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Supplementary Table from Reengineering Ponatinib to Minimize Cardiovascular Toxicity

Supplementary Figure from Reengineering Ponatinib to Minimize Cardiovascular Toxicity

Small molecule tyrosine kinase inhibitors (TKI) have revolutionized cancer treatment and greatly improved patient survival. However, life-threatening cardiotoxicity of many TKIs has become a major concern. Ponatinib (ICLUSIG) was developed as an inhibitor of the BCR-ABL oncogene and is among the most cardiotoxic of TKIs. Consequently, use of ponatinib is restricted to the treatment of tumors carrying T315I-mutated BCR-ABL, which occurs in chronic myeloid leukemia (CML) and confers resistance to first- and second-generation inhibitors such as imatinib and nilotinib. Through parallel screening of cardiovascular toxicity and antitumor efficacy assays, we engineered safer analogs of ponatinib that retained potency against T315I BCR-ABL kinase activity and suppressed T315I mutant CML tumor growth. The new compounds were substantially less toxic in human cardiac vasculogenesis and cardiomyocyte contractility assays in vitro. The compounds showed a larger therapeutic window in vivo, leading to regression of human T315I mutant CML xenografts without cardiotoxicity. Comparison of the kinase inhibition profiles of ponatinib and the new compounds suggested that ponatinib cardiotoxicity is mediated by a few kinases, some of which were previously unassociated with cardiovascular disease. Overall, the study develops an approach using complex phenotypic assays to reduce the high risk of cardiovascular toxicity that is prevalent among small molecule oncology therapeutics.Significance:Newly developed ponatinib analogs retain antitumor efficacy but elicit significantly decreased cardiotoxicity, representing a therapeutic opportunity for safer CML treatment.

Small molecule tyrosine kinase inhibitors (TKI) have revolutionized cancer treatment and greatly improved patient survival. However, life-threatening cardiotoxicity of many TKIs has become a major concern. Ponatinib (ICLUSIG) was developed as an inhibitor of the BCR-ABL oncogene and is among the most cardiotoxic of TKIs. Consequently, use of ponatinib is restricted to the treatment of tumors carrying T315I-mutated BCR-ABL, which occurs in chronic myeloid leukemia (CML) and confers resistance to first- and second-generation inhibitors such as imatinib and nilotinib. Through parallel screening of cardiovascular toxicity and antitumor efficacy assays, we engineered safer analogs of ponatinib that retained potency against T315I BCR-ABL kinase activity and suppressed T315I mutant CML tumor growth. The new compounds were substantially less toxic in human cardiac vasculogenesis and cardiomyocyte contractility assays in vitro. The compounds showed a larger therapeutic window in vivo, leading to regression of human T315I mutant CML xenografts without cardiotoxicity. Comparison of the kinase inhibition profiles of ponatinib and the new compounds suggested that ponatinib cardiotoxicity is mediated by a few kinases, some of which were previously unassociated with cardiovascular disease. Overall, the study develops an approach using complex phenotypic assays to reduce the high risk of cardiovascular toxicity that is prevalent among small molecule oncology therapeutics. Significance: Newly developed ponatinib analogs retain antitumor efficacy but elicit significantly decreased cardiotoxicity, representing a therapeutic opportunity for safer CML treatment.

Development of tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL oncogene constitutes an effective approach for the treatment of chronic myeloid leukemia (CML) and/or acute lymphoblastic leukemia. However, currently available inhibitors are limited by drug resistance and toxicity. Ponatinib, a third-generation inhibitor, has demonstrated excellent efficacy against both wild type and mutant BCR-ABL kinase, including the "gatekeeper" T315I mutation that is resistant to all other currently available TKIs. However, it is one of the most cardiotoxic of the FDA-approved TKIs. Herein, we report the structure-guided design of a novel series of potent BCR-ABL inhibitors, particularly for the T315I mutation. Our drug design paradigm was coupled to iPSC-cardiomyocyte models. Systematic structure-activity relationship studies identified two compounds

