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Diffuse midline gliomas (DMGs) including diffuse intrinsic pontine gliomas (DIPGs) bearing lysine-to-methionine mutations in histone H3 at lysine 27 (H3K27M) are lethal childhood brain cancers. These tumors harbor a global reduction in the transcriptional repressive mark H3K27me3 accompanied by an increase in the transcriptional activation mark H3K27ac. We postulated that H3K27M mutations, in addition to altering H3K27 modifications, reprogram the master chromatin remodeling switch/sucrose nonfermentable (SWI/SNF) complex. The SWI/SNF complex can exist in two main forms termed BAF and PBAF that play central roles in neurodevelopment and cancer. Moreover, BAF antagonizes PRC2, the main enzyme catalyzing H3K27me3. We demonstrate that H3K27M gliomas show increased protein levels of the SWI/SNF complex ATPase subunits SMARCA4 and SMARCA2, and the PBAF component PBRM1. Additionally, knockdown of mutant H3K27M lowered SMARCA4 protein levels. The proteolysis targeting chimera (PROTAC) AU-15330 that simultaneously targets SMARCA4, SMARCA2, and PBRM1 for degradation exhibits cytotoxicity in H3.3K27M but not H3 wild-type cells. AU-15330 lowered chromatin accessibility measured by ATAC-Seq at nonpromoter regions and reduced global H3K27ac levels. Integrated analysis of gene expression, proteomics, and chromatin accessibility in AU-15330-treated cells demonstrated reduction in the levels of FOXO1, a key member of the forkhead family of transcription factors. Moreover, genetic or pharmacologic targeting of FOXO1 resulted in cell death in H3K27M cells. Overall, our results suggest that H3K27M up-regulates SMARCA4 levels and combined targeting of SWI/SNF ATPases in H3.3K27M can serve as a potent therapeutic strategy for these deadly childhood brain tumors.

Osimertinib is a third-generation epidermal growth factor receptor and tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of lung adenocarcinoma patients harboring EGFR mutations. However, acquired resistance to this targeted therapy is inevitable, leading to disease relapse within a few years. Therefore, understanding the molecular mechanisms of osimertinib resistance and identifying novel targets to overcome such resistance are unmet needs of cancer patients. Here, we investigated the efficacy of two novel CDK12/13 inhibitors, AU-15506 and AU-16770, in osimertinib-resistant EGFR mutant lung adenocarcinoma cells in culture and xenograft models in vivo. We demonstrate that these drugs, either alone or in combination with osimertinib, are potent inhibitors of osimertinib-resistant as well as -sensitive lung adenocarcinoma cells in culture. Interestingly, only the CDK12/13 inhibitor in combination with osimertinib, although not as monotherapy, suppresses the growth of resistant tumors in xenograft models in vivo. Taken together, the results of this study suggest that inhibition of CDK12/13 in combination with osimertinib has the potential to overcome osimertinib resistance in EGFR mutant lung adenocarcinoma patients.

Supplementary table S1 shows the effect of ODM-203 in a large kinase panel. Supplementary figure S1 shows how ODM-203 is synthesized. Supplementary figure S2 shows the effect of ODM-203 on FRS2 phosphorylation. Supplement figure S3 shows the effect of ODM-203 in additional in vivo xenograft models. Supplement figure S4 shows the effect of ODM-203 on pFGFR in the SNU16 xenograft model. Supplement figure S5 shows the effect of ODM-203 on pFGFR in the KSM11 xenograft model. Supplement figure S6 shows the effect of ODM-203 angiogenesis in vivo. Supplement figure S7 shows the effect of ODM-203 on PD-L1 expression in vitro.

