Efficient identification of new small molecules targeting succinate dehydrogenase

Abstract

Efficient lung cancer treatment is challenging due to the lack of therapeutic targets. The succinate dehydrogenase (SDH) enzyme plays a critical metabolic role as an intermediate between the citric acid cycle and the electron transport chain associated with the cancer disease state. Although several existing compounds have been applied to target metabolic diseases in vitro, modulating SDH for lung cancer treatment has been elusive. By integrating the AtomNet® technology for compound identification with mitochondria- and cell-based enzyme activity assays, this disclosure demonstrates identification of four new small molecules that bind directly to SDH subunits in lung cancer cells. Mechanistically, treatment with these small molecules increased reactive oxygen species and DNA damage in lung cancer cell lines. Functionally, these small molecules reduced short-term and long-term malignant growth of lung cancer cell lines in vitro. This study provides a new therapeutic approach for targeting lung and other types of cancers through modulation of SDH.

Description

Attorney Docket No.: 10669-407PC0 Patent EFFICIENT IDENTIFICATION OF NEW SMALL MOLECULES TARGETING SUCCINATE DEHYDROGENASE BACKGROUND 1. Field of the Invention

[0001] The invention relates to the general field of medicine and in particular to small molecule drug identification and methods of treatment by modulating the succinate dehydrogenase enzyme complex. 2. Background of the Invention

[0002] Metabolic homeostasis is critical to maintaining normal cell proliferation. Aberrant changes in metabolic enzymes and metabolites cause various diseases including cancer. The succinate dehydrogenase enzyme complex (SDH) is a heterotetrameric, mitochondrial inner membrane-bound protein with constituent roles in both the citric acid cycle (TCA) and the electron transport chain (ETC). The enzyme consists of four major subunits: SDHA, SDHB, SDHC, and SDHD, in addition to several cofactors necessary for the assembly of the functional complex. In the TCA cycle, SDH catalyzes the reduction of succinate to fumarate, coupling the transfer of electrons from this reaction to the ETC where ubiquinone is reduced to ubiquinol.

[0003] Efficient lung cancer treatment remains challenging to treat due to the lack of therapeutic targets. Non-small cell lung cancer (NSCLC) is among the most common types of cancer worldwide. Lung cancers also represent the leading cancer-related cause of death. NSCLC and small-cell lung cancers pose great difficulty for targeted therapeutic intervention in a clinical setting as patients often present with one or more heterozygous genetic mutations which affect drug response. NSCLC often develops resistance to standard chemotherapeutic agents; resistance has even been observed to third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors which have traditionally represented a frontline standard of treatment. SUMMARY OF THE INVENTION

[0004] Thus, there exists a need in the art for new compounds that target succinateAttorney Docket No.: 10669-407PC0 Patent dehydrogenase. This invention provides an invention that has identified four new small molecules that can effectively modulate succinate dehydrogenase (SDH) enzymatic activity, degrade protein abundance, and generate cytotoxic reactive oxygen species (ROS). This can provide a new therapeutic approach to target lung and other types of cancers. The data supports that these molecules are more effective in inhibiting lung cancer colony formation than the known SDH inhibitor dimethyl malonate (DMM).

[0005] In particular embodiments, the present invention relates to a succinate dehydrogenase modifying compound selected from the group consisting of

[0006] dehydrogenase inhibitor small molecule compounds, comprising screening a chemical library at the interface of chains B, C, and D of 4YTP. This screening preferably is a screen of the interface of chains B, C, and D of 4YTP is defined by the residues Chain B; P169, W172 & W173; Chain C: I30, W35, M39, S42, I43, R46 & I50; Chain D: V87, D90 & Y91.Attorney Docket No.: 10669-407PC0 Patent

[0007] In certain embodiments, the invention relates to a method of treating a disease or condition involving mutation or dysregulation of the succinate dehydrogenase pathway in a subject in need thereof, comprising administrating one or more of the compounds of claim 1. The disease or condition can be cancer and preferably is a cancer selected from the group consisting of paraganglioma, pheochromocytoma, renal carcinoma, gastrointestinal stroma tumor, thyroid cancer, colorectal cancer, ovarian cancer, and lung cancer. Most preferably, the cancer is lung cancer. In other embodiments, the disease or condition is selected from the group consisting of Leigh syndrome, ataxia, leukodystrophy, and premature aging. In preferred embodiments, the method of treatment is more effective than treatment with dimethyl malonate. BRIEF SUMMARY OF THE DRAWINGS

[0008] Certain embodiments are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings.

[0009] FIG.1 shows a western blot for SDHA, SDHB, and SDHD expression across various non-small cell lung cancer and immortalized (AALE) cell lines with or without KRAS and EGFR mutations.

[0010] FIG.2A, FIG.2B, and FIG.2C are graphs showing the blot intensity, normalized to that in AALE cells. β-Actin was used as a loading control (n = 3).

[0011] FIG.3 presents immunofluorescent staining and quantification for SDHC in NSCLC and AALE cells. The fluorescence was quantitated by ImageJ and the fluorescence intensity in AALE cells was calibrated as 1. See FIG.4.

[0012] FIG.4 is a bar graph showing the fluorescence quantitated by ImageJ and the fluorescence intensity in AALE cells.

[0013] FIG.5A, FIG.5B, FIG.5C, and FIG.5D are graphs that show the differential gene expression for SDHA, SDHB, SDHC, and SDHD, respectively, in lung adenocarcinoma patients-derived tumor and tumor-adjacent normal tissues from The Cancer Genome Atlas database.

[0014] FIG.6A, FIG.6B, FIG.6C, and FIG.6D shows quantitative reverse transcription PCR analysis of gene expression for SDHA, SDHB, SDHC, and SDHD in NSCLC and AALE cells.

[0015] FIG.7 shows the overall structure of PDB 4YTP chains A, B, C, and D.Attorney Docket No.: 10669-407PC0 Patent

[0016] FIG.8 shows a zoomed-in version (FIG.8A) and surface representation (FIG.8B) of the binding site of interest.

[0017] FIG.9 is a flow diagram showing the workflow overview of screening and selecting small molecules using the AtomNet® model.

[0018] FIG.10 is a diagram showing the principles of measuring the SDH enzyme activity.

[0019] FIG.11 is a flow chart demonstrating the small molecule selection process and research design.

[0020] FIG.12 shows the differential SDH enzyme activity between H358 and AALE cells.

[0021] FIG.13 is a graph that shows the screening of effects of small molecules on absolute and relative SDH enzyme activities in isolated mitochondria from AALE cells.

[0022] FIG.14 shows the relative quantification of per cell SDH activity modulation by small molecules (H02, C06, and E08) in AALE cells treated at 50 mM for four hours.

[0023] FIG.15 shows the relative quantification of per cell SDH activity modulation by small molecules (H02, C06, H07, E08, and DMM) in H358 cells treated at 30~50 mM for four hours.

[0024] FIG.16 presents a thermal shift assay by western blotting for SDHA (FIG.16A), SDHB (FIG.16B), and SDHD (FIG.16C) on H358 cells treated with or without small molecules H2 / Z14, H7 / Z17, E8 / Z71, C6 / Z96, and DMM.

[0025] FIG.17A, FIG.17B, FIG.17C, FIG.17C, and FIG.17E show immunofluorescent staining for PBS, SDHA, SDHB, SDHC, and SDHD, respectively, of H1975 cells treated with or without small molecules (H2 / Z14, H7 / Z17, E8 / Z71, C6 / Z96) for six hours. Cells were co-stained with primary anti-SDHB and anti-SDHC antibodies or anti-SDHA and anti- SDHD antibodies, followed by secondary IgG conjugated with Alexa Fluor 488 or 555. The cells were then counterstained with DAPI and imaged by confocal microscopy (scale bar = 20 mm; n = 6 replicates).

[0026] FIG.18A, FIG.18B, FIG.18C, and FIG.18D show the quantitation of immunofluorescence intensity for each SDH subunit in cells treated with H2 / Z14, H7 / Z17, E8 / Z71, and C6 / Z96, respectively, compared to PBS (n = 6 replicates).

[0027] FIG.19 presents a western blot for HIF-1a and NRF2.

[0028] FIG.20 presents data on the quantitation of the results in FIG.19 across 12 NSCLC and immortalized AALE cell lines.

[0029] FIG.21 presents a western blot for HIF-1a and NRF2.Attorney Docket No.: 10669-407PC0 Patent

[0030] FIG.22 presents data on the quantitation in H1975 cells treated with small molecules (H2 / Z14, H7 / Z17, E8 / Z71, C6 / Z96, DMM, DMOG, DES) and vehicle (DMSO).

[0031] FIG.23 shows confocal imaging for ROS by immunofluorescent staining in H1975 cells treated with H2 / Z14 for 0, 2, 4, and 6 hours).

[0032] FIG.24 shows confocal imaging for ROS by immunofluorescent staining in H1975 cells treated with H7 / Z17, E8 / Z71, and C6 / Z96 for 6 hours.

[0033] FIG.25 presents immunofluorescence staining for p21Cip1in H1975 cells treated with C6 / Z96, H2 / Z14, E8 / Z71, H7 / Z17, or a vehicle (PBS) for four hours as indicated.

[0034] FIG.26 presents quantitation of the staining in FIG.25.

[0035] FIG.27 presents immunofluorescence staining for g-H2AX in H1975 cells treated with C6 / Z96, H2 / Z14, E8 / Z71, H7 / Z17, or a vehicle (PBS) for four hours as indicated.

[0036] FIG.28A and FIG.28B present quantitation of the staining in FIG.27.

[0037] FIG.29 shows the relative cell viability of cell lines (H1975 (FIG.29A), H358 (FIG. 29B), H460 (FIG.29C), H441 (FIG.29D), and H3255 (FIG.29E)) treated with C6 / Z96, H2 / Z14, E8 / Z71, H7 / Z17, or a vehicle (PBS) for three days.