Introduction: Ponatinib is one of the most cardiotoxic Tyrosine Kinase Inhibitors (TKIs), but continues to be used in clinical practice as it is the only TKI effective against the most common ABL T315I mutation in Chronic Myeloid Leukemia (CML). Long-term exposure to ponatinib increases cardiovascular events including myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. Novel therapeutics are needed to provide treatment for this common form of CML while avoiding cardiovascular side effects. Hypothesis: Chemical reengineering can create novel TKIs effective against T315I mutant CML but with reduced cardiovascular toxicity. Methods: Using fragment-based approach, we generated new, safer analogues of ponatinib. The anti-tumor efficacy of these analogues was tested in 2 different CML cell lines (K562 T315I and KCL22 T315I) and in CML patient samples. We assayed for myocardial toxicity by measuring contractile function in human iPSC-cardiomyocytes (hiPSC-CMs) using high-throughput functional imaging, and assayed for vascular toxicity by measuring vasculogenesis in human microvascular endothelial cells (HMVECs). Finally, we confirmed the safer cardiovascular profile and adequate anti-tumor efficacy in an in vivo xenograft mouse model of CML. Results: The new analogues inhibited T315I BCR-ABL kinase activity similar to ponatinib and suppressed T315I mutant CML tumor growth in vitro and in vivo . Compared to ponatinib, the new compounds showed markedly decreased adverse effects on contractility of hiPSC-CMs and vasculogenesis in HMVECs in vitro . The therapeutic window was increased in vivo , leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increased levels of cardiac troponin. Additionally, we identified multiple kinases, including FGFR1, that were inhibited by ponatinib but not the analogues, suggesting that there is a specific set of kinases responsible for ponatinib toxicity. Conclusions: This study demonstrates that chemical reengineering can generate novel, cardiovascular-safe TKIs that retain effective therapeutic properties against CML carrying ABL T315I mutation, but that exhibit minimal cardiovascular toxicity compared to ponatinib.

Introduction: Prostate cancer (PCa) is the second leading cause of cancer deaths among men in the United States. Approximately 50% of patients with PCa harbor an oncogenic TMPRRS2- ERG gene fusion in their primary tumor and 35% of patients with metastatic castration resistant prostate cancers have the gene fusion. We have identified a potent small molecule inhibitor, ERGi-USU-6 salt derivative 7b, that selectively inhibits the growth of ERG positive tumor. This small molecule inhibitor is also effective in inhibiting the growth of benign and cancerous mouse prostate organoids expressing TMPRSS2-ERG (ERG positive organoids). To gain insights into the cancer-selective properties of ERGi-USU-6 salt 7b we evaluated pathways associated in ERGi-USU induced inhibition. Methods: The biological activities of salt derivative 7b, were assessed in hormone- refractory metastatic tumor derived ERG positive prostate cancer cell line, VCaP along with organoids. We monitored the pathways associated in the mechanism of drug action through, cell cycle-regulator proteins by immunoblot assays, cell cycle and ferroptosis related analyses in response to 7b treatment. We also monitored the levels of the RIOK2 kinase, previously shown to bind the parental ERGi-USU compound. The normal primary endothelium derived HUVEC cells were used as normal control due to the normal endogenous expression of ERG in endothelial cells including HUVEC. Results: Cell growth and immunoblot analysis indicated the inhibition of ERG positive prostate organoid upon ERGi-USU treatments resulting in the downregulation of ERG and RIOK2 protein levels. The cell cycle analyses, pathway mapping by protein assessment and ferroptotic assays suggests that salt derivative 7b treatment inhibits the ERG positive prostate cancer through ferroptosis along with RIOK2 inhibition. Conclusions: Our results showed that the ferroptosis inducer ATF3 gene is involved in the cancer-selective activity of ERGi-USU-6 salt derivative 7b. Further, based on our observations we hypothesize that ferroptosis, the iron-dependent form of programmed cell-death, may be the mechanism of cancer selective activity of salt derivative 7b. Disclaimer: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of Uniformed Services University of the Health Sciences (USUHS), The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., the Department of Defense (DoD), the Departments of the Army, Navy, or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Binil Eldhose, Katherine Beck, Cyrus Eghtedari, Gartrell C. Bowling, Mallesh Pandrala, Sanjay V. Malhotra, Xiaofeng A. Su, Albert Dobi. Targeting of ERG positive prostate cancers with ERGi-USU-6 salt derivative. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4868.