Alterations in the gene encoding for the FGFR and upregulation of the VEGFR are found often in cancer, which correlate with disease progression and unfavorable survival. In addition, FGFR and VEGFR signaling synergistically promote tumor angiogenesis, and activation of FGFR signaling has been described as functional compensatory angiogenic signal following development of resistance to VEGFR inhibition. Several selective small-molecule FGFR kinase inhibitors are currently in clinical development. ODM-203 is a novel, selective, and equipotent inhibitor of the FGFR and VEGFR families. In this report we show that ODM-203 inhibits FGFR and VEGFR family kinases selectively and with equal potency in the low nanomolar range (IC50 6–35 nmol/L) in biochemical assays. In cellular assays, ODM-203 inhibits VEGFR-induced tube formation (IC50 33 nmol/L) with similar potency as it inhibits proliferation in FGFR-dependent cell lines (IC50 50–150 nmol/L). In vivo, ODM-203 shows strong antitumor activity in both FGFR-dependent xenograft models and in an angiogenic xenograft model at similar well-tolerated doses. In addition, ODM-203 inhibits metastatic tumor growth in a highly angiogenesis-dependent kidney capsule syngenic model. Interestingly, potent antitumor activity in the subcutaneous syngenic model correlated well with immune modulation in the tumor microenvironment as indicated by marked decrease in the expression of immune check points PD-1 and PD-L1 on CD8 T cells and NK cells, and increased activation of CD8 T cells. In summary, ODM-203 shows equipotent activity for both FGFR and VEGFR kinase families and antitumor activity in both FGFR and angigogenesis models.

Mutations in MEK1/2 have been described as a resistance mechanism to BRAF/MEK inhibitor treatment. We report the discovery of a novel ATP-competitive MEK1/2 inhibitor with efficacy in wildtype (WT) and mutant MEK12 models. Starting from a HTS hit, we obtained selective, cellularly active compounds that showed equipotent inhibition of WT MEK1/2 and a panel of MEK1/2 mutant cell lines. Using a structure-based approach, the optimization addressed the liabilities by systematic analysis of molecular matched pairs (MMPs) and ligand conformation. Addition of only three heavy atoms to early tool compound

The switch/sucrose non-fermentable (SWI/SNF) complex has a crucial role in chromatin remodelling1 and is altered in over 20% of cancers2,3. Here we developed a proteolysis-targeting chimera (PROTAC) degrader of the SWI/SNF ATPase subunits, SMARCA2 and SMARCA4, called AU-15330. Androgen receptor (AR)+ forkhead box A1 (FOXA1)+ prostate cancer cells are exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to normal and other cancer cell lines. SWI/SNF ATPase degradation rapidly compacts cis-regulatory elements bound by transcription factors that drive prostate cancer cell proliferation, namely AR, FOXA1, ERG and MYC, which dislodges them from chromatin, disables their core enhancer circuitry, and abolishes the downstream oncogenic gene programs. SWI/SNF ATPase degradation also disrupts super-enhancer and promoter looping interactions that wire supra-physiologic expression of the AR, FOXA1 and MYC oncogenes themselves. AU-15330 induces potent inhibition of tumour growth in xenograft models of prostate cancer and synergizes with the AR antagonist enzalutamide, even inducing disease remission in castration-resistant prostate cancer (CRPC) models without toxicity. Thus, impeding SWI/SNF-mediated enhancer accessibility represents a promising therapeutic approach for enhancer-addicted cancers., PROTAC degrader–induced SWI/SNF inactivation abolishes DNA accessibility at enhancer elements of oncogenes and also tempers supra-physiologic expression of driver transcription factors, resulting in potent inhibition of tumour growth in mouse models.

In the recent past, proteolysis-targeting chimera (PROTAC) technology has received enormous attention for its ability to overcome the limitations of protein inhibitors and its capability to target undruggable proteins. The PROTAC molecule consists of three components, a ubiquitin E3 ligase ligand, a linker, and a target protein ligand. The application of this technology is rapidly gaining momentum, especially in cancer therapy. In this review, we first look at the history of degraders, followed by a section on the ubiquitin proteasome system (UPS) and E3 ligases used in PROTAC development. PROTACs are dependent on the UPS for degradation of target proteins. We further discuss the scope and design of degraders and mitigation strategies for overcoming the hook effect seen with degraders. As PROTACs do not follow Lipinski's 'Rule of 5', these molecules face drug metabolism and pharmacokinetic challenges. A detailed section on absorption, distribution, metabolism, and excretion of degraders is provided wherein we discuss methodologies and strategies to surmount the challenges faced by these molecules. For understanding PROTAC-mediated degradation, the characterization and measurement of protein levels in cells is important. Currently used techniques and recent advancements in assessment tools for degraders are discussed. Furthermore, we examine the challenges and emerging technologies that need to be focused on in order to competently develop potent degraders. Many companies are working in this area of emerging new modality and a few PROTACs have already entered clinical trials; the details of the trials are included in this review.