[0038] FIG.30 presents data for fluorescent microscopy of GFP-labelled H358 cells incubated with increasing concentrations of small molecules C6 / Z96, H2 / Z14, E8 / Z71, or H7 / Z17 (0, 10, 300 µM) for 14 days.

[0039] FIG.31 presents quantitation of the staining in FIG.30. DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION 1. Overview

[0040] The present invention relates to identification of new small molecules from 96 predicted candidates by integrating in-silico compound design with mitochondria- and cell- based enzyme activity assays. A cellular thermal shift assay confirmed that certain of the small molecules bound directly to SDH subunits in lung cancer cells. Mechanistically, treatment with the small molecules increased reactive oxygen species and DNA damage in lung cancer cell lines in vitro. Functionally, the small molecules reduced short-term and long-term malignant growth of lung cancer cell lines in vitro.

[0041] Due to the nature of complex interactions as an intermediate between these two distinct metabolic processes, SDH is uniquely positioned as a gatekeeper for the metabolic dysfunction observed in various cancers and genetic diseases. SDHA mutations areAttorney Docket No.: 10669-407PC0 Patent associated with neurological disorders due to mitochondrial diseases such as Leigh Syndrome and mitochondrial encephalopathy. Loss-of-function mutations in SDHB, SDHC, and SDHD are correlated with hereditary paraganglioma and pheochromocytoma, renal carcinoma, and gastrointestinal stromal tumor (GIST). Accumulation of succinate is associated with tumor invasiveness and drug resistance.

[0042] These findings have supported the classification of SDH as a tumor suppressor and the succinate substrate as an oncometabolite, to which excess accumulation mediates tumorigenesis through inhibition of hypoxia-inducible factor (HIF)-1a prolyl hydroxylase (PHD). Under normal oxygen conditions, PHD is known to induce the degradation of HIF- 1a. Under elevated levels of cytosolic succinate, PHD is inactivated leading to the activation and stabilization of HIF-1a, which results in the activation of HIF response elements (HRE) in the genome and induction of a pseudohypoxic state. Succinate accumulation is typically due to loss of function mutations in one or more subunits comprising the SDH enzyme; whether post-translational modifications or endogenous inhibition of SDH play a role in the enzymatic efficiency of SDH is of major interest to our ongoing research in this area.

[0043] The four primary subunits of the SDH complex are nuclear encoded, with human SDHA on chromosome 5, SDHB and SDHC on chromosome 1, and SDHD on chromosome 11. The subunits must therefore be transcribed and translocated separately, followed by maturation and assembly into the mitochondria membrane with the help of several auxiliary cofactors. Interestingly, many disease patients presenting with apparent SDH deficiencies do not harbor any mutations of the SDH subunits, further implicating the role of auxiliary factors in mediating SDH-associated pathologies. Further regulation of the complex through RNA editing, RNA modification, and alternative splicing has been shown to induce differential effects on SDH assembly, maturation, and enzymatic function. Therefore, regulation of the complex can be achieved at multiple levels.

[0044] One way that we can improve treatment efficacy and patient outcomes for NSCLC is by conducting basic research into the metabolic dysfunction underlying tumorigenesis. Elucidating the molecular mechanisms of the tumorigenic transformation of healthy to cancerous cells, as well as the mechanisms of acquired drug resistance, may be of great benefit to better understanding the complexity of the lung cancer metabolic disease state as we continue to develop more effective treatments. Furthermore, if small molecules targeting the SDH can effectively restore its enzymatic function in disease states where it has beenAttorney Docket No.: 10669-407PC0 Patent disrupted, it may prevent the buildup of succinate and further downstream metabolic dysfunction. Conversely, if SDH is inhibited by small molecules in normal noncancerous cells, it can provide insight into the role of this protein in the mechanism of oncogenic transformation. Additionally, inhibition of the SDH complex by the anticancer agent lonidamine has been shown to promote cell death in melanoma via suppression of the pentose phosphate pathway and induction of critical reactive oxygen species (ROS) levels. This indicates that sufficient disruption of complex II may have potential as a therapeutic mechanism.

[0045] To explore this possibility, Enamine’s library, consisting of 2.5 million small molecules was used for screening with the AtomNet® model to identify small molecules that target the ubiquinone binding pocket of the SDH complex. The virtual screen was performed using the AtomNet neural network model at the interface of chains B, C, and D of 4YTP. The chemical library, Enamine, was used as a small molecule library. The results from this screen were filtered using Lipinski's rule of five, the scoring from the AtomNet model, physio-chemical property filters, and manual removal of potential electrophiles. Experimentally, these small molecules are capable of modulating SDH complex enzymatic activity at the cell-based and mitochondrial-based level. Ninety-six top candidates were identified and ordered for in vitro screening. Primary screening was performed directly in isolated mitochondria via 2,6-dichlorophenolindophenol (DCPIP) colorimetric assay to quantify the SDH activity in immortalized cells. This was followed by a screening in cell- based assays for immortalized and cancer cell lines. Our data showed that 67 out of 96 candidates affect electron transfer in SDH-catalyzed reactions. Using the cutting-edge technology of thermal shift assay, we found that these small molecules bind directly to SDHD and SDHB and promote stabilization of these subunits.

[0046] Unexpectedly, four small molecules also increase thermal stability of SDHA, indicating protein-protein interactions might be a primary component of the mechanism of action. A cellular thermal shift assay confirmed that these small molecules bind directly to SDH subunits in lung cancer cells. Mechanistically, treatment with these small molecules increased reactive oxygen species and DNA damage in lung cancer cell lines. Functionally, these small molecules reduced short-term and long-term malignant growth of lung cancer cell lines in vitro. These top four molecules also induced dysregulated oxidative stress and DNA damage, leading to reduced cell survival in NSCLC cell lines in vitro.Attorney Docket No.: 10669-407PC0 Patent 2. Definitions

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although various methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. However, the skilled artisan understands that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention. Moreover, as measurements are subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration and any other measurements, quantities or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary.

[0048] In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the exclusion of any other item, element or step or group of items, elements or steps. Furthermore, the indefinite article “a” or “an” is meant to indicate one or more of the item, element or step modified by the article.

[0049] As used herein, the term “about” means plus or minus 20 percent of the recited value, so that, for example, “about 0.125” means 0.125 ±0.025, and “about 1.0” means 1.0 ±0.2. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in specific non-limiting examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements at the time of this writing. Furthermore, unless otherwise clear from the context, a numerical value presented herein has an implied precision given by the least significant digit. Moreover, all ranges disclosed herein are to be understood to encompass any and all sub-ranges subsumed therein. For example, a range of "less than 10"Attorney Docket No.: 10669-407PC0 Patent can include any and all sub-ranges between (and including) the minimum value of zero and the maximum value of 10, that is, any and all sub-ranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 4.

[0050] As used herein, the term “cancer” relates to any hyperproliferative condition, and particularly includes paraganglioma, pheochromocytoma, renal carcinoma, gastrointestinal stromal tumor, thyroid cancer, colorectal cancer, ovarian cancer, and lung cancer.

[0051] The terms “subject,” “individual,” “host,” and “patient,” are used interchangeably herein to refer to an animal being treated with one or more enumerated compounds as taught herein, including, but not limited to, simians, humans, avians, felines, canines, equines, rodents, bovines, porcines, ovines, caprines, mammalian farm animals, mammalian sport animals, and mammalian pets. A suitable subject for the invention can be any animal, preferably a human, that is suspected of having, has been diagnosed as having, or is at risk of developing a disease that can be ameliorated, treated or prevented by administration of one or more enumerated compounds.

[0052] The term “treating” or “treatment of’ as used herein refers to providing any type of medical management to a subject. Treating includes, but is not limited to, administering a composition comprising one or more active agents to a subject using any known method for purposes such as curing, reversing, alleviating, reducing the severity of, inhibiting the progression of, or reducing the likelihood of a disease, disorder, or condition or one or more symptoms or manifestations of a disease, disorder or condition.

[0053] A “therapeutically effective amount” refers to an amount which, when administered in a proper dosing regimen, is sufficient to reduce or ameliorate the severity, duration, or progression of the disorder being treated (e.g., cancer), prevent the advancement of the disorder being treated (e.g., cancer), cause the regression of the disorder being treated (e.g., cancer), or enhance or improve the prophylactic or therapeutic effects(s) of another therapy. The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations per day for successive days. 3. Embodiments of the invention

[0054] Cancer cells have disordered metabolic pathways. This characteristic can be used as aAttorney Docket No.: 10669-407PC0 Patent target for anticancer therapy. One pivotal enzyme involved in these metabolic pathways is the succinate dehydrogenase (SDH) pathway, which regulates cell respiration in mitochondria. Unfortunately, progress in SDH-based targeted therapy has been limited due to a lack of effective small molecules that target the enzyme. This disclosure relates to methods for halting or reducing tumor growth by modulating the SDH enzyme. Through in silico and in vitro screening assays, four promising compounds were identified and studied.

[0055] Modulating enzyme activity with these compounds caused protein dysregulation in SDH by inducing cytotoxic reactive oxygen species and DNA damage. This resulted in a promising approach to block cancer cells from using SDH to support their growth. This approach may have immense potential for companies interested in developing antibody-drug conjugates (ADCs) by combining our newly discovered small molecules with antibodies to deliver them specifically to tumor sites. The implications of this study on SDH-based therapy for lung cancer extend far beyond our specific focus. The findings presented here possess immense potential for effectively targeting SDH in other cancers, mitochondrial disease, and lung fibrosis, thereby making a significant contribution to the wider field.

[0056] SDH is uniquely poised as a master regulator of cellular metabolism. Dysfunction of this enzyme may contribute to tumorigenicity of NSCLC. AALE cells were used to compare to cancer models to understand the role of SDH dysfunction. Gene expression levels of SDH subunits (SDHA, SDHB, and SDHC) in tissues from the TCGA dataset are higher in lung adenocarcinoma than in tumor-adjacent normal lung, inconsistent with the current classification of SDH as a tumor suppressor in some types of cancers. In contrast, the majority of lung adenocarcinoma cell lines tested in our study exhibited lower mRNA expression than the immortalized cell line AALE. This indicates that SDH abundance in the lung adenocarcinoma might confer to stromal cells in the tumor microenvironment. For example, dysregulated lung fibroblasts support oncogenic transformation and progression in epithelial cells by upregulating hepatocyte growth factor and modifying extracellular matrix.

