-G. Supernatants collected 2 days later were used to infect B16 cells. After 2 days, 20 pg / mL blasticidin (InvivoGen) was added to select for GFP or GSDMD-GFP expressing cells. For some experiments, GSDMD-GFP-expressing clones were selected; clones were verified by flow cytometry. Mouse experiments

[0140] CT26 cells (10scells / BALB / c mouse), KP tumor cells (2x10scells / C57BL / 6 mouse) or B16-GFP pooled cells, or B16-GSDMD-GFP clones 9 or 10 (5x10scells / C57BL / 6 mouse) were injected in 50 pL of PBS subcutaneously into the right flank of mice. For orthotopic tumor challenge, EMT6 (5xl04cells / mouse) or 4T1E (2.5xl04cells / mouse) were injected into the 4thmammary fat pad of BALB / c mice. DMB working solution was prepared by diluting the stock solution 1:10 in 10% 2-hydroxyl-propyl-b-cyclodextrin / PBS (Sigma-Aldrich). Diluted DMSO was used as the vehicle control. When EMT6, CT26 or KP tumors were palpable, mice were injected intraperitoneally with the indicated concentrations of DMB once per week. For the B16 experiments, treatment was initiated either when tumors became palpable (5 days post implantation) or treatment was delayed until 10 days post-implantation. DMB was administered every 3 days x 6 treatments. In some experiments, anti-PD-1 (200 mg / mouse, clone 29F. 1A12, BioXCell) was given intraperitoneally starting on day 9 after tumor challenge and every third day thereafter.

[0141] Tumor growth was monitored by measuring the perpendicular diameters of tumors every other day. For the vaccination study, EMT6 tumor cells were treated with each drug at concentrations that induced -60-70% cell death (60 pM, 20 h for MMC and 60 pM, 5 h for DMB). Both live and dead MMC- or DMB-treated cells were collected, and each mouse was immunized by injecting 106drug-treated tumor cells subcutaneously in the left flank and challenged 8 days later by injecting 1.5x10suntreated EMT6 cells in the right 4thmammary fat pad. For PI uptake assay, BALB / c or NSG mice bearing palpable EMT6 tumors were injected with PBS or 10 mg / kg DMB intraperitoneally once per week. One week following the second DMB injection, mice were injected intravenously with 2.5 mg / kg PI in 100 mb per mouse. Mice were euthanized 10 min later, and tumors were isolated and homogenized into single cell suspensions for analysis. TAM were defined as CD45+CD3 CD1 lb+F4 / 80+cells, tumor cells (including tumor stroma) were defined as CD45 CD3 cells.

[0142] Isolation of tumor-infiltrating immune cells

[0143] Tumors were collected, cut into small pieces and treated with 2 mg / mL collagenase D, 100 pg / mL DNase I (both from Sigma) and 2% FBS in RPMI with agitation for 20 min. Tumor fragments were homogenized and filtered through 40 pm strainers, and immune cells were purified by PercolLgradient centrifugation and washed with Leibovitz’s L-15 medium.

[0144] Antibody staining and flow cytometry

[0145] Immune cells isolated from mice were stained with anti-CD45-PerCPCy5.5 or -PacBlue, CD8-PacBlue, -PerCPCy5.5, -Alexa700, -FITC or -APC, CD4-PE-Cy7, -APC or PerCPCy5.5, CD44-PerCPCy5.5 or PacBlue, Ly-6G / Ly-6C(Gr-l)-FITC or -PE, CDllb-Alexa700, CD49b- PacBlue or FITC, NKp46-APC, F4 / 80-PE-Cy7, EpCAM-PE-Cy7 or PerCPCy5.5. Dead cells were excluded using the live / dead fixable aqua dead cell stain. For intracellular staining of GzmB or PFN, cells were first stained with antibodies to cell-surface markers for 30 min at 4 °C, then fixed and permeabilized with fixation / permeabilization buffer and stained with anti-GzmB- PacBlue, and anti-Perforin- PE. For intracellular cytokine staining of ex vivo stimulated lymphocytes, ~106cells per sample were cultured in RPMI medium containing 2% FBS and stimulated with PMA (50 ng / ml), ionomycin (2 pg / mL) and Golgiplug (1.5 pg / mL) for 4 h. Cells cultured with medium and Golgiplug alone served as negative control. Cells were then stained with antibodies to IFNg-PacBlue or -APC and TNF-PE-Cy7 after fixation / permeabilization. Cells were analyzed by BD FACSCanto II and data were analyzed with FlowJo V.10.

[0146] Immunofluorescence imaging and quantification of B16 tumors

[0147] Tumors were harvested and fixed in fixation buffer (20% sucrose, 4% paraformaldehyde) at 4 °C overnight. The next day they were embedded in OCT compound (Sakura), solidified on dry ice. and sectioned into 10 pm sections using a microtome (Leica). Sections were blocked in 1:100 TruStain FcX (BioLegend) blocking buffer (TBS containing 10% FBS, 2% normal mouse serum, 0.1% Tween- 20) for 1 h at room temperature and incubated overnight at 4 °C with fluorescently labeled anti-CD3, anti-CD8, anti-MHCII or anti-CDllc antibodies. Antibodies were used at 1 :100 dilution in blocking buffer. Nuclear staining was performed with DAPI (ThermoFisher) at 1: 1000 dilution in TBST for 15 min. Images were acquired using a Micron Olympus microscope.