AUR-109 is an orally bioavailable clinical stage receptor tyrosine kinase inhibitor (RTK) that inhibits the activity of proangiogenic and oncogenic pathway-related RTKs including DDR1, FGFR, VEGFR, PDGFR, and RET. DDR1 instigates immune exclusion by promoting collagen fiber alignment thereby altering extracellular matrix (ECM) components in tumor immune microenvironment (TIME). DDR1 expression inversely correlated with reduced immune cell infiltration in TIME leading to exhaustion of tumor-fighting immune cells, tumor escape, EMT and metastasis. FGFRs induce the expression the PD-L1 and VEGFRs promote the proliferation of regulatory T cells (Treg), inhibit T-cell development and maturation of dendritic cells. These findings suggest that the spectrum-selective inhibition of these RTKs by AUR-109 has the potential to strongly modulate the anti- tumor immune response. Clinically successful immune checkpoint inhibitors (ICI) (anti-PD1, anti-PD-L1, and anti-CTLA4 antibodies) block the pathways that inhibit immune cell activation thus stimulating immune responses against the tumor cells. Although ICIs show improved survival in patients with many types of cancers, they suffer from the lack of response in majority of patients along with the development of resistance to therapy. To overcome the resistance and improve the efficacy, a number of combinations of PD-1 blockade with other anticancer therapies are being evaluated. We have undertaken a detailed characterization of the anti-tumor immune response by AUR-109 either as a single agent or in combination with anti-PD1 antibodies. The effect of AUR-109 on CD4 T cells and Treg cells was evaluated in the Renca subcutaneous tumor model. Immune cell population in the blood and tumor microenvironment were analyzed by FACS. Anti-tumor efficacy was analyzed in both subcutaneous and orthotopic Renca syngeneic tumor models. AUR-109 treatment resulted in an increase of total and CD4 T cells. Higher ratio of total T cells to regulatory T cells was observed indicating that there is an increase in effector T cells. In the TILs, there was decrease in the expression of PD-1 and PD-L1 with a concomitant increase in IFN-γ expression on CD8 T cells and NK cells indicating a profound immune activation caused by AUR-109 within the tumor. These results demonstrate the therapeutic potential of AUR-109 in combination with PD-1 blockade in preclinical models and the results from the ongoing combination studies will be presented. Citation Format: Girish Daginakatte, Saravanan Thiyagarajan, Kiran Aithal, Mamon Dey, Reshma Reghu, Monalisha Mandal, Kavitha Nellore, Susanta Samajdar, Murali Ramachandra. Generation of profound anti-tumor immunity by AUR-109, a spectrum-selective tyrosine kinase inhibitor, either as a single agent or in combination with immune checkpoint inhibitors. [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 4030.

Multiple Splice variants of AR have been reported in the past few years. These splice variants are upregulated in most cases of CRPC resulting in poor prognosis. Most of these variants lack the ligand binding domain (LBD) but still bind to DNA resulting in constitutive activation of downstream targets. The AR-V7 splice variant has been characterized extensively and current clinical trials in CRPC are exploring the use of AR-V7 as a biomarker. New therapeutic molecules that selectively target AR-V7 are also being explored. However, there is a dearth of information available on the selectivity, phenotypic responses in AR-V7 dependent cell lines and pharmacokinetic properties of such molecules. Using our proprietary computational algorithms and rational SAR optimization, we have developed a potent and selective AR-V7 degrader from a known AR DNA binding domain (DBD) binder. This molecule effectively degraded AR-V7 in a CRPC cell line and demonstrated good oral bioavailability in mouse PK studies. This tool compound can be used to evaluate the pharmacological effects of AR-V7 degraders. Further exploration of SAR can be pursued to develop more optimized lead compounds.