[0057] Overgrowth of fibroblasts leading to lung fibrosis is highly related to lung cancer development. Targeting lung fibrosis might be a new strategy to prevent pathogenesis of lung fibrosis toward malignancy, although the therapeutic target against fibrosis is still not fully understood. A recent study showed that upregulated SDHA contributed to lung fibrosis. Targeting SDHA with a new SDHA small molecule dye inhibitor (IR-780) prevents fibrosis formation and respiratory dysfunction by increasing ROS levels in myoblast and inducingAttorney Docket No.: 10669-407PC0 Patent cellular apoptosis. These can be relevant as a new strategy to target SDH to prevent fibrosis- induced lung cancer. Recent cutting-edge technology for single-cell transcriptomic analysis will increase our understanding of stromal SDH in lung cancer. Furthermore, our data suggests that in NSCLC, the role of SDH may not be that of a simple and straightforward tumor suppressor, but rather an enzymatic fine tuner of the metabolic processes that promote cancer cell survival and reprogramming.

[0058] Recent advances in in-silico drug design methods have enabled substantial advancements in therapeutic development for cancer and other disorders. Here, small molecules targeting SDH were screened via proprietary computational software, enabling the identification of 96 small molecules for subsequent enzyme and cell-based screens. Typically, small molecule drug candidates are categorized as either agonists or antagonists, based on pharmacological effects at the target receptor. Recent studies have identified additional complexities of cellular signaling, enabling additional modalities for small molecules in the stabilization or degradation of the target.

[0059] In the present study, we screened SDH-targeting small molecules by initially quantifying SDH enzyme activity in the presence of these molecules. The vast majority of these compounds inhibited SDH enzymatic function. Surprisingly, several molecules increased SDH activity. We advanced a handful of molecules with the most potent modulatory effects on SDH activity for subsequent cell-based screening and characterization. Despite relatively normal SDHA protein abundance and RNA expression levels, the enzymatic activity of SDH in H358 cells was drastically reduced compared to AALE cells, indicating functional impairment of the complex through an additional mechanism, such as post-translational modifications, degradation, or regulation of subunit transport and assembly.

[0060] The transport and assembly of the complete SDH complex is a highly coordinated cellular process requiring support from multiple cofactors and transport proteins, providing numerous points for potential dysfunction. In H358 cells, compounds H2 / Z14 and C6 / Z96 showed potent inhibition of SDH activity, while H7 / Z17 and E8 / Z71 increased SDH activity. In the present study, SDHA displayed the lowest melting temperature (41.2°C) of the subunits, suggesting this may be a rate-limiting component of functional SDH complex assembly. All SDH-targeting lead compounds increased the Tm of SDHA, leading to enhanced stability. SDHB thermal stability was enhanced by H7 / Z17 only, while SDHD thermal stability was enhanced by all compounds other than E8 / Z71. Together, these resultsAttorney Docket No.: 10669-407PC0 Patent suggest direct binding of SDH by small molecules resulting in the stabilization of complex subunits.

[0061] Immunofluorescent imaging of the subunits revealed an increase of SDHB and SDHC protein levels upon H2 / Z14 treatment and decrease in SDHA and SDHD protein levels. The small molecules could be binding to the SDHA and SDHD components causing their degradation and apparent low protein abundance. Moreover, H2 / Z14 resulted in elevation of DNA damage proteins and ROS levels in the cells. These results suggest that the small molecules could be binding to two particular subunits, leading to their degradation, which might be disrupting the function of the entire complex resulting in other toxic effects in the cells like enhanced DNA damage protein. Therefore, compounds which demonstrated inhibitory effects on SDH activity may in fact reduce SDH acutely through other mechanisms such as polyubiquitination and targeted degradation or inhibition of functional complex maturation and assembly. Likewise, compounds with a stimulatory effect on SDH activity may act to enhance complex assembly and stabilization or prevent degradation.

[0062] Hypoxia is considered both a driver and a consequence of oncogenesis. Expression of HIF-1α is correlated to invasiveness, drug resistance, and worse prognosis in lung cancer. Some inhibitors of HIF-1α have demonstrated efficacy in reducing tumor cell proliferation, while other studies found that HIF-1α silencing led to increased necrosis and did not reduce tumor proliferation. The differential effects observed in these studies highlight the complexity of HIF-1α as a master regulator of hypoxia in lung cancer. Here, we demonstrate the basal induction of HIF-1α in all NSCLC lines sampled. Notably, this increase in HIF-1α was associated with a decrease in NRF2, a master regulator of cellular resistance to oxidative stress, in all cancer cell lines apart from H460. H460 cells have been shown to exhibit pathologic NRF2 gain of function, and RNAi-mediated lowering of NRF2 resulted in increased ROS and sensitivity to radiotherapy. In H1975 cells, all four SDH modulating compounds induced HIF-1α. DMOG induced HIF-1α as expected, while known SDH substrate DES and inhibitor DMM had no effect on HIF-1α. All SDH modulators and DMOG induced HIF-1α without a corresponding increase in NRF2 levels, suggesting that lung cancer cells may be unable to properly activate antioxidant pathways in response to acute hypoxia resulting from pharmacological targeting of SDH. H2 / Z14 treatment led to an increase of ROS. A potential mechanism could be direct binding to SDH subunits mayAttorney Docket No.: 10669-407PC0 Patent induce hypoxia resulting in increased ROS. Small molecule induced ROS and DNA damage may affect the viability and survival of the cancer cells.

[0063] Inhibition of SDH has been previously demonstrated to display efficacy in cancer. Here, SDH modulators displayed an acute reduction of cell viability in multiple NSCLC cell lines of varying mutation status. Long term effects on cell viability were assessed by colony formation assay, a common in vitro technique to assess the ability of a single cell to form clusters. This demonstrates the intrinsic ability for the cell to self-renew, proliferate, and resist cell death. Our adapted GFP colony formation assay data validates the ability of SDH modulators to disrupt NSCLC proliferation and colony formation, possibly through induction of ROS accumulation and SDH subunit degradation, suggesting a possible future therapeutic application either standalone or in combination with existing chemotherapies. Interestingly, only inhibitory compounds H2 / Z14 and C6 / Z96 reduced colony formation at 10 µM, while all four SDH modulators reduced colony formation at 300 µM. Established SDH inhibitor, DMM, had no significant effect on colony formation at 300 µM, indicating an enhanced therapeutic response of these newly characterized SDH modulators.

[0064] In the circumstances of an increase of SDH subunits, some cancer subtypes upregulate these proteins to create a dysregulated metabolic state that increases their survival and growth. Targeting and decreasing these upregulated components can be important for combination cancer drug therapy. In the circumstances of decreasing SDH subunits, these small molecules also could provide insight into possible research for diseases that have an innate low SDH expression (SDH deficiency in neurological disorders). The use of the stimulatory small molecules could provide researchers with new tools to elucidate additional possible mechanisms.

[0065] Overall, this study introduces four small molecules that effectively target the SDH by integrating AtomNet neural network and experimentally filtering the top candidates. This aims to create a foundation for small molecule approach in targeting the SDH for cancer metabolic research. Treatment involves administration of one or more of the compounds to a subject suffering from a disease or condition that involves mutation or dysregulation of SDH or the SDH pathway.

[0066] This invention provides new small molecules identified for treating lung and other types of cancers by effectively modulating succinate dehydrogenase (SDH) enzymatic activity. These compounds inhibit lung cancer colony formation better than the SDHAttorney Docket No.: 10669-407PC0 Patent inhibitor dimethyl malonate (DMM) and can be used to treat any cancers with mutations or dysregulation in the SDH components. Examples of such cancers include paraganglioma, pheochromocytoma, renal carcinoma, gastrointestinal stromal tumor, thyroid cancer, colorectal cancer, ovarian cancer, and lung cancer. In addition, the compounds identified here can reduce anti-cancer drug resistance by causing protein degradation of the SDH subunits and modulating the SDH complex’s enzymatic activity at the cell- and mitochondrial-based levels. Thus, the compounds can be used in standalone cancer treatment or in combination with existing chemotherapies for those cancers, and also for treatment of any noncancerous (non-hyperproliferative) disease conditions that exhibit SDH mutations or dysregulated expression, including Leigh syndrome, ataxia, leukodystrophy, and premature aging.

[0067] The succinate dehydrogenase (SDH) enzyme plays a critical metabolic role as an intermediate between the citric acid cycle and the electron transport chain associated with the cancer disease state. Although several existing compounds have been applied to target metabolic diseases in vitro, modulating SDH for lung cancer treatment is still elusive. The compounds discussed here preferably are used in a treatment for cancer, and most particularly lung cancer. For such treatments, the dosages can be easily determined by the person of skill in the art.

[0068] The compounds of the invention include the base, and any pharmaceutically acceptable hydrate, solvate, acid or salt, and can be amorphous or in any crystalline form, or as an oil or wax. Any pharmaceutically acceptable salt can be used, as may be convenient. Generally, these salts are derived from pharmaceutically and biologically acceptable inorganic or organic acids and bases or metals. Examples of such salts include, but are not limited to: acetate, adipate, alginate, ammonium, aspartate, benzoate, benzenesulfonate (besylate), bicarbonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, carbonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, magnesium, maleate, malonate, methanesulfonate (mesylate), 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, potassium, propionate, salicylate, sodium, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate (tosylate) and undecanoate salts.Attorney Docket No.: 10669-407PC0 Patent

[0069] In preferred method embodiments, the compounds described herein are formulated and are administered as a pharmaceutical composition that includes a pharmaceutically acceptable carrier and one or more pharmaceutical agent, including one or more of the inventive compounds described herein, and including one or more of the compounds described and claimed herein with an additional agent, such as an anticancer or other drug of another class. A pharmaceutically acceptable carrier refers to any convenient compound or group of compounds that is not toxic and that does not destroy or significantly diminish the pharmacological activity of the therapeutic agent with which it is formulated. Such pharmaceutically acceptable carriers or vehicles encompass any of the standard pharmaceutically accepted solid, liquid, or gaseous carriers known in the art, such as those discussed in the art.