Borrelia burgdorferi (Bb) infection causes Lyme disease, for which there is need for more effective therapies. Here, we sequenced the antibody repertoire of plasmablasts in Bb-infected humans. We expressed recombinant monoclonal antibodies (mAbs) representing the identified plasmablast clonal families, and identified their binding specificities. Our recombinant anti-Bb mAbs exhibit a range of activity in mediating macrophage phagocytosis of Bb. To determine if we could increase the macrophage phagocytosis-promoting activity of our anti-Bb mAbs, we generated a TLR9-agonist CpG-oligo-conjugated anti-BmpA mAb. We demonstrated that our CpG-conjugated anti-BmpA mAb exhibited increased peak Bb phagocytosis at 12-24 h, and sustained macrophage phagocytosis over 60+ hrs. Further, our CpG-conjugated anti-BmpA mAb induced macrophages to exhibit a sustained activation morphology. Our findings demonstrate the potential for TLR9-agonist CpG-oligo conjugates to enhance mAb-mediated clearance of Bb, and this approach might also enhance the activity of other anti-microbial mAbs.

TP53 tumor suppressor gene is a commonly mutated gene in cancer. p53 mediated senescence is critical in preventing oncogenesis in normal cells. Since p53 is a transcription factor, mutations in its DNA binding domain result in the functional loss of p53-mediated cellular pathways. Similarly, nuclear factor erythroid 2–related factor 2 (Nrf2) is another transcription factor that maintains cellular homeostasis by regulating redox and detoxification mechanisms. In glioblastoma (GBM), Nrf2-mediated antioxidant activity is upregulated while p53-mediated senescence is lost, both rendering GBM cells resistant to treatment. To address this, we identified novel Nrf2 inhibitors from bioactive compounds using a molecular imaging biosensor-based screening approach. We further evaluated the identified compounds for their in vitro and in vivo chemotherapy enhancement capabilities in GBM cells carrying different p53 mutations. We thus identified an Nrf2 inhibitor that is effective in GBM cells carrying the p53 (R175H) mutation, a frequent clinically observed hotspot structural mutation responsible for chemotherapeutic resistance in GBM. Combining this drug with low-dose chemotherapies can potentially reduce their toxicity and increase their efficacy by transiently suppressing Nrf2-mediated detoxification function in GBM cells carrying this important p53 missense mutation.

Prostate cancer is among the leading causes of cancer related death of men in the United States. The ERG gene fusion leading to overexpression of near full-length ERG transcript and protein represents most prevalent (50-65%) prostate cancer driver gene alterations. The ERG oncoprotein overexpression persists in approximately 35% of metastatic castration resistant prostate cancers. Due to the emergence of eventual refractoriness to second- and third-generation androgen axis-based inhibitors, there remains a pressing need to develop drugs targeting other validated prostate cancer drivers such as ERG. Here we report the new and more potent ERG inhibitor ERGi-USU-6 developed by structure-activity studies from the parental ERGi-USU. We have developed an improved procedure for the synthesis of ERGi-USU-6 and identified a salt formulation that further improves its activity in biological assays for selective targeting of ERG harboring prostate cancer cells.

Iridium-catalyzed transfer hydrogenation (TH) of carbonyl compounds using HCOOR (R = H, Na, NH4) as a hydrogen source is a pivotal process as it provides the clean process and is easy to execute. However, the existing highly efficient iridium catalysts work at a narrow pH; thus, does not apply to a wide variety of substrates. Therefore, the development of a new catalyst which works at a broad pH range is essential as it can gain a broader scope of utilization. Here we report highly efficient polypyridyl iridium(III) catalysts, [Ir(tpy)(L)Cl](PF6)2 {where tpy = 2,2′:6′,2′'-Terpyridine, L = phen (1,10-Phenanthroline), Me2phen (4,7-Dimethyl-1,10-phenanthroline), Me4phen (3,4,7,8-Tetramethyl-1,10-phenanthroline), Me2bpy (4,4′-Dimethyl-2–2′-dipyridyl)} for the chemoselective reduction of aldehydes to alcohols in aqueous ethanol and sodium formate as the hydride source. The reaction can be carried out efficiently in broad pH ranges, from pH 6 to 11. These catalysts are air stable, easy to prepare using commercially available starting materials, and are highly applicable for a wide range of substrates, such as electron-rich or deficient (hetero)arenes, halogens, phenols, alkoxy, ketones, esters, carboxylic acids, cyano, and nitro groups. Particularly, acid and hydroxy groups containing aldehydes were reduced successfully in basic and acidic reaction conditions, demonstrating the efficiency of the catalyst in a broad pH range with high conversion rates under microwave irradiation.