Background: E1A binding protein (p300) and its paralog CREB binding protein (CBP or CREBBP) are ubiquitously expressed acetyl transferases (HAT) that also act as co-activators for number of transcription factors including HIF1a, BRCA-1, p53, c-Myc and androgen receptor (AR). Both CBP and p300 possess bromodomain (BRD) and a lysine acetyltransferase (KAT) domain. These two closely related epigenetic modulators are known to play an oncogenic role in a variety of cancers. Functional synthetic lethal screens have identified preferential killing in CBP-deficient and/or MYC-dependent hematological cancer cells by suppression of the paralogue p300. CBP/p300 BRD inhibitor could also prevent AR and ER signaling, thereby potentially inhibiting growth of AR and ER -dependent cancer cells. Thus, targeting CBP/p300 represents an attractive approach for developing personalized cancer therapies. Experimental Results: Multiple potent and selective CBP/p300 BRD inhibitors that are structurally unrelated to known inhibitors were identified by iterative medicinal chemistry and SAR based approaches. The lead compound AU-18069 was optimized towards attaining good potency, selectivity, physicochemical properties and DMPK profile. AU-18069 potently inhibited proliferation of a wide range of cell lines derived from prostate cancer, breast cancer and CBP mutant/MYC-dependent hematological cancers. The lead compound demonstrated good PK profile in rodents as well as in dogs. Excellent anti-tumor efficacy was achieved in leukemia and lymphoma xenograft models at well-tolerated doses. Significant downregulation of c-Myc was observed in a single dose PK-PD study. Conclusion: Lead candidate AU-18069 demonstrated that selective CBP/p300 bromodomain inhibitors are efficacious in models of hematologic malignancies and solid cancers in vitro and in vivo. Further evaluation of efficacy in other xenograft models, as a single agent as well as in combination with other agents is planned. Long term toxicological and safety pharmacology evaluation in different species and other IND enabling studies are in progress to support progression of this compound to clinical trials. Citation Format: Chandrasekhar Abbineni, Saravanan Thiyagarajan, Aravind A B, Amit A Dhudashiya, Sivapriya Marappan, Naveen Kumar R, Raghavendra N R, Mamon Dey, Avinash Kumar, Uma Bharathi B V, Girish Aggunda Renukappa, N Venkata Siva Reddy, Asha Babu, Aakanksha J Shetty, Amith A, Narasimha Rao K, Suraj T Gore, Mahaboobi Jaleel, Ramesh S Senaiar, Vijaya Shankar Nataraj, Subhendu Mukherjee, Samiulla D S, Thomas Antony, Girish Daginakatte, Rajesh Eswarappa, Kavitha Nellore, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Evaluation of AU-18069, a novel small molecule CBP/p300 bromodomain inhibitor for the treatment of cancers [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 4114.