[0070] As is known in the art, a suitable carrier depends on the route of administration contemplated for the pharmaceutical composition. Routes of administration are determined by the person of skill according to convenience, the health and condition of the subject to be treated, and the location and stage of the condition to be treated.

[0071] Such routes can be any route which the practitioner deems to be most effective or convenient using considerations such as the patient, the patient’s general condition, and the specific condition to be treated. For example, routes of administration can include, but are not limited to: local or parenteral, including: oral, intravenous, intraarterial, intrathecal, subcutaneous, intradermal, intraperitoneal, rectal, vaginal, topical, nasal, local injection, buccal, transdermal, sublingual, inhalation, transmucosal, wound covering, direct injection into a tumor or the area surrounding a tumor, and the like. The administration can be given by transfusion or infusion, and can be administered by an implant, an implanted pump, or an external pump, or any device known in the art.

[0072] Therefore, the forms which the pharmaceutical composition can take will include, but are not limited to: tablets, capsules, caplets, lozenges, dragees, pills, granules, oral solutions, powders for dilution, powders for inhalation, vapors, gases, sterile solutions or other liquids for injection or infusion, transdermal patches, buccal patches, inserts and implants, rectal suppositories, vaginal suppositories, creams, lotions, oils, ointments, topical coverings (e.g., wound coverings and bandages), suspensions, emulsions, lipid vesicles, and the like. Additionally, methods such as antibody conjugated therapy, liposomes, polymer- drug conjugates, inorganic-organic hybrid nanoparticle, PEGylated liposome, inorganicAttorney Docket No.: 10669-407PC0 Patent scaffolds, or polymeric micelles can be used.

[0073] Treatment regimens include a single administration or a course of administrations lasting two or more days, including a week, two weeks, several weeks, a month, two months, several months, a year, or more, including administration for the remainder of the subject’s life. The regimen can include multiple doses per day, one dose per day or per week, for example, or a long infusion administration lasting for an hour, multiple hours, a full day, or longer.

[0074] Dosage amounts per administration include any amount determined by the practitioner, and will depend on the size of the subject to be treated, the state of the health of the subject, the route of administration, the condition to be treated or prevented, and the like. In general, it is contemplated that for the majority of subjects, a dose in the range of about 0.01 mg / kg to about 100 mg / kg is suitable, preferably about 0.1 mg / kg to about 50 mg / kg, more preferably about 0.1 mg / kg to about 10 mg / kg, and most preferably about 0.2 mg / kg to about 5 mg / kg are useful. This dose can be administered weekly, daily, or multiple times per day. A dose of about 0.1 mg, 0.2 mg, 0.25 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 mg, 250 mg, 500 mg, or 1000 mg can be administered. 4. Examples

[0075] This invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein, are incorporated by reference in their entirety; nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0076] Example 1: General Methods.

[0077] A. Reagents

[0078] See Table 1. Reagents or Resource Source IdentifierAttorney Docket No.: 10669-407PC0 Patent Antibodies Mouse (C4) anti-β-Actin Santa Cruz Biotechnology Cat# sc-47778 Lot# G0820Attorney Docket No.: 10669-407PC0 Patent H441 A.F. Gazdar, M. Brower and N / A D. Carney and associates.

[0080] Each cell line was maintained in a 5% CO2atmosphere at 37oC. Human lung EGFR- mutant and TP53-mutant cell line HCC827, H3255, PC-9, PC-ER (Erlotinib resistant), and H1975 (provided by Susumu Kobayashi), human lung EGFR-mutant, TP53-mutant and CDKN2A-mutant cell line H1650, human lung KRAS-mutant and TP53 null cell line H358 (derived by A.F. Gazdar, H.K. Oie, J.D. Minna and associates), human lung KRAS-mutant and P53-mutant cell line H23, Calu-6 and H441 (H441 derived by A.F. Gazdar, M. Brower and D. Carney and associates), human lung KRAS-mutant and STK11-mutant cell line A549 (derived in 1972 by D.J. Giard, et al), and human lung KRAS-mutant, PIK3CA-mutant, and STK11-mutant cell line H460 (derived by A.F. Gazdar and associates in 1982) were cultured in DMEM (Cat#11995-40, Gibco) with 10% FBS, 2 mM L-glutamine and 1% penicillin- streptomycin. Immortalized tracheobronchial epithelial AALE cells (provided by William C. Hahn) were derived as previously described and maintained in SAGM media (Cat#CC-3118, Lonza). Cell line identities were confirmed by STR fingerprinting and all were found negative for mycoplasma using the MycoAler Kit (Lonza).

[0081] C. Antibodies and Reagents

[0082] For western blotting, primary anti-SDHA antibody (1:1000, Cat#11998S, Cell Signaling Technology), anti-SDHB (1:1000, Cat#10620-1-AP, Proteintech), anti-SDHC (Cat#14575-1-AP, Proteintech), and anti-SDHD (Cat#PA5-34387, Invitrogen) were used. Mouse anti-β-actin (1:1000, Cat#sc-47778, clone C4, Santa Cruz) was used as a loadingAttorney Docket No.: 10669-407PC0 Patent control. Goat anti-rabbit (1:1000, Cat#32460, Invitrogen) and goat-anti-mouse (1:1000, Cat#32430, Invitrogen) were used as secondary antibodies.

[0083] For Immunofluorescence, anti-p21Cip1antibody (1:200, Cat#2947S, Cell Signaling Technology) and anti- g-H2AX (1:200, Cat#9718S, Cell Signaling Technology) were used primary antibodies. Goat anti-rabbit IgG with Alexa Fluor 555 (1:200, Cat#A32732, Thermo Fisher Scientific) and goat anti-rabbit IgG conjugated with Alexa Fluor 488 (1:200, Cat#A32731TR, Thermo Fisher Scientific) were used as secondary antibodies.

[0084] D. Small Molecules

[0085] DMOG (Cat#400091, Calbiochem), diethyl succinate (DES, Cat#112402, Sigma- Aldrich) and dimethyl malonate (DMM, Cat#136441, Sigma-Aldrich) were used. All small molecules screened for SDH activity were provided by Atomwise Inc. and purchased from Enamine (enamine.net).

[0086] E. Small molecule identification methodology

[0087] To determine if an SDH structure was available, a HMMER search was performed on the human sequences of interest. The uniprot IDs P31040 (SDHA_HUMAN), P21912 (SDHB_HUMAN), Q99643 (C560_HUMAN), and O14521 (DHSD_HUMAN) were determined to have a percent similarity of 97.3%, 98.2%, 97.0%, and 93.7% to chains A, B, C, and D of the X-ray crystal structure of 4YTP (3.10 Å) from Sus scrofa, respectively. Given the high percent similarity between the sequences of interest and the crystal structure chains, it was determined that 4YTP would be used for the virtual screen. Despite looking for agonists, the screening site was selected based on the inhibitor that was co-solved in 4YTP (Flutolanil) and defined by the following residues: Chain B; P169, W172 & W173; Chain C: I30, W35, M39, S42, I43, R46 & I50; Chain D: V87, D90 & Y91.

[0088] The virtual high throughput screening of the target, SDH enzyme, was performed using the AtomNet® neural network. The screening process to identify small molecules in the binding pocket of the SDH complex was performed as previously described by Hseih et al. Briefly, the binding site of interest from the crystal structure 4YTP was used for the virtual screen by the AtomNet® model and screened with approximately 2.5 million small molecules from the ENAMINE instock v200204 library (enamine.net). The results from this screen were filtered using Lipinski's rule of five, the scoring from the AtomNet® model, physio-chemical property filters, and manual removal of potential electrophiles. This resulted in 94 compounds that were then experimentally analyzed to determine SDH activity.Attorney Docket No.: 10669-407PC0 Patent The chemical identifications of the small molecules were blinded to the researchers. Dimethyl sulfoxide (DMSO) was included as a blinded internal control in place of two small molecules to give a total of 96 compounds that were analyzed.

[0089] F. Small Molecule Treatment

[0090] For small molecule treatment, human lung cancer cell line H1975 was plated equally across four 6-well plates in DMEM (Cat#11995-40, Gibco) with 10% fetal bovine serum (FBS, Cat#F4135, MilliporeSigma) (30,000 cells / 4mL / well, 10-15% confluence) and allowed to settle to adherence for six hours. After 96 hours, cells achieve 500,000 cells per well. Media was initially replaced with fresh DMEM with 10% FBS 30 minutes prior to treatment and afterwards the media was changed to serum-free DMEM containing respective small molecules and incubated for four hours. The concentration of small molecule treatment for C6 / Z96, H2 / Z14, E8 / Z71 and H7 / Z17 was 30 μM. For the SDH-known inhibitor, dimethyl malonate (DMM), the concentration of treatment was 30 μM. For the SDH known stimulator, diethyl succinate (DES), the concentration of treatment was 30 μM.

[0091] For thermal stability assay, H358 cells were plated, allowed to adhere, and treated with respective small molecules (30 µM) for 15 minutes. After treatment, cells were collected and heated for their respective temperature for 3 minutes and allowed to cool down for three minutes. This was followed by protein extraction and western blot protocol.

[0092] For cell based SDH assay, cells were plated at 150,000 cells per well, allowed to adhere, and treated with respective small molecules in serum-free DMEM for four hours. After treatment, Succinate Dehydrogenase Activity Colorimetric Assay protocol was performed to gather SDH activity.

[0093] For colony formation assay, 250 H358 conjugated with green fluorescence protein (GFP) cells were seeded in a 96-well black plate (BD Biosciences – Falcon). After adherence, respective treatment media was added and cells were incubated for 14 days. After 14 days, cells were visualized (EVOS FL, Life Technology) for GFP signal and quantified using ImageJ (Version 1.54d 30 March 2023).