Introduction and Purpose of the Study: The advent of small molecule Tyrosine Kinase Inhibitors (TKIs) has revolutionized cancer treatment and greatly improved patient survival. However, the life-threatening cardiovascular toxicity of many TKIs present major concerns. Ponatinib is the most effective treatment for patients harboring the common T315I mutation of BCR-ABL that drives Chronic Myeloid Leukemia (CML) yet is one of the most cardiotoxic of TKIs. Ponatinib-induced events include myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. The aim of this study is to reengineer the chemical structure of ponatinib to minimize its adverse cardiovascular effects by using in vitro and in vivo models of cardiovascular toxicity. Experimental Procedures: We developed in vitro cardiovascular toxicity assays using human iPSC-cardiomyocytes (hiPSC-CMs) and human microvascular endothelial cells (HMVECs), and assessed anti-tumor efficacy in T315I mutated K562 CML cells. The cardiotoxicity was determined by impairment of cardiomyocyte contractility function, the vascular toxicity was defined by inability to form capillary-like networks in vasculogenesis assays. In vitro screening of ponatinib analogues probed chemical moieties responsible for its cardiovascular toxicity that were confirmed using mice xenograft mice models with human T315I mutant K562 cells. Summary of Unpublished Data: Compared to ponatinib, refined analogues showed markedly decreased adverse effects on HMVECs vasculogenesis and hiPSC-CMs contractility in vitro. The therapeutic window was increased in vivo, leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increasing serum levels of cardiac troponin. The kinase inhibition profiles of the “safe” analogues compared to ponatinib identified candidate mediators of ponatinib-induced toxicity. Validation by selective knockdown revealed likely mediators of cardiotoxicity, including FGFR. Conclusions: We succeeded in engineering a cardiovascular safe TKI that retained effective therapeutic properties against BCR-ABL T351 mutated form of CML, but with substantially decreased cardiovascular toxicity. Citation Format: Anna Pavlovna Hnatiuk, Arne A. Bruyneel, Dhanir Taylor, Mallesh Pandrala, Arpit Dheeraj, Wendy Li, Ricardo Serrano Fernandez, Dries A. Feyen, Michelle M. Wu, Prashila Amatya, Isabel Morgado, Volker Wiebking, Matthew H. Porteus, Sanjay Malhotra, Mark Mercola. Reengineering ponatinib to minimize cardiovascular toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB077.

Triple-negative breast cancer (TNBC) is one of the aggressive forms of breast cancer and frequently relapses and metastases. Currently, very few options are available for TNBC treatment. Cancer cells exploit glycolytic machinery for the Warburg effect and aerobic glycolysis. Enolase 1 (ENO1) is the glycolytic enzyme expressed in the majority of tissues and many cancer cells have its higher expression. Apart from the glycolytic role in the cytosol, ENO1 also plays different roles in cancer cells including function as a surface receptor. We have developed a chemical small molecule (CET12) that strongly binds to ENO1 and restrain its activity and subcellular localization. CET12 treatment arrests the TNBC cells in the mitotic phase and leads the apoptotic cell death via AMPK activation. Global proteome profiling suggested that CET12 pushes the cells toward oxidative phosphorylation. It also inhibits cell migration and invasion in vitro. In vivo studies using 4T1 and EMT6 syngeneic mouse models are also in-line with in vitro results and revealed that SU0212 treatment inhibits tumor progression and metastasis. These results were further supported by tail vain lung metastasis assay and intracardiac injection mouse model. This study provides compelling preclinical data for further development of CET12 for the treatment of TNBC. Citation Format: Dhanir Tailor, Arpit Dheeraj, Fernando Jose Garcia-Marques, Mallesh Pandrala, Abel Bermudez, Sharon Pitteri, Sanjay V. Malhotra. Inhibition of triple negative breast cancer metastasis via Enolase-1 modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1799.