Background: The BAF (SWI/SNF) chromatin remodeling complex comprises of two mutually exclusive ATPases, SMARCA2 (BRM) and SMARCA4 (BRG1), that affect the mobilization and positioning of nucleosomes on DNA and thereby regulates important cellular functions including transcription, DNA recombination, DNA repair and chromosome decatenation during mitosis. SMARCA4 is frequently overexpressed in several types of cancers. Overexpression has been linked to increased proliferation and survival, as well as aggressive tumors and poor prognosis. SMARCA4 knockdown in these tumors lead to inhibition of proliferation and increased sensitivity to known chemotherapeutic agents, supporting the validity of targeting SMARCA4. Genetic silencing studies have established that the oncogenic activity of tumors lacking SMARCA4 is primarily driven by SMARCA2-containing residual SWI/SNF complex, suggesting the importance of dual inhibition of SMARCA2 and SMARCA4. While SMARCA4 is known to play a vital role in maintaining the oncogenic transcription program and driving proliferation in leukemia, the impact of dual SMARCA2 and SMARCA4 inhibition/degradation in acute myeloid leukemia (AML) is largely unexplored. Methods and Results: As part of the initial design plan, selective SMARCA2/4 Bromodomain inhibitors and specific ligands of several E3 ligases were chosen to arrive at different degrader designs. A choice of linkers and different exit vectors were considered to construct a variety of hetero bifunctional molecules. Our proprietary ternary complex modeling algorithm, ALMOND (ALgorithm for MOdeling Neosubstrate Degraders) helped in prioritizing the designs. Short listed compounds were synthesized and profiled in multiple cellular assays to understand their degradation potential. Several compounds that degrade SMARCA2, SMARCA4 & PBRM1 with pico molar DC50 were identified. These compounds have shown very potent anti-proliferative activity in both SMARCA2/4 proficient (MV-4-11, VCaP etc) and SMARCA4 mutant cell lines (SK-MEL-5 & RERF-LC-A1 etc). Further, potent compounds were optimized for their pharmacokinetic properties. Multiple lead compounds with low IV clearance and good oral bioavailability in rodents were identified. Advanced lead compounds are currently being evaluated in rodent tolerability and PK-PD experiments to select doses for the efficacy study. Conclusions: Highly potent degraders of SMARCA2, SMARCA4 & PBRM1 were identified by conjugating selective SMARCA2/4 Bromodomain inhibitors and several E3 ligase specific ligands. Further optimization of the linkers resulted in compounds with improved pharmacokinetic profile and very good oral bioavailability in rodents. Highly potent and orally available degraders of SMARCA2, SMARCA4 are efficacious in AML xenograft models and advanced profiling of candidate molecule is in progress. Citation Format: Chandrasekhar Abbineni, Leena Khare, Bilash Kuila, Abdul Rawoof Khaji, Dhaytadak Bhagwan Mahadeo, Sandeep Vitthal Dukare, Bhagya M S Kumar, Suraj T Gore, Vijay Kamal Ahuja, Amit A Dhudashiya, Raghavendra N R, Nagesh Gowda, Charamanna K B, Kiran Aithal B, Samiulla D S, Subhendu Mukherjee, Thomas Antony, Sanjeev Giri, Shekar Chelur, Kavitha Nellore, Girish Daginakatte, Murali Ramachandra, Susanta Samajdar. Discovery of orally bioavailable SMARCA2/4 dual degraders for treatment of acute myeloid leukemia [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 3729.

The CD47/signal regulatory protein alpha axis is a critical regulator of myeloid cell activation and serves as an immune checkpoint for macrophage mediated phagocytosis. Targeting CD47-SIRPα axis is emerging as one of the promising new immunotherapy approaches that targets innate immune response. Number of clinical trials are in progress to evaluate CD47/SIRPα blocking therapies. Most of these molecules are either anti-CD47 antibodies or SIRPα-Fc recombinant proteins. We have developed a novel small molecule CD47 antagonist, AUR103 as a therapeutic agent for solid and hematological cancers. AUR103 is efficacious as a single agent in tumors with high expression of “eat-me” signals and synergizes well in combination with tumor-targeting antibodies. We hypothesized that agents capable of inducing “eat-me” signals such as calreticulin (CRT) a pro-phagocytic signal, will synergize with AUR103 to enhance phagocytosis and antitumor activity. Here, we report the anti-tumor efficacy of AUR103 in combination with azacytidine and bortezomib in acute myeloid leukemia and multiple myeloma model of cancer, respectively.In the phagocytosis assay, HL-60 human myeloid leukemia or NCI-H929 human multiple myeloma cells were treated with azacytidine or bortezomib for 48 hours. Cells were stained with CFSE and were further treated with AUR103. Cells were then added to human macrophages and allowed to undergo phagocytosis. Phagocytosis of target cells was measured by detecting cells that were double positive for APC and CFSE by FACS. HL-60 cells were orthotopically implanted in NOD SCID mice while NCI-H929 multiple myeloma cells were sub-cutaneously implanted in NOD SCID mice. Tumor bearing mice were treated with AUR103 (10 and 30 mg/kg, b.i.d, and po) as a single agent or in combination with azacytidine (5 mg/kg i.p., twice weekly) or bortezomib (0.4 mg/kg i.v., twice weekly). AUR103 in combination with azacytidine and bortezomib significantly enhanced phagocytosis of HL-60 and NCI-H929 tumor cells, respectively when compared to individual treatments. In the combination efficacy studies, AUR103 combination treatments with azacytidine and bortezomib were well tolerated without any signs of toxicity. In the HL-60 orthotopic model, AUR103 combination with azacytidine significantly reduced the engraftment of HL-60 tumor cells. AUR103 combination with bortezomib resulted in enhanced tumor growth inhibition in NCI-H929 tumor model when compared to individual treatments. These results demonstrate the therapeutic potential of AUR103 in combination with agents that are capable of inducing “eat-me” or pro-phagocytic signals. Citation Format: Girish C. Daginakatte, Sasikumar Pottayil, Sudarshan Naremaddepalli, Gundala Chennakrishnareddy, Prasad Bilugudi, Sai Krishna Tangella, Sandeep Patil, Nagesh Gowda, Kiran Aithal, Wesley Roy Balasubramanian, Amit Dhudashiya, Samiulla Dodheri, Kavitha Nellore, Susanta Samajdar, Murali Ramachandra. AUR103 an oral small molecule CD47 antagonist in combination with azacytidine and bortezomib exhibits potent anti-tumor activity in myeloma and leukemia models in vitro and in vivo [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 3500.