[0094] For immunofluorescence staining, cells were plated at 50,000 cells per coverslip. After overnight incubation for adherence, small molecules were added with DMEM at a concentration of 125 μM. After six hours of treatment, the treatment media was aspirated to proceed with cell permeabilization, fixation, and antibody incubation.Attorney Docket No.: 10669-407PC0 Patent

[0095] For ROS assay, cells were plated at a density of 70,000 cells per coverslip. Once the cells became adhered, cells were incubated with DCFDA reagent for 45 minutes in the dark at 37°C after which treatment media was added, and cells were incubated for six hours. This was followed by visualization under a confocal microscope for fluorescent signal and quantified using ImageJTM.

[0096] G. Thermal Shift Assay

[0097] H358 cells were plated in a 6-well plate to reach 80% confluency upon treatment and extraction. Media was replaced with complete DMEM (Gibco) 30 minutes prior to treatment with small molecule treatment (30 µM). Treatment with the small molecules was performed for 15 minutes in complete DMEM (1.5 mL), and cells were immediately scraped with a cell scraper (Cat#TC7023, CellPro). Wells were washed with phosphate buffer saline (PBS, Cat#10010023, Gibco) and collected. Cells were then pelleted, and supernatant was removed until approximately 20 µl volume. Cell pellets were resuspended by flicking and heating proceeded. Cells were heated for three minutes at their respective temperature in a mini dry bath incubator (Four E’s Scientific) and then a three-minute cool down followed. 80 µl of RIPA Buffer (Radio Immuno Precipitation Assay buffer, Cat#PI89901, Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitor cocktail (Cat#A32963, Thermo Fisher Scientific) was added to the cells to lyse. Samples were shaken in a 4°C cold room for two hours followed by a centrifugation step at 4°C for 40 minutes at 14,000 RPM. Supernatant was collected and protein quantification followed.

[0098] H. Harvesting of Mitochondria

[0099] AALE cells were plated across twenty 10-cm dishes and grown to 90% confluency (~ 3X107cells). Harvesting of the mitochondria was performed using the Mitochondria Isolation Kit (Cat#89874, Thermo Fisher Scientific) for cultured cells. Cells were pelleted in a 2-mL microcentrifuge tube at ~850 x g for 2 minutes. Supernatant was discarded. 800 μl of Mitochondria Isolation Reagent A was added. The tube was vortexed at medium speed for 5 seconds and incubated on ice for exactly 2 minutes. 10 μl of Mitochondrial Isolation Reagent B was added afterward. Samples were then vortexed at maximum speed for 5 seconds followed by incubation on ice for 5 minutes. Within that 5-minute period, the tube was vortexed every minute at maximum speed. 800 μl of Mitochondria Isolation Reagent C was added and tube was inverted several times. Sample was then centrifuged at 700 x g for 10 minutes at 4°C and the supernatant was transferred to a fresh 2.0 mL centrifuge tube andAttorney Docket No.: 10669-407PC0 Patent centrifuged at 12,000 x g for 15 minutes at 4°C. The supernatant was discarded. 500 μl of the Mitochondria Isolation Reagent C was added to the pellet and centrifuged at 12,000 x g for 5 minutes. Supernatant was discarded and the resulting pellet was frozen at -80°C following SDH enzyme activity assay.

[0100] I. Succinate Dehydrogenase Activity Colorimetric Assay

[0101] SDH activity quantification was performed as described in the Succinate Dehydrogenase Activity Colorimetric Assay Kit (Cat#K660-100, BioVision). Extracted mitochondrial pellets were resuspended in PBS (2 tubes containing 1.5 mL each) and stored at -80°C. When ready to use, one pellet was centrifuged and resuspended in 1100 ml PBS, then divided into 10 ml aliquots, each containing mitochondria from 1.5X105cells. Each of these aliquots represent (1) sample well for SDH activity screening. 10 mM stock concentration of small molecules (w / DMSO) were added directly to the aliquots and brought to a final concentration of 10 μM with addition of PBS. Small molecules were tested in duplicate and compared to PBS. Mitochondrial samples were incubated with selected small molecules for 4 hours at 4°C. Samples were then added in duplicate volumes of 50 ml to clear 96 well plates and brought up to a final volume of 100 ml with SDH Activity Colorimetric Assay Reaction Mix. Samples were read in Envision plate reader for one hour at 595 nm and 25°C with absorbance readings every 5 minutes for AALE cells and every 30 seconds for H358 cells. Values were transformed to SDH activity by the formula (nmol / min / µL = mU / µl = U / mL). These values were then further normalized to SDH Activity per cell for comparison across cell lines.

[0102] Top stimulatory and inhibitory candidates were selected for further screening in whole cells. Small molecules were selected based on cross reference of 5-minute and 30- minute AV / RQ values from the previous screen in AALE mitochondria. AALE cells were plated across ten 6 well plates dishes in SAGM (150,000 cells / 2mL / well, 90% confluence) and allowed to settle approximately 8 hours. Media was then changed to low-bovine serum albumin (BSA) SABM and incubated for 14 hours. After this incubation, media was then changed back to complete SAGM. Small molecules were added in triplicate to the 6 well plates in 20 µM concentration and incubated for 18 hours. To compare the results between noncancerous and cancerous cells, the same shortlisted small molecule candidates were screened in H358 cells. H358 cells were plated across 6 well plates in DMEM with 10% FBS (60,000 cells / 2mL / well, 80% confluence) and allowed to settle approximately 24 hours.Attorney Docket No.: 10669-407PC0 Patent Media was then changed to DMEM with 2% FBS and 1% penicillin / streptomycin and incubated for three hours. After this incubation, media was then changed back to complete DMEM with 10% FBS. Small molecules were added in triplicates to the 6 well plates in 20 μM concentration and incubated 18 hours. Cells were then pelleted and processed downstream for SDH Activity assay. Scatterplots were generated by comparing SDH absolute values and relative quantification (RQ) values on the same graph, allowing for elimination of variance between assays that were performed on different days.

[0103] J. Cell Viability Assay

[0104] Cell viability was performed on white opaque 96-well plates (Cat#13485, SPL Life Sciences). Respective cells were plated at 3,000 cells per well and allowed to adhere for at least four hours before respective treatment. After adherence, cells were treated with respective small molecules for 72 hours. After treatment, the cell viability was measured at the treatment endpoint using the CellTiter-Glo® luminescent cell viability assay kit (Cat#G7570, Promega) according to the manufacturer’s instructions.

[0105] K. Colony Formation Assay

[0106] Long-term colony formation assay was performed as described previously. Briefly, H358 conjugated with GFP cells were plated at a density of 250 cells per well in a 96-well black plate (Cat#08772225, Fisher Scientific) with three replicates per group. H358-GFP cells were treated in a titrating dosage manner from 0 μM to 300 μM with indicated compounds for 14 days. Cell viability was determined by GFP signal intensity using a fluorescence microscope. After 14 days, fluorescence images were taken on a fluorescence microscope (Cat#AMF5000, EVOS FL, Life Technology) and the number of colonies was quantified by ImageJ (Version 1.54d 30 March 2023).

[0107] L. Western Blotting

[0108] Protein extraction was performed on various cell lines, after respective treatment, the treatment media was aspirated. The plate was washed with cold phosphate buffered saline (PBS) once. PBS was aspirated and the plates immediately placed on a flat bed of ice. 600 µl of RIPA Lysis and Extraction Buffer (Cat#PI89901, Thermo Fisher Scientific) containing Pierce Protease and Phosphatase Inhibitor Mini Tablets (Cat #PIA32959, Thermo Fisher Scientific) was added for protein extraction. The plate was incubated on ice for 5 minutes. A pre-chilled cell scraper (Cat#TC7023, CellPro) was used to collect the lysed cells and transferred into a pre-chilled 1.5 mL Eppendorf tube. Tubes were sealed with one layer ofAttorney Docket No.: 10669-407PC0 Patent Parafilm. Tubes were shaken at 4°C for two hours with a Roto-Mini™ Rotator Series (Cat#RS3020, Alkali Scientific). Samples were centrifuged for 40 minutes at max speed (15,000 RPM) at 4°C. After centrifugation, the samples were placed on ice and carefully aspirated the supernatant to a fresh 1.5 mL tube. The collected supernatant was placed in ice. The pellet was discarded. Samples were frozen at -80°C for long-term storage. Protein concentration was normalized using Pierce BCA Protein Assay (Cat#23225, Thermo Fisher Scientific) and measured on Envision Manager. SDS-Page was performed using standard protocol for NuPAGE Bis-Tris Mini Gels (Cat#NP0321BOX, Thermo Fisher Scientific). Samples were combined with NuPAGE LDS Sample Buffer (4x) (Cat#NP0007, Invitrogen), NuPAGE Reducing Agent (10x) (Cat#NP0004, Invitrogen), and deionized water. 1x Running buffer was made using 20x NuPAGE MES Running Buffer (Cat#NP0002, Invitrogen) to 950 mL deionized water. 200 mL of 1x Running Buffer was added to 500 µL NuPAGE Antioxidant (Cat#NP0005, Invitrogen) for upper chamber buffer. SDS-Page was ran using PowerEase (Cat#PSC350MB, Invitrogen) at 45 min on 200V constant. Membrane transfer was performed overnight at 17V. Blocking was performed with 5% BSA in TRIS buffer saline in Tween 20 (TBS-T) for one hour at room temperature followed by overnight primary antibody incubation. Secondary incubation occurred for one hour at room temperature. The membrane was washed with TBS-T between incubations. The protein detection was performed using Supersignal West PICO chemiluminescent substrate (Cat#34580, Thermo Fisher Scientific) and visualized on Chemilmager (Biorad). Data was analyzed using Image Lab (v6.0.1, Biorad Laboratories Inc).