[0148] Images from GSDMD-GFP expressing tumors that had been stained were processed with Fiji (v2.0.0-rc-69 / 1.52i). For every tumor 3-4 regions of interest (ROI) were selected based on GFP expression blindly to the other channels. For every ROI, DAPI channel was used for segmentation using the Trainable Weka Segmentation (v3.2.29) plugin. The generated mask was used to measure the mean intensity in each cell of every marker. Cell populations were called based on level of expression of each marker (2000 au for CD3 and CD8 and 3000 au for MHCII and CD11c). Total number of cells was normalized by ROI area in mm2.

[0149] Statistical analysis

[0150] A Student’ s t-test (two-tailed) or Mann- Whitney test was used to determine differences between two groups. One- or two-way ANOVA was used to calculate differences among multiple populations. Differences between tumor growth curves were compared by first calculating the area-under-curve values for each sample and then comparing different groups using the Student’s t-test or one-way ANOVA. Type I errors were corrected by the Holm-Sidak or Dunn’ s multiple comparisons test. Nonparametric test was used when data did not follow a normal distribution. Significance was set at p-value < 0.05. For all figures, *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001. All statistical analyses were conducted using GraphPad Prism 8.

[0151] Results

[0152] High-throughput screen identified DMB as a potent activator of GSDMD

[0153] A high-throughput screening was used to search for GSDMD agonists using a time- resolved Anorogenic (TRF) liposome leakage assay, which detects leakage of terbium (Tb3+) from Tb3+-loaded liposomes incubated with GSDMD by Tb3+complexation with dipicolinic acid (DPA) in a buffer (FIG. 1A). In comparison with a steady-state tluorogenic assay, a TRF assay afforded by the long fluorescence lifetime of the Tb3+chelate enhances sensitivity by avoiding background interference. Using detergent-permeabilized liposomes as a positive control, the Z’ factor was determined, a value that measures reproducible separation of hits from controls, of this screen to be ~0.8, indicating its suitability for high-throughput screening. Over 100,000 small molecules from a Harvard ICCB-Longwood collection were screened for hit compounds that activated GSDMD pore formation to trigger liposome leakage that was at least 50% of the total leakage caused by detergent (FIG. IB). After excluding pan-assay-interference compounds that nonspecifically react with many biological targets, GSDMD-independent liposome-disrupting compounds, auto-Auorescent compounds and those without saturable dose response curves, 24 active compounds were identified. One of the most potent hits was C-185, which had an ECso of 0.7 + 0.0 pM (FIGs. 1C-1E). C-185 is quinoxaline 6,7-dichloro-2-methylsulfonyl-3-N-tert- butylaminoquinoxaline (DMB) (FIG. ID), a potential oral drug for modulating glucagon-like peptide- 1 receptor (GLP-1R) in diabetic patients that was not further developed for clinical use because it was less effective than corresponding biologies. DMB directly bound to GSDMD by microscale thermophoresis (MST) with a dissociation constant (KD) of 1.1 ± 0.4 pM (FIG. IF).

[0154] DMB induces pyroptosis in immortalized and primary cells in a GSDMD-dependent and cleavage-independent manner

[0155] To evaluate whether DMB activates pyroptosis in a GSDMD-dependent manner, the human monocytic THP-1 cancer cell line was used that constitutively expresses GSDMD. Adding DMB to WT or GSDMD knockout (KO) THP-1 cells induced significant cell death within 2 hours in WT cells, but minimally in GSDMD KO cells, measured by propidium iodide (PI) uptake, similar to treatment with nigericin after lipopolysaccharide (LPS) priming (FIG. 2A, FIG. 8B). This distinction between WT and GSDMD KO cells was maintained throughout the time course of 24 hours for either 5 pM or 20 pM DMB. The concentration dependence of DMB-mediated cell death was then measured, and an EC50 value of 4.8 ± 0.4 pM was obtained (FIG. 2B). To determine whether GSDMD cleavage was required for DMB-induced pyroptosis, it was examined whether DMB can activate the D275A mutant uncleavable by inflammatory caspases or caspase-8 in GSDMD KO THP-1 cells reconstituted with WT or D275A GSDMD. After treatment with 5 pM or 20 pM DMB, THP-1 KO cells rescued with WT and D275A GSDMD showed comparable cell death by PI uptake at 1, 2, and 4 hours (FIG. 2C). These results showed that DMB activates GSDMD independently of its cleavage.

[0156] The kinetics of killing of THP-1 cells by DMB, measured by PI uptake, LDH release and ATP release was similar to that by nigericin (FIG. 2A, 2D, 2E), indicating similar mechanisms of cell death. ATP release peaked earlier than PI uptake or LDH release, suggesting that cells were dead before NINJ 1 activation and plasma membrane rupture that increase dye update and LDH release. Indeed, NINJ1 was oligomerized and released to the THP-1 supernatant upon DMB treatment, like LPS plus nigericin treatment, and as expected, glycine inhibited NINJ 1 oligomerization and release (FIG. 8C). Although membrane damage activates the NLRP3 inflammasome in monocytes or macrophages, DMB did not significantly activate NLRP3 in THP- 1 cells despite inducing both PI and S YTOX Green uptake, signs of membrane permeabilization, since it did not trigger ASC speck formation or IL-ip release, while LPS electroporation to activate the noncanonical inflammasome did (FIG. 8D). The reason for this observation is unclear, which could suggest more complexity in the relationship between membrane damage and NLRP3 activation.