Cancer remains a leading cause of death worldwide, resulting in continuous efforts to discover and develop highly efficacious anticancer drugs. High-throughput screening of heterocyclic compound libraries is one of the promising approaches that provided several new lead molecules with a novel mechanism of action. On the basis of the promising anticancer potential of imidazoquinoxaline as well as the structurally similar imidazoquinoline-derived scaffold, we prepared a set of C6-substituted benzimidazo[1,2-a]quinoxaline derivatives via two novel synthetic routes using commercially available starting materials, with good to excellent yields and evaluated their anticancer activity against the NCI-60 cancer cell lines. The one-dose (10 µM) anticancer screening of the synthesized compounds in the NCI-60 cell line panel revealed that the substituents have a significant role in the activity. In particular, the indole (7f), imidazole (7g), and benzimidazole (7h) derivatives showed significant activity against the triple-negative breast cancer cell line, MDA-MB-468. The lead compounds also exhibited notable IC50 values against another breast cancer cell line, MCF-7. Furthermore, it was observed that these compounds were relatively nontoxic to normal cell lines: HEK293 (human embryonic kidney cell line) and MCF12A (nontumorigenic human breast epithelial cell line). The IC50 values against healthy cells were at least 5- to 11-fold higher, offering a new class of heterocycles that can be further developed as promising therapeutics for cancer treatment.

Department of Chemistry and Centre of Advanced Studies in Chemistry, Panjab University, Chandigarh-160 014, India Department of Cell, Development and Cancer Biology, Center for Experimental Therapeutics, Knight Cancer Institute, Oregon Health and Science University, Portland, OR 97201 Division of Medicinal Chemistry, CSIR-Institute of Microbiology Technology Chandigarh, Sector-39A, Chandigarh-160 036, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201 002, Uttar Pradesh, India National Interdisciplinary Centre of Vaccine, Immunotherapeutic and Antimicrobials, Panjab University, Chandigarh-160 014, India E-mail: salunke@pu.ac.in Manuscript received online 10 July 2020, accepted 28 July 2020 Biphenyl is a privileged scaffold observed in several marketed drugs and is known to predominantly bind to a wide range of proteins with high specificity. Fused imidazole is another privileged structure which is found in several bioactive compounds. The present investigation describes the design and synthesis of a biprivileged compound library comprising biphenyl linked fused imidazoles and their activity against NCI-60 cell line to identify potential ‘hits’ for further anti-cancer drug discovery. In the preliminary investigation, imidazo[1,2-a]pyridine based heterocycles having tert-alkyl amine and a biphenyl substituent demonstrated promising results against some of the leukaemia, colon cancer, ovarian cancer as well as breast cancer cell lines. The active compounds were also found to be non-toxic to several other cancer cell lines, warranting further structure activity relationship (SAR) investigation. A systematic structural modifications and bioactivity evaluation against NC-I60 cell line resulted in the identification of 2-aryl-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]pyrazin-3-amine scaffold with biphenyl, benzo[d][1,3]dioxole and 4-(trifluoromethyl)benzene as substituents at C-2 position showing anticancer activity.

Summary Y box binding protein 1 (YB-1) is a multifunctional protein associated with tumor progression and the emergence of treatment resistance (TR). Here, we report an azopodophyllotoxin small molecule, SU056, that potently inhibits tumor growth and progression via YB-1 inhibition. This YB-1 inhibitor inhibits cell proliferation, resistance to apoptosis in ovarian cancer (OC) cells, and arrests in the G1 phase. Inhibitor treatment leads to enrichment of proteins associated with apoptosis and RNA degradation pathways while downregulating spliceosome pathway. In vivo, SU056 independently restrains OC progression and exerts a synergistic effect with paclitaxel to further reduce disease progression with no observable liver toxicity. Moreover, in vitro mechanistic studies showed delayed disease progression via inhibition of drug efflux and multidrug resistance 1, and significantly lower neurotoxicity as compared with etoposide. These data suggest that YB-1 inhibition may be an effective strategy to reduce OC progression, antagonize TR, and decrease patient mortality.