[0109] M. RNA Extraction and qRT-PCR

[0110] RNA extraction was performed using mirVana miRNA Isolation Kit (Cat#AM1560, Invitrogen) according to known methods. A total of 10 ng RNA each sample was input for the Reverse Transcription reaction to acquire the cDNA. Real-time PCR was performed using TaqMan probes on QuantStudio Real-Time PCR. TaqMan probes (Applied Biosystem) included the following: SDHA-Hs00188166_m1 (Cat#4331182), SDHB-Hs00268117_m1 (Cat#4331182), SDHC-Hs01698067_s1 (Cat#4331182), SDHD-HS00829723_g1 (Cat#4331182), and GAPDH-HS02786624_g1 (Cat#4331182).

[0111] N. Immunofluorescence staining

[0112] The cells were plated on a PLL coated coverslip placed in a 35mm Petri dish, and they were allowed to grow and attach to the coverslip overnight. To visualize the DNAAttorney Docket No.: 10669-407PC0 Patent damage proteins (p21Cip1and g-H2AX), the cells were treated with 30 µM of the small molecule for 4 hours. To visualize the SDH subunits, the cells were treated with 125 µM for 6 hours. After treatment, the media was aspirated, and the cells were briefly washed with PBS. Cells were then incubated for 10 minutes with 500 µL of 4% paraformaldehyde (PFA) to fix the cells followed by a 10-minute incubation with 1 mL of 0.1% Triton X-100 in PBS to facilitate antibody permeabilization. Blocking was performed with 5% BSA in TRIS buffer saline in Tween 20 (TBS-T) for 30 minutes at room temperature followed by overnight primary antibody incubation at 4°C. Secondary incubation is done by incubating the cells in the dark at 37°C for 45 minutes. The slides were prepared by adding 20 µL of DAPI on them and the coverslips were placed on them such that the cells were facing downwards.

[0113] For co-staining, after incubation at 4°C with the first primary antibody overnight, the cells were washed briefly with PBS and then were allowed to incubate with the secondary antibody conjugated with Alexa Fluor 488 for 45 minutes in the dark at 37°C. Then the second primary antibody was added and incubated at room temperature at 37°C followed by incubation with another fluorescence emitting secondary antibody conjugated with Alexa Fluor 555. The images were taken under a confocal microscope (Zeiss) and then quantified using ImageJ (Version 1.54d 30 March 2023) software in which the mean of the fluorescent signal intensity was used as a measure to analyze the expression levels.

[0114] O. ROS measurement

[0115] Intracellular ROS expression was determined using the DCFDA / H2DCFDA – Cellular ROS Assay kit (Cat#ab113851, Abcam) and performed in accordance with the manufacturer’s protocol for confocal imaging. The cells were treated with 125 µM of the small molecules for six hours, and then incubated with 1 µg / ml of Hoechst stain (Cat#62249, Thermo Fisher Scientific) for 10 minutes in the dark. Then cells were visualized under a confocal microscope (Zeiss). Each group had six biological replicates which were then quantified using ImageJ software in which the mean of the fluorescent signal intensity was used as a measure to analyze the expression levels.

[0116] P. Statistical Analyses

[0117] All experiments were performed in 2 to 6 biological replicates and independently reproduced as indicated in figure captions. Data are presented as the means ± SEM. Unless otherwise stated, statistical significance was determined by a student’s two-tailed t-test by GraphPad Prism (v8.4.3). P<0.05 was considered statistically significant. Two tailed t-testAttorney Docket No.: 10669-407PC0 Patent with Welch’s correction was applied for two samples with unequal variances. For three and more samples that are normally distributed, one-way ANOVA was used for multiple comparisons.

[0118] Q. Data and software availability

[0119] The results shown in this manuscript were partially based upon data generated by the Lung Cancer Explorer portal: lce.biohpc.swmed.edu / lungcancer / . The genetic mutation status was confirmed by cansar portal (v3.0 beta) (cansar.icr.ac.uk / ) and cancer Catalogue of Somatic mutations in cancer (cancer.sanger.ac.uk / cosmic / sample / overview?id=722040). The data that support the plots within this paper and other finding of this study are available from the corresponding author upon reasonable request.

[0120] Example 2: SDH expression is dysregulated in lung cancer.

[0121] To characterize SDH components across NSCLC and to determine the relationship between NSCLC mutation status (KRAS, EGFR) and SDH complex protein expression, immunoblotting was performed in cancerous H358, H23, H441, Calu-6, A549, H460, HCC827, H3255, PC-9, PC-9ER, H1975, H1650, and immortalized lung epithelial (AALE) cells. See FIG.1 and FIG.2. FIG.1 shows a western blot for SDHA, SDHB, and SDHD expression across various non-small cell lung cancer and immortalized (AALE) cell lines with or without KRAS and EGFR mutations. The blot intensity was normalized to that in AALE cells, as shown in FIG.2 (n = 3). β-Actin was used as a loading control.

[0122] The relative protein levels of the primary subunits of the enzymatic complex, SDHA, SDHB, and SDHD were quantitated to discriminate changes in the regulation of each individual component. Due to variance observed in the pathogenic consequence of individual SDH subunit mutations, it is reasonable to conclude that changes in the protein levels of one or more subunits of the SDH complex play a unique role in metabolic dysfunction and tumorigenicity.

[0123] Surprisingly, NSCLC adenocarcinoma cell line H23 (KRAS + / EGFR -) showed reduced protein expression of all SDH subunits. In general, most NSCLC cell lines trended towards either a decrease or increase of SDHA, SDHB, and SDHD collectively, indicating unidirectional regulation of the SDH complex at the protein level. However, other cell lines such as Calu-6 (KRAS + / EGFR -) and H1650 (KRAS - / EGFR +) showed heterogenicity of protein levels across the subunits of the SDH complex. Interestingly, for both Calu-6 andAttorney Docket No.: 10669-407PC0 Patent H1650, SDHA was upregulated while SDHB and SDHD were downregulated. SDHA comprises the binding site which allows for the oxidation of succinate. Collectively, these results do not suggest a simplified direct relationship between KRAS / EGFR mutation status and protein levels of individual SDH subunits in NSCLC. We found detecting SDHC for immunoblotting to be unsuccessful, likely due to the reduced stability of SDHC protein during the immunoblotting process. We therefore opted to visualize SDHC expression by immunofluorescent imaging, which has less impact on protein stability than immunoblotting. See FIG.3 and FIG.4. FIG.3 presents immunofluorescent staining and quantification for SDHC in NSCLC and AALE cells. Cells were stained with primary anti-SDHC antibody followed by anti-rabbit IgG conjugated with Alexa Fluor 488. The cells were then counterstained with DAPI. Scale bar = 20 mm. n = 6 replicates. The fluorescence was quantitated by ImageJ and the fluorescence intensity in AALE cells was calibrated as 1. See FIG.4.

[0124] We chose six cell lines representative of a broad spectrum of NSCLC cell lines and visualized SDHC protein expression. Similar to the SDHD protein expression profile by western blotting, the five NSCLC cell lines exhibited lower SDHC expression than AALE cells. Among the five NSCLC cell lines, H1975 cells had the lowest SDHC protein expression.

[0125] Using data collected from the Cancer Genome Atlas Program (TCGA), the mRNA expression levels between adenocarcinoma and tumor-adjacent lung tissues were compared for each SDH subunit. SDHA (p<0.0001), SDHB (p<0.05), and SDHC (p<0.0001) showed significant upregulation across tumor samples, while SDHD was slightly downregulated in tumor samples (p<0.05). See FIG.5, which shows the differential gene expression for SDHA, SDHB, SDHC, and SDHD in lung adenocarcinoma patients-derived tumor and tumor- adjacent normal tissues from The Cancer Genome Atlas database (n = 517 (tumor); n = 59 (normal).

[0126] Using quantitative reverse transcription PCR (qRT-PCR),12 NSCLC cell lines with various mutational statuses were analyzed for SDH gene expression. Surprisingly, all subunits of the SDH complex trended towards a decrease in gene expression when normalized to immortalized AALE cells. See FIG.6, which shows quantitative reverse transcription PCR analysis of gene expression for SDHA, SDHB, SDHC, and SDHD in NSCLC and AALE cells. The gene expression was normalized to that in AALE cells (n = 3Attorney Docket No.: 10669-407PC0 Patent replicates). GAPDH was used as an endogenous control. Data are mean ± s.e.m. and were analyzed with two-tailed unpaired student’s t-test (c) and one-way ANOVA (a,b,d). *, p<0.05; **, p<0.01; ****, p<0.0001; ns, not significant.

[0127] The differential SDH expression between lung tissues and cell lines indicates heterogenous SDH abundance in the various cell types present in lung tissues. In addition, H1975 and PC9 showed consistent SDHA, SDHB, and SDHD at protein and RNA levels except for SDHC protein expression, indicating differential post-transcriptional regulation in these two cell lines.

[0128] Example 3: Small molecule identification workflow and structure of SDH subunits.

[0129] The genes encoding for complex II are classified as tumor suppressor genes and targeting complex II could give another route in combating cancer with therapeutics. The crystal structure 4YTP contains an inhibitor in the ubiquinone binding site of complex II, located at the mitochondrial inner membrane, consisting of chains B, C, and D. See FIG.7, which shows the overall structure of PDB 4YTP chains A, B, C, and D. The binding site of interest can be seen in the box with residues seen as sticks and a ligand (seen in white) present to define the site for the AtomNet® model.

[0130] Therefore, the virtual screen was performed using the AtomNet® model at the interface of chains B, C, and D of 4YTP where Flutolanil, the inhibitor, was located. See FIG 8A and FIG.8B. FIG.8 shows a zoomed-in version (FIG.8A) and surface representation (FIG.8B) of the binding site of interest. When performing the virtual screen against the chemical space of the Enamine small molecule library, 94 compounds were identified as small molecule candidates to target the ubiquinone site of SDH. See FIG.9 for methods. Of those 94 compounds, four compounds showed promising results when SDH activity was experimentally determined.

[0131] Example 4: Small molecules modulate SDH enzyme activity.