[0157] To evaluate the GSDMD dependence of DMB’ s activity in more types of cells, human peripheral blood mononuclear cells (PBMCs) were treated with DMB. DMB robustly induced PI positivity, LDH release and ATP release in PBMCs, and pretreatment of PBMCs with the GSDMD inhibitor disulfiram (DSF) strongly suppressed cell death (FIGs. 2F, 2G, 8E, and 8F). DMB treatment of WT and GSDMD KO primary mouse bone-marrow derived macrophages (BMDMs), also showed that DMB induction of cell death depended on GSDMD (FIG. 2H, 21, 8G, 8H). Similarly DMB at both 5 ,uM and 20 ,uM concentrations triggered cell death in WT immortalized mouse BMDMs (iBMDMs), which was not affected by genetic ablation of both caspase- 1 and caspase- 11 (FIG. 2J, 2K, 81, 8J), showing that GSDMD activation by DMB does not depend on cleavage. Because DMB reacts with a Cys residue in GSDMD (see below) and caspase- 1 is a cysteine protease, it was verified that DMB neither inhibited nor activated recombinant caspase- 1 in vitro (FIG. 8K). DMB binds to GSDMD and induces cleavage-independent GSDMD oligomerization and pore formation

[0158] To examine whether DMB interacts with GSDMD in cells, two forms of biotinylated DMB were designed and synthesized, one of which (DMB-biotin) activated GSDMD in the liposome leakage assay with activity comparable to DMB (FIGs. 3A, 9A, 9B). DMB-biotin pulled down endogenous GSDMD from THP-1 cell lysates and much less GSDMD was pulled down in the presence of an excess of unlabeled DMB (FIG. 3A).

[0159] The ability of DMB to induce recombinant GSDMD to form pores in liposomes was examined. Recombinant GSDMD was incubated with DMB or an activating protease as a control in the presence of liposomes. The liposomes were then solubilized in C12E8 detergent and examined by negative-staining electron microscopy. Large pores were observed when GSDMD was cleaved by the protease or activated by DMB (FIG. 3B), confirming that DMB directly induced GSDMD pore assembly. Moreover, the kinetics and extent of liposome leakage were similar whether GSDMD was activated by DMB or cleavage (FIG. 3C). A bioluminescence resonance energy transfer (BRET) assay that is sensitive to the distance between a yellow fluorescent protein (YFP) fused at the N-terminus of GSDMD and luciferase (RLuc) fused at the C-terminus was used to examine the effect of DMB on GSDMD autoinhibition. DMB decreased the BRET signal compared with the DMSO control (FIG. 3D). Thus, these data show that DMB directly interacts with GSDMD in vitro and in cells to weaken NT-CT autoinhibition.

[0160] DMB modifies GSDMD at C191

[0161] DMB covalently modifies GLP-1R at C347 as shown by cryo-electron microscopy (cryo- EM). To understand how DMB activates GSDMD, nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS / MS) was used to analyze DMB-treated recombinant human GSDMD. Tryptic fragments indicated a quinoxaline adduct of Cysl91 through the carbon position 2 of DMB and the thiol of Cysl91, with limited modification of other Cys residues (FIG. 3E, 3F, 9C, and 9D). In fact, Cysl91 is a key unpaired Cys residue required for GSDMD oligomerization and pore formation that was recently shown to be palmitoylated. This Cys residue is conserved in GSDMD from different species, but not in other gasdermin family members (FIG. 9E). Consistently, DMB activated mouse GSDMD in the liposome leakage assay, albeit with ~5-fold reduced efficacy (FIG. 9F), and activated human GSDMD, but not other human gasdermins, suggesting that DMB acts selectively on GSDMD (FIG. 3G).

[0162] To confirm that DMB targets GSDMD at Cys 191, GSDMD alanine mutations of Cysl91, and of Cys38 as a control were generated evaluated in the liposome leakage assay. Although the DMB EC 50 values for WT and C38A GSDMD were both approximately 0.9 pM for activating GSDMD pore formation, C191A GSDMD was not activated by DMB (FIG. 9G). To confirm that DMB’s targeting of Cysl91 of GSDMD is responsible for its effect in cells, we constructed additional Cysl91 mutants of GSDMD and examined whether they caused pyroptosis, as assessed by PI uptake, when expressed in HEK293T cells. Although C191R and C191F GSDMD-NT induced cell death in HEK293T cells comparably to WT GSDMD-NT, the C191R and C191F full-length GSDMD mutants could not be activated by DMB (FIG. 3H, 31, S2H). DMB activated WT GSDMD, the D275 A mutant uncleavable by inflammatory caspases and caspase-8, or the C268G / D275A double mutant that is also uncleavable by ELANE (FIG. 3H, 31, 9H). Consistently, DMB activation of GSDMD to permeabilize liposomes was abrogated by GSDMD pretreatment with DSF, which covalently binds to Cysl91 (FIG. 3J). Thus, DMB and DSF compete for the same Cysl91 binding site and DMB activates full-length GSDMD pore formation without cleavage by selectively and covalently modifying Cysl91.

[0163] To dissect which regions of DMB are important in agonizing GSDMD, we performed a limited structure-activity relationship (SAR) study of some DMB analogs (FIGs. 10A-10C). These data showed that an electron withdrawing group (such as the methylsulfonyl moiety) at position 2 of the quinoxaline ring system strongly influenced the activity of the compounds, consistent with covalent bonding of DMB to GSDMD Cysl91 (FIG. 10D). Chemical groups at carbon positions 6 and 7 of DMB also had a strong influence on binding (FIG. 10E), indicating that noncovalent interactions between DMB and GSDMD also contribute to GSDMD activation.