Despite the ubiquitous nature of titanium(iv) and several examples of its beneficial behavior in different organisms, the metal remains underappreciated in biology. There is little understanding of how the metal might play an important function in the human body. Nonetheless, a new insight is obtained regarding the molecular mechanisms that regulate the blood speciation of the metal to maintain it in a nontoxic and potentially bioavailable form for use in the body. This review surveys the literature on Ti(iv) application in prosthetics and in the development of anticancer therapeutics to gain an insight into soluble Ti(iv) influx in the body and its long-term impact. The limitation in analytical tools makes it difficult to depict the full picture of how Ti(iv) is transported and distributed throughout the body. An improved understanding of Ti function and its interaction with biomolecules will be helpful in developing future technologies for its imaging in the body.

Small molecules that disrupt leukocyte trafficking have proven effective in treating patients with multiple sclerosis (MS). We previously reported that chemerin receptor chemokine-like receptor 1 (CMKLR1) is required for maximal clinical and histological experimental autoimmune encephalomyelitis (EAE); and identified CMKLR1 small molecule antagonist 2-(α-naphthoyl) ethyltrimethylammonium iodide (α-NETA) that significantly suppressed disease onset in vivo. Here we directly compared α-NETA versus FDA-approved MS drug Tecfidera for clinical efficacy in EAE; characterized key safety/toxicity parameters for α-NETA; identified structure-activity relationships among α-NETA domains and CMKLR1 inhibition; and evaluated improved α-NETA analogs for in vivo efficacy. α-NETA proved safe and superior to Tecfidera in suppressing clinical EAE. In addition, we discovered structurally differentiated α-NETA analogs (primarily ortho- or para-methoxy substitutions) with significantly improved target potency in vitro and improved efficacy in vivo. These findings suggest that α-NETA-based CMKLR1 inhibitors may prove safe and effective in treating demyelinating diseases and potentially other autoimmune disorders.

Today, CisPt is one of the most successful chemotherapeutic metallodrugs. Unfortunately, the use of CisPt is restricted by the dose-limiting toxic effects and the acquired resistance, especially in lung cancer. As an alternatively, FDA-approved drugs for different diseases which target cancer metabolic pathways, are considered an attractive strategy to diminish systemic toxic effects. In the same way, the mechanistic pathways activated/inactivated by this drug could potentially overcome the acquired resistance induced by CisPt. For this reason in our approach, we incorporated deferasirox (Def), a clinical drug for iron (Fe) overload, as the ligand in the development of novel Pt complexes. In cancer progression, Fe plays a key role as a nutrient for cell growth and energy production. In consequence, we have synthesized and characterized our first library of three Pt-Def complexes: Pt(Def-COOH), Pt(Def-CONH2), Pt(Def-COOCH3). In this strategy, we have modified the OH of the carboxylic group of the Def to see the effects in the metabolic pathways. Viability assay results showed that Pt(Def-CONH2) induced a dramatic decrease in the IC50 in non-small lung adenocarcinoma cells after 24 h of incubation compared to CisPt, Def and Def-CONH2. Gene expression studies were conducted for cell growth, resistance and metastases related genes, where Pt(Def-CONH2) showed a significant downregulation of all of them. In addition, insight into target sites and metabolic pathways (apoptosis, caspase 3, ROS, cell cycle) inhibited or activated was garnered. This study will afford the optimization of the rationale to fine tune the structure of novel potential drugs. Citation Format: Yamixa Delgado, Mallesh Pandrala, Daraishka Perez, Eddian Velazquez, Jaisy Vega, Melissa Milian, Anamaris Torres. Development of novel Pt-based drugs using Deferasirox as ligand to diminish systemic toxicity and resistance induced by CisPt [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6375.