[0132] Quantification of complex II enzymatic activity corresponding to an amount of reduced 2, 6-dichlorophenolindophenol (DCPIP) due to electron transfer in an in vitro reaction21is a common method employed in the study of mitochondrial function and the electron transport chain. See FIG.10, showing the principles of measuring the SDH enzyme activity. The electrons generated from SDH-catalyzed reactions are transferred to DCPIPAttorney Docket No.: 10669-407PC0 Patent (blue dye), converting DCPIP to the colorless product DCPIPH2. The reduced optical density is measured by a microplate reader to quantify the SDH enzyme activity.

[0133] The small molecules that target the SDH complex were identified using the drug discovery approach. See FIG.11, which is a flow chart demonstrating the small molecule selection process and research design. Ninety-four small molecules targeting SDH and DMSO (n=2) were initially gathered by screening with the AtomNet® model. Subsequently, single-blinded screening with mitochondria- and cell-based SDH activity and cell viability assays were performed, and four lead small molecules were finally shortlisted.

[0134] Here we employ several mitochondria- and cell-based SDH activity screens to identify small molecule modulators of the SDH complex. First, SDH activity in cancerous H358 and immortalized AALE cells were compared. H358 cells were found to have lower SDH enzyme activity than AALE, which is consistent with reduced expression of SDHA, SDHB, SDHC and SDHD in H358 cells than in AALE cells. See FIG.12, which shows the differential SDH enzyme activity between H358 and AALE cells. A mitochondrial fraction from each cell line was isolated to quantify the SDH enzyme activity. The relative SDH enzyme activity in AALE cells was normalized as 1 (n = 3 replicates).

[0135] This suggests functional impairment of the SDH complex in H358 cells. The small molecules were then screened for initial target engagement by measuring SDH activity in isolated AALE mitochondria. After 30-minute incubation in the presence of succinate, only E8 / Z71, H7 / Z17, D8, and E9 showed stimulatory effects on SDH activity (fold change >1.15, n=4 of 96), while the majority of small molecules were inhibitory (fold change <0.85, n=67 of 96) (Table 2). Compounds H2 / Z14 and C6 / Z96 had the strongest inhibitory effects. FIG. 13, which shows the screening of effects of small molecules on absolute and relative SDH enzyme activities in isolated mitochondria from AALE cells. The relative SDH activities of all tested small molecules (x-axis) were normalized to that treated with PBS (n = 2 replicates).

[0136] Following unblinding of small molecule chemical identities, the internal controls (DMSO) showed relatively similar SDH activity as our original quality control, phosphate buffered saline (PBS), supporting the validity of the SDH activity assay for assessment of small molecule effects. Upon selecting the top four compounds that showed potent modulation of SDH enzyme activity values (C6 / Z96, H2 / Z14, H7 / Z17, and E8 / Z71) (Table 3), we then moved to secondary screening of SDH activity in intact and live AALE cells.Attorney Docket No.: 10669-407PC0 Patent Compounds H2 / Z14 and C6 / Z96 did not significantly reduce SDH activity, while E8 / Z71 significantly increased SDH activity by 17.7%. See FIG.14, which shows the relative quantification of per cell SDH activity modulation by small molecules (H02, C06, and E08) in AALE cells treated at 50 mM for four hours. N = 2 replicates.

[0137] Known SDH inhibitor dimethyl malonate (DMM)22reduced SDH activity in H358 cells by 13.1% after four-hour treatment in 50 mM concentration. Additionally, two lead small molecule candidates reduced SDH activity significantly after four hours (H2 / Z14: 42.2%, p<0.05; C6 / Z96: 37.2%, p<0.05), while the other top two lead small molecules significantly increased SDH activity (H7 / Z17: 1.84-fold, p<0.05; E8 / Z71: 1.56-fold, p<0.05). See FIG.15, which shows the relative quantification of per cell SDH activity modulation by small molecules (H02, C06, H07, E08, and DMM) in H358 cells treated at 30~50 mM for four hours (n = 4 replicates). Data are mean± s.e.m. and were analyzed with unpaired two- tailed t-test (c) and Welch’s t-test (e,f). *, p<0.05; **, p<0.01; ***, p<0.001, ****, p<0.0001, ns, not significant.

[0138] The top candidates showing a 15% increase or decrease in enzyme activity were listed. See Table 2, below. See Table 3 and below for more information on the four best candidates. Table 2. Changes of SDH enzyme activities in isolated mitochondria from AALE cells treated with small molecules. E5 1.18E-08 0.81 A11 1.18E-08 0.81 Treatment SDH activity Fold (10-8change )Attorney Docket No.: 10669-407PC0 Patent E11 1.10E-08 0.72 H3 1.00E-08 0.64 E3 1.10E-08 0.72 E10 9.99E-09 0.64 D11 109E-08 072 C3 992E-09 064Table 3. Top four small molecule chemical identification. Name Chemical Simplified Molecular-Input Line-Entry System (SMILES) Formula F)The four top candidates are referred to as Z17, Z14, Z96, and Z71: Z17 (Z1742743002): MW 392.374.2-methyl-N-(1-[2-(trifluoromethyl)pyrimidine-5- carbonyl]piperidin-4-yl)benzamide; Z14 (Z1449835140): MW 308.377.3-methyl-2-[4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidin- 1-yl]quinoline; Z96 (Z96611497): MW 369.725. 2-([3-chloro-2-oxo-5-(trifluoromethyl)-1,2-dihydropyridin- 1-yl]methyl)-8-methyl-3,4-dihydroquinazolin-4-one; Z71 (Z71062692): MW 354.469. N-[(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methyl]- N,2,4-trimethyl-1,3-thiazole-5-carboxamide.Attorney Docket No.: 10669-407PC0 Patent

[0139] Example 5: Small molecules H2 / Z14, H7 / Z17, E8 / Z71, and C6 / Z96 bind SDH directly to and modulate subunit protein abundance.

[0140] The cellular thermal shift assay analyzes the direct binding of small molecules by demonstrating decreased protein denaturation across an array of increasing temperatures, as quantified by the melting temperature (Tm) of a protein. We assessed if these small molecules bind directly to the SDH subunits through this assay. As expected, we found an increase in the Tm of SDHA by 10.9°C upon DMM treatment in H358 cells (DMSO Tm: 41.2°C, DMM Tm: 52.1°C). See FIG.16A: small molecules H2 / Z14, C6 / Z96, H7 / Z17, and E8 / Z71 increased Tm of SDHA by 10.9~11.4°C (H2 / Z17 Tm: 52.6°C, C6 / Z96 Tm: 57.6°C, H7 / Z17 Tm: 52.6°C, E8 / Z71 Tm: 60.6°C). For SDHB, only small molecule H7 / Z17 slightly increased Tm by 4.7°C (DMSO Tm: 54.7°C, H7 / Z17 Tm: 59.4°C). See FIG.16B. For SDHD, small molecules H2 / Z14, C6 / Z96, and H7 / Z17 elevated Tm by 4.3~25.7°C (DMSO Tm: 46.4°C, H2 / Z14 Tm: 55.4°C, C6 / Z96 Tm: 50.7°C, H7 / Z17 Tm: 72.1°C). See FIG.16C.Attorney Docket No.: 10669-407PC0 Patent

[0141] FIG.16 presents a thermal shift assay by western blotting for SDHA (FIG.16A), SDHB (FIG.16B), and SDHD (FIG.16C) on H358 cells treated with or without small molecules H2 / Z14, H7 / Z17, E8 / Z71, C6 / Z96, and DMM. Melting temperature (Tm) was calculated using nonlinear regression analysis (n = 3 replicates).

[0142] Surprisingly, SDHA was bound by small molecule treatment elucidating affinity to that subunit. C6 / Z96 and E8 / Z71 had greater affinity to SDHA while H2 / Z14 and H7 / Z17 had a greater affinity to SDHD. These data suggest that these top small molecules directly bind to one or more subunits of SDH and are more effective in inhibiting lung cancer colony formation than the known SDH inhibitor dimethyl malonate (DMM).

[0143] Immunofluorescent confocal imaging was employed to assess individual SDH subunit localization and abundance in H1975 cells after small molecule treatment. SDHA levels were significantly reduced following treatment with the inhibitor H2 / Z14 (p<0.001), while H7 / Z17 treatment increased SDHA levels (p<0.05) more than DMSO treatment. C6 / Z96 and E8 / Z71 had no significant effect on SDHA levels. However, SDHB staining shows that H2 / Z14 treatment resulted in a significant increase (p<0.05), while the other small molecules did not have any significant effect. See FIG.17 and FIG.18.

[0144] Interestingly, SDHC levels were significantly increased following treatment with H2 / Z14 (p<0.05) and H7 / Z17 (p<0.001), while C6 / Z96 and E8 / Z71 treatment showed no significant effects. SDHD abundance was significantly decreased by H2 / Z14 (p<0.01) and significantly increased by E8 / Z71 (p<0.05). H7 / Z17 and C6 / Z96 showed no significant effects on SDHD abundance (FIG.17 and FIG.18). Collectively, these results point to the potent reduction of SDHA and SDHD subunit abundance by H2 / Z14 and stimulation of SDHD expression by E8 / Z71 and SDHA and SDHC expression by H7 / Z17.

[0145] FIG.17A through FIG.17E show immunofluorescent staining for PBS (control), SDHA, SDHB, SDHC, and SDHD, respectively, of H1975 cells treated with or without small molecules (H2 / Z14, H7 / Z17, E8 / Z71, C6 / Z96) for six hours. Cells were co-stained with primary anti-SDHB and anti-SDHC antibodies or anti-SDHA and anti-SDHD antibodies, followed by secondary IgG conjugated with Alexa Fluor 488 or 555. The cells were then counterstained with DAPI and imaged by confocal microscopy (scale bar = 20 mm; n = 6 replicates). FIG.18A, FIG.18B, FIG.18C, and FIG.18D show the quantitation of immunofluorescence intensity for each SDH subunit in cells treated with H2 / Z14, H7 / Z17, E8 / Z71, and C6 / Z96 compared to PBS (n = 6 replicates). Data are mean ± s.e.m. and wereAttorney Docket No.: 10669-407PC0 Patent analyzed with one-way ANOVA (i). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant.