[0164] DMB induces pyroptosis in mouse cancer cell lines in a GSDMD-dependent manner To study the effectiveness of DMB in mice and its GSDMD dependence, we generated GSDMD KO and GSDMD / GSDME double KO (dKO) clones of the GSDMD-expressing, cold triple negative breast cancer line EMT6 using CRISPR / Cas9 (FIGs. 11A, 11B). DMB and the apoptosis-inducing chemotherapy drug mitomycin C (MMC) were similarly potent at killing WT EMT6 cells in vitro as assessed by Cell-Titer Gio assay (FIG. 11C). However, DMB induced the characteristic “bubble-like” cell morphology and swelling of pyroptosis and SYTOX Green uptake, while MMC induced apoptotic blebbing with little SYTOX Green uptake (FIGs. 4A, 4B). DMB treated GSDMD KO EMT6 clones exhibited much less SYTOX green uptake than WT EMT6, shown for three independent clones (FIGs. 4A, 4B, 11D, HE). DMB also triggered release of LDH, a marker of cell membrane rupture during pyroptosis, beginning within an hour of treatment, in WT but not GSDMD KO EMT6 cells (FIG. 4C). GSDMD / GSDME dKO EMT6 clones were also resistant to pyroptosis by DMB measured by Sytox Green uptake and LDH release, shown for three independent clones (FIGs. 11F, 11G). By contrast, MMC caused much less and delayed LDH release in WT or GSDMD KO EMT6 (FIG. 4C), which might be due to secondary necrosis after apoptosis or GSDME-related conversion of apoptosis to pyroptosis since EMT6 cells express GSDME. Consistently, GSDMD / GSDME dKO EMT6 cells did not release LDH upon MMC treatment (FIGs. 11F, 11G).

[0165] Pyroptotic cells release ATP, a damage-associated molecular pattern (DAMP). Transient ATP release into supernatants after treatment with DMB , which peaked 2 and 3 h after adding DMB to EMT6 cells, but MMC did not trigger ATP release (FIG. 4D). Similarly, DMB, but not MMC, induced Sytox Green positivity, LDH release and ATP release in the GSDMD+colorectal carcinoma cell line CT26 (FIGs. 4E, 4F, 11H, 111). To examine if DMB-induced pyroptosis was dependent on caspases, SYTOX Green uptake in the presence or absence of the inflammatory caspase inhibitor AC-FLTD-CMK was compared (FIGs. 4G, 11J). Caspase inhibition did not affect DMB-induced pyroptosis in EMT6 cells. Thus, DMB rapidly induced pyroptosis and release of immunogenic DAMPs from a variety of GSDMD-expressing tumor types, independently of inflammatory caspase cleavage of GSDMD.

[0166] DMB induces tumor regression and enhances antitumor immunity that depends on GSDMD expression in the tumor

[0167] Whether DMB could reduce tumor mass in syngeneic mice orthotopically implanted with EMT6 tumors was then tested. To evaluate the role of gasdermin expression in the tumor, wsyngeneic BALB / c mice were implanted with WT, Gsdmd' / ', or Gsdmd^Gsdme' ' EMT6 cells, and treated or not with 10 mg / kg DMB weekly for two weeks when tumors became palpable. Mice were sacrificed when any tumor exceeded the allowed size. While DMB significantly suppressed WT EMT6 tumors, Gsdrnd^' and Gsdmd~ / 'Gsdme' / EM.T6 tumor growth was not significantly reduced by DMB (FIG. 5A). A longer experiment on Gsdmd'^ EMT6 tumors also confirmed the lack of significance in the effect by DMB (FIG. 12A). Although DMB did not change the numbers of tumor infiltrating lymphocytes (TILs) in WT EMT6 tumors (FIG. 12B), it significantly increased GzmB and perforin (PFN) expression and PMA + ionomycin- stimulated IFN-g and TNF-a production of CD8+and natural killer (NK) TILs (FIGs. 5B-5D). By contrast, DMB -treatment of G. dmd'' EMT6 tumors did not significantly alter TIL number or function. A similar reduction in tumor growth and increase in CD8+and NK TIL functions without a change in TIL numbers were seen after DMB treatment of syngeneic mice bearing subcutaneous implants of CT26 colorectal cancer (FIGs. 5E-5F, 12C).

[0168] At the dose used, chosen based on preclinical studies of DMB for diabetes, DMB treatment of EMT6-bearing mice did not cause systemic inflammation since plasma levels of inflammatory cytokines IL-10, IL-18, IL-6, or TNF-a were not elevated (FIG. 12D), or overt toxicity as evidenced by normal blood counts, even though monocytes and neutrophils express GSDMD (FIG. 12E).

[0169] To study whether GSDMD in the host contributes to the antitumor activity of DMB, KP lung adenocarcinoma tumors were implanted subcutaneously in syngeneic WT and Gsdmd ' C57BL / 6 mice since Gsdmd ' mice were available in this background. KP tumor growth was similarly inhibited and CD8+and NK TIL functions improved in both WT and Gsdmd mice (FIGs. 5G-5J, 12F), showing that GSDMD expression in host cells is dispensable for DMB’s effectiveness.

[0170] DMB’s direct killing is low and its effect requires the immune system

[0171] DMB suppressed the growth of multiple tumor cell lines and activated tumor immunity in vivo. To examine DMB’s direct cell killing and the role of immunity in tumor control, the effect of DMB in immunocompetent WT and immunodeficient NOD scid gamma (NSG) mice that lack functional lymphocytes was compared. Unlike in syngeneic WT mice, DMB did not suppress the growth of EMT6 tumors in NSG mice, indicating the DMB’s effectiveness was immune-mediated (FIGs. 6A, 6B, 13A). Next pyroptosis was compared in EMT6 tumors of NSG and WT BALB / C mice by examining in vivo PI uptake in tumor cells and tumorinfiltrating immune cells in mice sacrificed 10 min after injecting PI intravenously. Strikingly, DMB caused more cell death than control in CD45" CD3 tumor cells in WT mice, but not NSG mice, while CD45+CD1 lb+F4 / 80+tumor associated macrophages (TAMs) did not have significant increase in PI uptake even in WT mice (FIGs. 6C, 6D). These data suggest that DMB, at the dose used, directly induced a very low level of cell death, and selectively leads to tumor cell death and tumor growth control due to the action of lymphocytes. It is believed that this is a feedforward process initiated by DMB-induced tumor cell pyroptosis, and fueled by immune cell recruitment and activation that result in more pyroptosis, which in turn recruits and activates more immune cells.