In mammalian cells, DNA is wrapped around histone octamers (collectively referred to as nucleosomes) which form a physical barrier to all DNA-based processes. The switch/sucrose non-fermentable (SWI/SNF) is a multi-subunit chromatin remodeling complex that uses energy from ATP hydrolysis to reposition or eject nucleosomes at non-coding regulatory elements, thereby enabling access to the underlying DNA for transcriptional activation. Notably, the SWI/SNF complex plays a crucial role in chromatin remodeling and is recurrently altered in over 20% of human cancers, with the revised complex in cancer cells enabling central oncogenic gene programs. Yet, no studies have assessed the therapeutic efficacy of complete SWI/SNF inactivation across human cancers. Here, we developed a proteolysis targeting chimera (PROTAC) degrader of ATPase subunits of the SWI/SNF complex, SMARCA2 and SMARCA4. In a panel with over 90 normal and cancer cell lines from 18 different lineages, we found MYC-driven multiple myeloma and androgen receptor (AR)/forkhead box A1 (FOXA1)-positive prostate and breast cancers to be preferentially sensitive to dual SMARCA2 and SMARCA4 degradation relative to benign prostate as well as other cancer cell lines, including cancer cell lines with inactivating SMARCA4 mutations. We found complete SWI/SNF ATPase degradation to instantaneously compact the cis-regulatory elements that are bound and activated by transcription factors that drive cancer proliferation, namely MYC, IRF4, TCF3, AR, FOXA1, and ERG. This ensued in parallel untethering of these oncogenic drivers from the chromatin, with subsequent chemical decommissioning of their core enhancer circuitry and attenuation of downstream gene programs. Furthermore, using chromatin conformation assays we found SWI/SNF inactivation to disrupt super-enhancer and promoter DNA looping interactions that wire supra-physiologic expression of the MYC, AR, ERG, IRF4, and TCF3 oncogenes themselves, thereby tempering their expression in cancer cells. Treatment with the SMARCA2/4 degrader alone induced potent inhibition of tumor growth in cell line-derived xenograft models of multiple myeloma, as well as prostate cancer, and synergized with AR antagonists, inducing disease remission in several drug-resistant disease models. Notably, no major toxicities were seen in mice upon prolonged treatment with the SMARCA2/4 degrader, including no indications of thrombocytopenia, gastrointestinal goblet cell depletion, or germ cell degeneration—all being major toxicities associated with the BRD4-targeting therapeutics. To our knowledge, this study is the first preclinical proof of concept that targeted obstruction of chromatin accessibility at non-coding regulatory elements can be a potent therapeutic strategy in enhancer-addicted tumors, warranting the safety and efficacy assessments of SWI/SNF inhibitors and degraders in human clinical trials. Citation Format: Abhijit Parolia, Lanbo Xiao, Yuanyuan Qiao, Pushpinder Bawa, Sanjana Eyunni, Eleanor Young, Rahul Mannan, Sandra E. Carson, Yu Chang, Yuping Zhang, James George, Mustapha Jaber, Fengyun Su, Rui Wang, Sanjita Sasmal, Leena Khare, Subhendu Mukerjee, Chandrasekhar AbbinenI, Kiran Aithal, Xuhong Cao, Yuzhuo Wang, Susanta Samajdar, Murali Ramachandra, Arul M. Chinnaiyan. Targeting SWI/SNF ATPases in enhancer-addicted human cancers [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 3592.