[0146] Example 6: H2 / Z14 destabilizes NRF2 leading to ROS generation.

[0147] We first investigated the relationship between HIF-1α and NRF2 protein levels in 12 NSCLC cell lines and immortalized AALE cells. See FIG.19, which presents a western blot for HIF-1a and NRF2.

[0148] We observed significant disruption of redox balance, evidenced by increased levels of HIF-1α in the absence of increased NRF2, in cancer cell lines , HCC827, H23, H1975, H1650, H358, and H3255 compared with AALE cells. See FIG.20, which presents data on the quantitation of the results in FIG.19 across 12 NSCLC and immortalized AALE cell lines. GAPDH was used as a loading control. The expression levels of HIF-1a and NRF2 in AALE cells were calibrated as 1 (n = 2 replicates (HIF-1a); n = 1 replicate (NRF2).

[0149] H1975 cells were selected for studying the effects of SDH modulators on hypoxia signaling due to the SDH component RNA expression of this cell line closely recapitulating what was observed in the TCGA patient samples. Treatment of H1975 cells with three lead SDH-targeting small molecules (H2 / Z14, H7 / Z17, and E8 / Z71) and a known HIF-1α activator (dimethyloxalylglycine, DMOG) resulted in a significant increase in HIF-1α without compensatory increases in NRF2, suggesting the induction of a hypoxic state (p<0.05) (FIG.21 and FIG.22). Treatment with SDH modulator C6 / Z96 resulted in a 2.5- fold increase in HIF-1α levels, but this did not reach significance in post-hoc analysis. Known SDH-binding molecules diethyl succinate (DES) and dimethyl malonate (DMM) had no significant effect on HIF-1α or NRF2 levels in H1975 cells. See FIG.21 and FIG.22. FIG.21 presents a western blot for HIF-1a and NRF2. FIG.22 presents data on the quantitation in H1975 cells treated with small molecules (H2 / Z14, H7 / Z17, E8 / Z71, C6 / Z96, DMM, DMOG, DES) and vehicle (DMSO). Beta-actin was used as a loading control. The expression levels of HIF-1a and NRF2 in cells treated with DMSO were calibrated as 1 (n = 3 replicates).

[0150] Since SDH plays a key role in the maintenance of redox balance, we asked if targeting SDH by small molecules will affect redox status. To answer this question, we measured ROS levels in H1975 cells upon small molecule treatment by immunofluorescence staining and confocal microscopy. We observed a time-dependent ROS elevation in cellsAttorney Docket No.: 10669-407PC0 Patent treated with H2 / Z14. See FIG.23, which shows confocal imaging for ROS by immunofluorescent staining in H1975 cells treated with H2 / Z14 for 0, 2, 4, and 6 hours). The cells were then counterstained with Hoechst 33342 (scale bar = 20 mm; n = 3 replicates).

[0151] H2 / Z14, C6 / Z96, and E8 / Z71 treatment for six hours showed a significant increase in ROS by 4.2-fold (p<0.0001), 2.3-fold (p<0.0001) and 1.8-fold (p<0.05) respectively. See FIG.24, which shows confocal imaging for ROS by immunofluorescent staining in H1975 cells treated with H7 / Z17, E8 / Z71, and C6 / Z96 for 6 hours (scale bar = 20 mm; n = 3 replicates).

[0152] To further understand the potential toxic mechanisms of SDH-targeting small molecule treatment, we investigated DNA damage proteins p21Cip1and g-H2AX using immunofluorescence staining. See FIG.25 through FIG.28. These figures present immunofluorescence staining and quantification for p21Cip1(FIG.25 and FIG.26) and g- H2AX (FIG.27 and FIG.28) in H1975 cells treated with C6 / Z96, H2 / Z14, E8 / Z71, H7 / Z17, or a vehicle (PBS) for four hours. The fluorescence intensity in cells treated with PBS was calibrated as 1 (scale bar, 20 mm; n = 3 replicates). Data are mean ± s.e.m. and were analyzed with one-way ANOVA (d,f,g,h). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0153] We observed that C6 / Z96 treatment resulted in 2.4-fold (p<0.05) and 1.2-fold (p<0.05) increase in p21Cip1and g-H2AX, respectively. Similarly, E8 / Z71 treatment led to 1.5-fold (p<0.05) and 1.3-fold (p>0.05) increase in p21Cip1and g-H2AX, respectively. These data suggest that modulating SDH induces DNA damage in lung cancer cells.

[0154] Example 7: Small molecules H2 / Z14, H7 / Z17, E8 / Z71, and C6 / Z96 inhibit short and long-term lung tumor cell growth in vitro.

[0155] Accumulation of cellular succinate is associated with tumor invasiveness and drug resistance. However, significant disruption of the SDH complex by antitumor agent lonidamine results in accumulation of succinate, inhibition of fumarate formation, and decreased cell viability due to toxic accumulation of reactive oxygen species. Therefore, the small molecules discussed here target the ubiquinone binding pocket of the SDH complex may act in either of these two opposing directions on cancer cell viability, contingent upon the ability to either contribute to oncogenic mitochondrial dysfunction or accrue critical levels of ROS, possibly through reverse electron transport resulting in apoptosis. ToAttorney Docket No.: 10669-407PC0 Patent investigate the dose-dependent effects of SDH modulation on cell viability, H460, H1975, H358, H441, and H3255 cell lines were treated with increasing doses of lead SDH-targeting small molecules and cell viability was determined within three days. See FIG.29. This figure shows the relative cell viability of cell lines (H1975 (FIG.29A), H358 (FIG.29B), H460 (FIG.29C), H441 (FIG.29D), and H3255(FIG.29E)) treated with C6 / Z96, H2 / Z14, E8 / Z71, H7 / Z17, or a vehicle (PBS) for three days (n = 3 replicates). Data are mean ± s.e.m. and were analyzed with one-way ANOVA (f). **, p<0.01. For FIG.29, PBS (control) group is the concentration farthest to the left on the graph. It represents the 100% cell viability point on the Y axis. Every value is relative to this sample. The data show that the tested small molecules reduce cell viability through increased DNA damage, leading to cell death.

[0156] All compounds reduced cancer cell viability in a dose-dependent manner. Compound C6 / Z96 drastically reduced cell viability in all tested cell lines, with a major inflexion occurring at 62.5 µM. H460 cells showed greater overall resistance to SDH modulation, while H358 and H3255 cells were the most sensitive to SDH modulation. See FIG.29A through FIG.29E. H1975, H441, and H460 did not display decreased cell viability at concentrations below 125 µM.

[0157] Surprisingly, SDH inhibitor H2 / Z14 increased cell viability up to 1.5-fold in H358, H441, and H3255 at lower concentrations (below 5 µM), but decreased cell viability at higher concentrations. These data suggest that the acute response of tumor cells to SDH modulation is dose-dependent.

[0158] To address if targeting SDH will cause a chronic response in cancer cells, we investigated the long-term effects of SDH-targeting small molecule treatment on cancer cells by colony formation assay. H358 cells expressing green fluorescence protein (GFP) were incubated with increasing concentrations of small molecules (0~300 µM), and fluorescent images were collected after 14 days. See FIG.30 and FIG.31, which show data for fluorescent microscopy and quantification of GFP-labelled H358 cells incubated with increasing concentrations of small molecules C6 / Z96, H2 / Z14, E8 / Z71, or H7 / Z17 (0, 10, 300 µM) for 14 days. The relative GFP+ colony number in cells treated with PBS was calibrated as 1 (scale bar, 750 µm). Data are mean ± s.e.m. and were analyzed with one-way ANOVA. **, p<0.01.

[0159] At 10 µM concentration, C6 / Z96 and H2 / Z14 partially reduced colony formation by 46.7% (p<0.01) and 52.3% (p<0.01), respectively. However, cells treated with E8 / Z71,Attorney Docket No.: 10669-407PC0 Patent H7 / Z17 or DMM at 10 µM did not display obvious changes in colony formation (p>0.05). At 300 µM, all four compounds resulted in widespread cytotoxicity and near-complete reduction of colony formation. This is in contrast to DMM, which at 300 µM, had no distinguishable effects on colony formation. Together, these results suggest that these newly identified small molecule modulators of SDH significantly reduce NSCLC proliferation and colony formation. REFERENCES

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Claims

Attorney Docket No.: 10669-407PC0 Patent CLAIMS 1. A succinate dehydrogenase modifying compound selected from the group consisting of2. A method of identifying succinate dehydrogenase inhibitor small molecule compounds, comprising screening a chemical library at the interface of chains B, C, and D of 4YTP.

3. The method of claim 2 wherein the interface of chains B, C, and D of 4YTP is defined by the residues Chain B; P169, W172 & W173; Chain C: I30, W35, M39, S42, I43, R46 & I50; Chain D: V87, D90 & Y91.

4. A method of treating a disease or condition involving mutation or dysregulation of the succinate dehydrogenase pathway in a subject in need thereof, comprising administrating a therapeutically effective amount of one or more of the compounds of claim 1 or a composition of claim 10.Attorney Docket No.: 10669-407PC0 Patent 5. The method of claim 4 wherein the disease or condition is cancer.

6. The method of claim 5 wherein the cancer is selected from the group consisting of paraganglioma, pheochromocytoma, renal carcinoma, gastrointestinal stroma tumor, thyroid cancer, colorectal cancer, ovarian cancer, and lung cancer.

7. The method of claim 5 wherein the cancer is lung cancer.

8. The method of claim 4 wherein the disease or condition is selected from the group consisting of Leigh syndrome, ataxia, leukodystrophy, fibrosis, inflammatory disease, and premature aging.

9. The method of claim 4 wherein the method is more effective than treatment with dimethyl malonate.

10. A composition comprising at least one compound of claim 1 and a pharmaceutically acceptable carrier.

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