[0172] DMB-treated tumor cells act as a tumor vaccine

[0173] To determine whether DMB induces immunogenic cell death, WT BALB / c mice were immunized with pyroptotic DMB-treated EMT6 cells or apoptotic MMC-treated EMT6 cells injected subcutaneously and then challenged them 8 days later with untreated EMT6 cells orthotopically injected into a mammary fat pad (FIG. 6E). Immunization with DMB-treated EMT6 markedly protected mice from tumor challenge (FIG. 6F). Three of 12 mice vaccinated with DMB-treated cells remained tumor- free for the duration of the study and the remaining mice had tiny tumors, while tumors grew in all the mice immunized with MMC-treated tumors. Moreover, the tumors that grew in mice immunized with DMB-treated cells were on average 6 times smaller than those in mice immunized with MMC-treated cells. Although there was interanimal variability in immune responses, CD8+, CD4+and NK TILs in the tumors of DMB- immunized mice showed significantly increased GzmB and PFN expression and stimulated IFN- g and TNF-a production (FIG. 6G-I). These results indicated that DMB can trigger immunogenic cell death that provides protective antitumor immunity.

[0174] DMB controls B16 tumors expressing human GSDMD

[0175] To further test the GSDMD-dependence of DMB-mediated tumor control and examine whether DMB activates human GSDMD in vivo, stable clones of B 16 melanoma were generated, which do not express endogenous Gsdmd (FIG. 13B), by expressing a human GSDMD-GFP fusion protein or GFP alone (FIGs. 7A and 13C). Human GSDMD-GFP-expressing, but not the control GFP-expressing, B 16 cells became sensitive to DMB-induced pyroptosis in vitro, as shown by PI uptake (FIGs. 7B and 13D). Pyroptosis occurred without GSDMD-GFP cleavage (FIG. 7C).

[0176] Next the B 16 clones were injected subcutaneously into syngeneic mice and treated with DMB every 3 days x 6 after tumors became palpable (5 days post-implantation, average tumor size 65.1 ± 21.7 mm3) (FIG. 7D). DMB strongly inhibited tumor growth and increased survival of mice implanted with B16 cells expressing human GSDMD-GFP (FIG. 7E), but had no significant effect on B16 cells that expressed GFP (FIG. 13E). All mice challenged with GFP- expressing B16 died (FIG. S6F), as did vehicle-treated mice challenged with B 16 expressing GSDMD-GFP but treated with vehicle (FIG. 7F). By contrast, 5 of 16 mice bearing B16 clones expressing GSDMD-GFP that were treated with DMB survived to the end of the experiment, and 3 of these mice were cured and had no palpable tumor (FIG. 7F). To examine the effect of DMB on immune cell infiltration into GSDMD-GFP-expressing tumors, sections of vehicle- or DMB-treated tumors were stained for CD3 and CD8 and for CD11c and MHCII to label infiltrating T cells and dendritic cells (DCs), respectively (FIG. 7G). The number of T cells and DCs per tumor area were highly enriched in DMB- vs vehicle-treated mice (8.3-fold more T cells, 25.2-fold more DCs). This strong effect of DMB in recruiting immune cell to B16 tumors expressing human GSDMD is in contrast to mouse tumor lines expressing endogenous mouse GSDMD (FIGs. 12B, 12C, 12F), but may be consistent with the higher efficacy of DMB to human than to mouse GSDMD (FIG. 9F). DMB treatment had no effect on mouse body weight (FIG. 13G). These data confirm that GSDMD is required for DMB’s enhanced tumor control and indicate that DMB could be effective against human tumors without causing unacceptable toxicity. Large tumors are more difficult to control in mouse models. To investigate whether DMB treatment could control larger tumors, B 16-GSDMD-GFP tumors were allowed to grow for 10 days when the average tumor size was 135.8 + 86.4 mm3to start DMB treatment once every 3 days for 6 times. Of the seven mice treated with DMB, two of the four mice bearing smaller tumors (45.9 and 73.0 mm3at time of initiating treatment) survived, whereas the remaining two mice with smaller tumors and the three mice with larger tumors (166.3 + 83.8 mm3at time of initiating treatment) died (FIG. 7H). A plot of the tumor sizes at the time of initiating treatment vs tumor sizes 3 days after the 6thDMB treatment, combining mice who initiated treatment 5 and 10 days post implantation, showed a strong correlation between tumor size and DMB responsiveness. This finding indicates that if warranted a GSDMD agonist may be combined with another therapy or used only after surgery or chemotherapy to reduce tumor burden for advanced disease.