Background: Prostatic adenocarcinoma is among the leading cause of cancer-related deaths among men in the United States. The Erythroblast Transformation-Specific-Related Gene, ERG is dormant in normal prostate epithelium. Due to gene fusions, the ERG oncogene is frequently activated by androgenic signals in prostate cancer. ERG disrupts the normal differentiation, promotes epithelial-mesenchymal transition, migratory and invasive properties of cancer cells. Approximately 35% of metastatic castration-resistant prostate cancers harbor ERG oncogene. Due to the failure of androgen axis directed therapies, there is an urgent need to develop inhibitors to targeting prostate cancer-causing genes, such as ERG. We have identified a potent small-molecule inhibitor ERGi-USU that is remarkably selective for inhibiting the growth of ERG positive cancer cells through direct binding to the RIOK2 atypical kinase, a putative upstream regulator of ERG. We completed a structure-activity relationship (SAR) study and compound development resulting in the potent derivative, ERGi-USU-6. Our current objective is to improve the therapeutic properties of ERGi-USU-6, by new salt formulations. Methods: Evaluation of the five selected salt formulations of ERGi-USU-6 were performed by assessing the growth of ERG positive prostate cancer cell line (VCaP) and by monitoring ERG and RIOK2 protein levels. Selectivity was assessed by monitoring the growth, endogenous ERG and RIOK2 levels in normal ERG positive human umbilical vein derived endothelial cells (HUVEC). The IC50 values for cell growth, compared to the parental compound were calculated in a 12-step dilution range performed in triplicates and independently repeated three times. Cell growth was measured by quantitative Cell Glow assay monitoring viable cells using Perkin Elmer Envision assay instrument and protein levels were quantified by measuring bioluminescence in IBright instrument in a wide dynamic range. Results: One new salt formula with improved activity were identified, demonstrating improved cell growth (IC50=89nM vs. parental IC50=139), ERG protein and RIOK2 protein inhibition. None of the salt formulas of ERGi-USU-6 showed any effect on the primary cultures of the ERG positive normal endothelial cells (HUVEC) in the effective concentration range. Conclusion: The first evaluation of salt formulas of ERGi-USU-6 may open the possibilities for preclinical assessments of this remarkably cancer-selective compound. Citation Format: Binil Eldhose, Charles P. Xavier, Mallesh Pandrala, Sanjay V. Malhotra, Albert Dobi. Effective inhibition of TMPRSS2-ERG positive prostate cancer cells by a new formula of ERGi-USU-6 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5240.

Triple negative breast cancer is an aggressive, heterogeneous disease with high recurrence and metastasis rates even with modern chemotherapy regimens and thus is in need of new therapeutics. Here, three novel synthetic analogues of chalcones, plant-based molecules that have demonstrated potency against a wide variety of cancers, were investigated as potential therapeutics for triple negative breast cancer. These compounds exhibit IC(50) values of ~ 5 μM in triple negative breast cancer cell lines and are more potent against triple negative breast cancer cell lines than against non-tumor breast cell lines according to viability experiments. Tandem mass tag-based quantitative proteomics followed by gene set enrichment analysis and validation experiments using flow cytometry, apoptosis, and western blot assays revealed three different anti-cancer mechanisms for these compounds. First, the chalcone analogues induce the unfolded protein response followed by apoptosis. Second, increases in the abundances of MHC-I pathway proteins occurs, which would likely result in immune stimulation in an organism. And third, treatment with the chalcone analogues causes disruption of the cell cycle by interfering with microtubule structure and by inducing G1 phase arrest. These data demonstrate the potential of these novel chalcone derivatives as treatments for triple negative breast cancer, though further work evaluating their efficacy in vivo is needed.

The anticancer activities of dinuclear iridium(III) complexes [{Ir(tpy)Cl}2{μ-bbn}]4+ {Cl-Irbbn; tpy = 2,2':6',2"-terpyridine, bbn = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 7, 12 and 16)} against MCF-7 and MDA-MB-231 breast cancer cell lines have been determined. The activities of the Cl-Irbbn complexes increased with increasing chain length, the Cl-Irbb16 complex showing the best anticancer properties. However, while Cl-Irbb16 was active against the metastatic MDA-MB-231 cells (3 μM), it was relatively inactive against the nonmetastatic MCF-7 line (29 μM). The three geometric isomers of Cl-Irbb16 were isolated and characterised by NMR spectroscopy and mass spectrometry, and their anticancer activities, lipophilicities (log P) and DNA-binding abilities were examined. The trans,trans (t,t) isomer (IC50 = 2 μM against MDA-MB-231) showed considerably greater anticancer activity than the cis,trans (c,t) and cis,cis (c,c) isomers (8 and 31 μM, respectively). From log P measurements, the t,t isomer of Cl-Irbb16 (log P = –0.62) was found to be slightly more lipophilic than the other geometric isomers (–0.95 and –0.98 for the c,c and c,t isomers, respectively). Calf-thymus (CT) DNA-binding experiments demonstrated that the t,t isomer did induce a different alteration to the helix conformation compared to the other isomers. The results of this study also highlight the significance of the differences in three-dimensional shape that can be obtained through geometric isomerism for octahedral coordination complexes.

Ovarian cancer, the most common gynecologic malignancy, is a leading cause of cancer deaths in women. Seventy percent of patients at diagnosis present with stage III or IV disease, in which the tumor has disseminated beyond the ovaries and pelvic organs to distant sites. Despite resection and and chemotherapy, 80% of patients diagnosed with advanced epithelial ovarian cancer develop recurrent disease and overall prognosis is poor. Resistance to chemotherapeutic agents such as carboplatin and paclitaxel (taxane-based drugs) accounts for the lack of effectiveness of current therapy. Characterization of taxane-resistant tumors has shown that Class III β-tubulin (βTUB3) is significantly overexpressed in ovarian cancer as well as other cancers. Also, molecular characterization of the causes of treatment resistance revealed that βTUB3 plays a prominent role in the incorporation of guanylate binding protein 1, a conditionally expressed GTPase normally involved in the inflammatory response, as one of the critical effectors of taxane and radiotherapy resistance in ovarian cancer. Once in the cytoskeleton, GBP1 binds to pro-survival kinases such as serine/threonine-protein kinase pim-1 (PIM1) and initiates a signaling cascade that induces treatment resistance. We hypothesize that inhibiting the activity of GBP1 will restore sensitivity to taxane therapy. This is now well supported by our preliminary proof-of-concept studies. We have demonstrated that natural product-derived small molecule SU093 stabilizes GBP1 in a nonbinding conformation, resulting in the inhibition of GBP1:PIM1 interaction. The mechanistic studies using confocal microscopy showed that SU093 inhibits GBP1 by blocking its nuclear translocation and disrupts the tubulin dynamics. Cell cycle analysis showed that SU093 leads to G1 phase arrest followed by apoptotic cell death. SU093 treatment also induced pro-apoptotic protein Bax and reduced anti-apoptotic protein Bcl2. Overall, our in vitro and in vivo investigations provide a compelling foundation to develop a novel therapy to treat ovarian cancer by inhibition of GBP1. Citation Format: Dhanir Tailor, Vineet Kumar, Mallesh Pandrala, Angel Resendez, Sanjay V. Malhotra. Inhibiting guanylate binding protein 1 (GBP1) impedes ovarian cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4951.

A series of polypyridyl-ruthenium(II) and -iridium(III) complexes that contain labile chlorido ligands, [{M(tpy)Cl}(2){μ-bb(n)}](2/4+) {Cl-Mbb(n); where M = Ru or Ir; tpy = 2,2':6',2''-terpyridine; and bb(n) = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 7, 12 or 16)} have been synthesised and their potential as antimicrobial agents examined. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the series of metal complexes against four strains of bacteria - Gram positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) - have been determined. All the ruthenium complexes were highly active and bactericidal. In particular, the Cl-Rubb(12) complex showed excellent activity against all bacterial cell lines with MIC values of 1 μg mL(-1) against the Gram positive bacteria and 2 and 8 μg mL(-1) against E. coli and P. aeruginosa, respectively. The corresponding iridium(III) complexes also showed significant antimicrobial activity in terms of MIC values; however and surprisingly, the iridium complexes were bacteriostatic rather than bactericidal. The inert iridium(III) complex, [{Ir(phen)(2)}(2){μ-bb(12)}](6+) {where phen = 1,10-phenanthroline) exhibited no antimicrobial activity, suggesting that it could not cross the bacterial membrane. The mononuclear model complex, [Ir(tpy)(Me(2)bpy)Cl]Cl(2) (where Me(2)bpy = 4,4'-dimethyl-2,2'-bipyridine), was found to aquate very rapidly, with the pK(a) of the iridium-bound water in the corresponding aqua complex determined to be 6.0. This suggests the dinuclear complexes [Ir(tpy)Cl}(2){μ-bb(n)}](4+) aquate and deprotonate rapidly and enter the bacterial cells as 4+ charged hydroxo species.