[0177] Low dose DMB synergizes with checkpoint blockade

[0178] Next it was investigated whether DMB could induce responses to CPB in immunologically cold CPB -unresponsive 4T1E tumors. For this purpose, a low dose of DMB (1 mg / kg) was used at which DMB does not inhibit tumor growth on its own. BALB / c mice bearing palpable orthotopic tumors were treated with vehicle or anti-PD- 1 every two days or low dose DMB every week, alone or together. Anti-PD- 1 combined with DMB, but not anti-PD- 1 or DMB alone, significantly reduced tumor volume in comparison with vehicle (FIG. 14A). The combined treatment significantly decreased PD- 1 and CTLA-4 expression on antigen- experienced CD44+CD8+TILs, but anti-PD- 1 on its own only decreased PD-1 , which might have been due in part to shielding by the therapeutic antibody which recognizes the same epitope as the staining antibody (FIG. 14B). The combination of DMB and anti-PD- 1 increased the percentage of CD8+and NK TILs expressing GzmB and PFN and the percentage of CD8+TILs producing IFN-g and TNF-a after ex vivo stimulation (FIG. 14C), without significantly changing the number of TILs or other infiltrating immune cells (FIGs. 14D, 14E). None of the treated mice lost weight throughout the treatment (FIG. 14F), indicating no serious toxicity. Thus, it is contemplated that combining DMB with CPB will be effective and well tolerated in some situations.

[0179] Inflammatory cell death is emerging to be an important immune mechanism that bridges innate and adaptive immunity to stimulate antitumor immunity and potentiate CPB or CAR-T cell therapy. In addition, the clinical activity of some conventional and targeted antineoplastic agents currently used in humans is attributed to their role in re-establishing immune surveillance. These agents often induce inflammatory or immunogenic cell death, which promotes the anti- tumor functions of TILs that are associated with more favorable therapeutic responses in patients with cancer. Treatment with antineoplastic agents that do not predictably induce inflammatory cell death or antitumor immunity may be more prone to develop tumor resistance, relapse and metastatic disease.

[0180] The studies provide a new paradigm in immunotherapy by inducing a low level of pyroptosis to stimulate antitumor immunity. DMB is, to our knowledge, the first identified small molecule agonist capable of inducing pyroptotic inflammatory cell death by directly activating a gasdermin family protein without cleaving it. DMB works by targeting Cysl91 selectively on human and mouse GSDMD to trigger oligomerization and pore formation, by overcoming autoinhibition. It induces GSDMD-dependent pyroptotic cell death in cancer lines within hours at pM potency, which results in plasma membrane rupture to release LDH and DAMPs, such as extracellular ATP. DMB reduces tumor burden in mice challenged with breast, colorectal and lung tumors and melanoma and increases the cytotoxicity and cytokine secretion of tumorinfiltrating CD8+and NK cells. Tumor reduction depends on GSDMD expression in the tumor but not the host, supporting direct killing of cancer cells and tumor-autonomous activation of antitumor immunity. Importantly, DMB induces immunogenic cell death since vaccination with DMB-treated cells protected mice against subsequent tumor challenge. In addition, low dose DMB synergized with anti-PD-1 to suppress growth of a CPB-resistant tumor. Importantly, DMB recognized human GSDMD and showed anti-tumor activity in vivo against a tumor line expressing human GSDMD.

[0181] Direct induction of pyroptosis by GSDMD agonists may have several advantages over secondary pyroptosis induction by chemo- and radiotherapy. First, tumor cell-killing by chemotherapy and radiotherapy is mostly by apoptosis unless the tumor cell expresses GSDME. Apoptosis can be converted to pyroptosis if a tumor expresses GSDME, but GSDME expression in tumors is typically repressed by DNA hypermethylation. Thus, pyroptosis induction by these therapies can be useful but does not occur predictably. Second, induction of pyroptosis in a small number of tumor cells can stimulate widespread antitumor immune control. A low degree of pyroptosis induced directly by DMB is shown by the data described herein, since tumors implanted in NSG mice showed no significant increase in pyroptosis after treatment with DMB compared to vehicle. The strong vaccination effects from DMB-treated pyroptotic tumor cells indicate that the combined release of tumor antigens and DAMPs from pyroptotic tumor cells may be potent immunogens. Third, while radiotherapy and chemotherapy induce somatic mutations in tumor and healthy cells that can lead to drug resistance or secondary malignancies, pyroptotic agonists might more precisely trigger immunogenic cell death. Fourth, knowing the direct target of the GSDMD agonists could facilitate patient stratification depending on GSDMD expression. Fifth, the synergy of pyroptosis with anti-PD-1 or other CPB inhibitors could expand the range of immunotherapy responsive tumors. Finally, the effectiveness of GSDMD agonists against smaller tumors indicates that it can be utilized in combination with therapies that reduce tumor burden such as surgery, chemo- and radiotherapy for advanced disease.

[0182] While DMB covalently modifies GLP-1R in vitro, because GLP-1R expression is mostly limited to the pancreas, and DMB has protective effects, it is believed that this cross-reactivity may not cause toxicity or inhibit DMB -mediated GSDMD agonism. Lack of activation by DMB of full-length GSDMD C191R and C191F, which were equally active as NT when expressed in HEK293T cells, suggested that the pyroptotic effect of DMB is mediated by GSDMD agonism at residue Cysl91. The Cysl91 / 192 (human / mouse) residue targeted by DMB is also modified by GSDMD antagonists, including disulfiram, necrosulfonamide or dimethyl fumarate, as well as by endogenous fumarate.

[0183] Since this residue is now known to be palmitoylated during inflammasome activation or high redox stress, these antagonists likely function at least in part by competing with GSDMD palmitoylation. DMB modifies Cysl91 to overcome autoinhibition to activate GSDMD, and because of its hydrophobic nature, may also partially mimic palmitoylation. Thus, there is no coincidence that Cysl91 of GSDMD is targeted by inhibitors and activators alike because it is the central residue for GSDMD activation. Nonetheless, as a Cys-reactive compound, DMB likely targets other unknown cellular targets, which need to be functionally characterized. DMB is a tool compound useful for probing the role of GSDMD activation in vitro and in vivo, and along with the hits identified in these studies serve platforms for identifying GSDMD agonists with improved activity and selectivity for potential preclinical development.

[0184] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We Claim:

1. A compound having a structure:Formula I or a pharmaceutically acceptable salt thereof, wherein:A and B are independently substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl; at least one Ra and at least one Rb are independently a first electron withdrawing group and a second electron withdrawing group, respectively, independently selected from -S(O)2R, - S(O)R, -C(O)OR, -C(O)R’, -C(0)NRIDR2D, -OR, nitrile, -NRIDR2D (primary, secondary, or tertiary), nitro, haloalkyl, and halogen (e.g., -F, -Cl, -Br, -I), wherein R, R’, RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2- C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3- C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, preferably wherein at least one of Ra and Rb is -S(O)2R;Rc and Rd are independently hydrogen, halogen, azide, -S(O)2R, -S(O)R, -C(O)OR, - C(O)R’, -C(0)NRIDR2D, -OR, -SR, hydroxyl, nitrile, -NRIDR2D (primary, secondary, or tertiary), nitro, haloalkyl, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2- C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein R, R’, RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, preferably wherein at least one of Rc and Rd is a halogen; m, n, o, and p are independently integers from 1 to 4, such as 1, 2, 3, and 4, and preferably wherein the compound is not2. The compound of claim 1, wherein A and B are independently substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3- C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl.

3. The compound of claim 1 or 2, wherein A and B are independently substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group.

4. The compound of any one of claims 1 to 3, wherein A and B are independently substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, wherein one of A and B is substituted C6-C12 aryl group or unsubstituted Ce-Ci aryl group, and the other of A and B is substituted C2-C12 heteroaryl group or unsubstituted C2-C12 heteroaryl group.

5. The compound of any one of claims 1 to 4, having the structure:Rs, Re, R7, and Rs are independently hydrogen, halogen, azide, -S(O)2R, -S(O)R, - C(O)OR, -C(O)R’, -C(0)NRIDR2D, -OR, -SR, hydroxyl, nitrile, -NRIDR2D (primary, secondary, or tertiary), nitro, haloalkyl, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group,substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein R, R’, RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of R5, Rs, R7, and Rg is a halogen.

6. The compound of claim 5, wherein that R, R’ , RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of RID, and R2D are independently selected from substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3- C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl.

7. The compound of claim 5 or 6, wherein R, R’, RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3- C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, orunsubstituted C2-C12 heterocyclyl, wherein at least one of RID, and R2D are independently selected from substituted C1-C5 alkyl group or unsubstituted C1-C5 alkyl group.

8. The compound of any one of claims 5 to 7, wherein R is substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl.

9. The compound of any one of claims 5 to 8, wherein RID and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3- C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of RID, and R2D are independently selected from substituted C1-C5 alkyl group or unsubstituted C1-C5 alkyl group.

10. The compound of any one of claims 5 to 9, wherein RID is hydrogen and R2D is independently substituted C1-C5 alkyl group or unsubstituted C1-C5 alkyl group.

11. The compound of any one of claims 5 to 10, wherein (i) two of xl, x2, x3, and x4 are nitrogen, and the other of xl, x2, x3, and x4 are carbon, (ii) xl and x4 are nitrogen, and x2 and x3 are carbon.

12. The compound of any one of claims 5 to 11, having a structure:Formula 11113. The compound of any one of claims 5 to 12, wherein:(i) at least one of Re and R7 are a halogen,(ii) Re is a halogen and R7 is hydrogen,(iii) Re is hydrogen and R7 is a halogen, or(iv) Re and R7 are both halogens that may be the same or different.

14. The compound of any one of claims 5 to 13, wherein:(i) at least one of R5 and Rs are independently hydrogen, substituted C1-C5 alkyl group,unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, or unsubstituted C2-C5 alkynyl group,(ii) at least one of R5 and Rs are independently hydrogen, substituted C1-C5 alkyl group, or unsubstituted C1-C5 alkyl group,(iii) at least one of Rs and Rs are hydrogen.

15. The compound of any one of claims 5 to 14, having a structure:Formula IV16. The compound of any one of claims 5 to 15, wherein at least one of Re and R7 is a halogen.

17. The compound of any one of claims 5 to 16, wherein:(i) Re is a halogen and R7 is hydrogen,(ii) Re is hydrogen and R7 is a halogen, or(iii) Re and R7 are both halogens that may be the same or different.

18. The compound of any one of claims 1 to 17, having a structure:

20. The compound of any one of claims 1 to 19 in the form of an antibody-compound conjugate, polymer-compound conjugate, or small molecule-compound conjugate.

21. The compound of any one of claims 1 to 20 encapsulated in, on the surface of, or both, a microparticle or nanoparticle.

22. A pharmaceutical composition comprising the compound of any one of claims 1 to 21 and a pharmaceutically acceptable excipient.

23. A method of treating a cancer, the method comprising providing the pharmaceutical compound of claim 22.

24. A method of treating a cancer, the method comprising providing a compound having a structure:Formula I or a pharmaceutically acceptable salt thereof, wherein:A and B are independently substituted Ce-Cn aryl group, unsubstituted Ce-Cn aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl; at least one Ra and at least one Rb are independently a first electron withdrawing group and a second electron withdrawing group, respectively, independently selected from -S(O)2R, - S(O)R, -C(O)OR, -C(O)R’, -C(0)NRIDR2D, -OR, nitrile, -NRIDR2D (primary, secondary, ortertiary), nitro, haloalkyl, and halogen (e.g. -F, -Cl, -Br, -I), wherein R, R’, Rm, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2- C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted Ce-Cn aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3- C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, preferably wherein at least one of Ra and Rb is -S(O)2R;Rc and Rd are independently hydrogen, halogen, azide, -S(O)2R, -S(O)R, -C(O)OR, - C(O)R’, -C(0)NRIDR2D, -OR, -SR, hydroxyl, nitrile, -NRIDR2D (primary, secondary, or tertiary), nitro, haloalkyl, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2- C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein R, R’, RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, preferably wherein at least one of Rc and Rd is a halogen; m, n, o, and p are independently integers from 1 to 4, such as 1, 2, 3, and 4.

25. The method of claim 24, wherein A and B are independently substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3- C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl.

26. The method of claim 24 or 25, wherein A and B are independently substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group.

27. The method of any one of claims 24 to 26, wherein A and B are independently substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, wherein one of A and B is substituted C6-C12 aryl group or unsubstituted C6-C12 aryl group, and the other of A and B is substituted C2-C12 heteroaryl group or unsubstituted C2-C12 heteroaryl group.

28. The method of any one of claims 24 to 27, wherein the compound has the structure:wherein: xl, x2, x3, and x4 are independently carbon or nitrogen;Ri, R2, R3, and R4 are independently absent, -S(O)2R, -S(O)R, -C(O)OR, -C(O)R’, - C(0)NRIDR2D, -OR, nitrile, -NRIDR2D (primary, secondary, or tertiary), nitro, haloalkyl, and halogen (e.g., -F, -Cl, -Br, -I), wherein R, R’, RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2- C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of Ri, R2, R3, and R4 is -S(O R, and the other of Ri, R2, R3, and R4 is -NRIDR2D, wherein Ri, R2, R3, and R4 are present or absent according to valency;R5, Re, R7, and Rs are independently hydrogen, halogen, azide, -S(O)2R, -S(O)R, - C(O)OR, -C(O)R’, -C(0)NRIDR2D, -OR, -SR, hydroxyl, nitrile, -NR1DR2D (primary, secondary, or tertiary), nitro, haloalkyl, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted Ce-Ci?aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein R, R’, RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of Rs, Re, R7, and Rs is a halogen.

29. The method of claim 28, wherein that R, R’ , RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C2-C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, unsubstituted C2-C5 alkynyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of RID, and R2D are independently selected from substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3- C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl.

30. The method of claim 28 or 29, wherein R, R’ , RID, and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C6-C12 aryl group, unsubstituted C6-C12 aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3- C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of RID, and R2D are independently selected from substituted C1-C5 alkyl group or unsubstituted C1-C5 alkyl group.

31. The method of any one of claims 28 to 30, wherein R is substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3-C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl.

32. The method of any one of claims 28 to 31 , wherein RID and R2D are each independently selected from hydrogen, substituted C1-C5 alkyl group, unsubstituted C1-C5 alkyl group, substituted Ce-Cn aryl group, unsubstituted Ce-Cn aryl group, substituted C2-C12 heteroaryl group, unsubstituted C2-C12 heteroaryl group, substituted C3-C12 cycloalkyl, unsubstituted C3-C12 cycloalkyl, substituted C3-C12 cycloalkenyl, unsubstituted C3-C12 cycloalkenyl, substituted C3- C12 cycloalkynyl, unsubstituted C3-C12 cycloalkynyl, substituted C2-C12 heterocyclyl, or unsubstituted C2-C12 heterocyclyl, wherein at least one of RID, and R2D are independently selected from substituted C1-C5 alkyl group or unsubstituted C1-C5 alkyl group.

33. The method of any one of claims 28 to 32, wherein RID is hydrogen and R2D is independently substituted C1-C5 alkyl group or unsubstituted C1-C5 alkyl group.

34. The method of any one of claims 28 to 33, wherein (i) two of xl , x2, x3, and x4 are nitrogen, and the other of xl, x2, x3, and x4 are carbon, (ii) xl and x4 are nitrogen, and x2 and x3 are carbon.

35. The method of any one of claims 28 to 34, wherein the compound has a structure:Formula III36. The method of any one of claims 28 to 35, wherein:(i) at least one of Rs and R7 are a halogen,(ii) Re is a halogen and R7 is hydrogen,(iii) Re is hydrogen and R7 is a halogen, or(iv) Re and R7 are both halogens that may be the same or different.

37. The method of any one of claims 28 to 36, wherein:(i) at least one of Rs and Rs are independently hydrogen, substituted Ci-Cs alkyl group, unsubstituted C1-C5 alkyl group, substituted C -C5 alkenyl group, unsubstituted C2-C5 alkenyl group, substituted C2-C5 alkynyl group, or unsubstituted C2-C5 alkynyl group,(ii) at least one of Rs and Rs are independently hydrogen, substituted C1-C5 alkyl group, or unsubstituted C1-C5 alkyl group,(iii) at least one of Rs and Rs are hydrogen.

38. The method of any one of claims 28 to 37, wherein the compound has a structure:Formula IV39. The method of any one of claims 28 to 38, wherein at least one of Re and R? is a halogen.

40. The method of any one of claims 28 to 39, wherein:(i) Re is a halogen and R? is hydrogen,(ii) Re is hydrogen and R? is a halogen, or(iii) Re and R? are both halogens that may be the same or different.

41. The method of any one of claims 24 to 40, wherein the compound has a structure:

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