The switch/sucrose non-fermentable (SWI/SNF) complex plays a crucial role in chromatin remodeling and is recurrently altered in over 20% of human cancers. Here, we developed a proteolysis targeting chimera (PROTAC) degrader of ATPase subunits of the SWI/SNF complex, SMARCA2 and SMARCA4. Intriguingly, we found androgen receptor (AR)/forkhead box A1 (FOXA1)-positive prostate cancer to be exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to benign prostate as well as other cancer cell lines, including those with inactivating SMARCA4 mutations. Mechanistically, SWI/SNF inhibition rapidly compacts the cis-regulatory elements that are bound and activated by transcription factors that drive cancer proliferation, namely AR, FOXA1, ERG, and MYC. This ensues in chromatin untethering of these oncogenic drivers, chemical decommissioning of their core enhancer circuitry, and attenuation of downstream gene programs. Furthermore, we found SWI/SNF inhibition to disrupt super-enhancer and promoter DNA looping interactions that wire supra-physiologic expression of the AR, FOXA1, and MYC oncogenes, thereby tempering their expression in cancer cells. Monotherapy with the SMARCA2/4 degrader induced potent inhibition of tumor growth in cell line-derived xenograft models of prostate cancer and remarkably synergized with AR antagonists, inducing disease remission in models of castration-resistant prostate cancer. We also found the combinatorial treatment to significantly inhibit the growth of enzalutamide resistant disease using in vitro as well as patient-derived xenograft models. Notably, no major toxicities were seen in mice upon prolonged treatment with the SMARCA2/4 degrader, including no indications of thrombocytopenia, gastrointestinal goblet cell depletion, or germ cell degeneration. Taken together, these results suggest that impeding enhancer accessibility through SWI/SNF ATPase inactivation represents a novel therapeutic approach in enhancer addicted human cancers. Citation Format: Lanbo Xiao, Abhijit Parolia, Yuanyuan Qiao, Pushpinder Bawa Pushpinder, Sanjana Eyunni, Rahul Mannan, Sandra E. Carson, Yu Chang, Xiaoju Wang, Yuping Zhang, Josh Vo, Steven Kregel, Stephanie A. Simko, Andrew D. Delekta, Mustapha Jaber, Heng Zheng, Ingrid Apel, Lisa McMurry, Fengyun Su, Rui Wang, Sylvia Wang, Sanjita Sasmal, Leena K. Satyam, Subhendu Mukherjee, Chandrasekhar AbbinenI, Kiran Aithal, Mital S. Bhakta, Jay Ghurye, Xuhong Cao, Nora M. Navone, Alexey Nesvizhskii, Rohit Mehra, Ulka Vaishampayan, Marco Blanchette, Yuzhuo Wang, Susanta Samajdar, Murali Ramachandra, Arul M. Chinnaiyan. Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer [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 5469.

Dysregulated fatty acid metabolism is thought to be a hallmark of cancer, wherein fatty acids function both as an energy source and as signals for enzymatic and transcriptional networks contributing to malignancy. Fatty acid-binding protein 5 (FABP5) is an intracellular protein that facilitates transport of fatty acids and plays a role in regulating the expression of genes associated with cancer progression such as cell growth, survival, and metastasis. Overexpression of FABP5 has been reported to contribute to an aggressive phenotype and a poor survival correlation in several cancers. Therefore, inhibition of FABP5 is considered as a therapeutic approach for cancers. Phenotypic screening of a library of covalent compounds for selective sensitivity of cancer cells followed by medicinal chemistry optimization resulted in the identification of AUR104 with desirable properties. Chemoproteomic-based target deconvolution revealed FABP5 as the cellular target of AUR104. Covalent adduct formation with Cys43 of FABP5 by AUR104 was confirmed by mass spectrometry. Target occupancy studies using a biotin-tagged AUR104 demonstrated potent covalent binding to FABP5 in both cell-free and cellular conditions. Ligand displacement assay with a fluorescent fatty acid probe confirmed the competitive binding mode of AUR104 with fatty acids. Binding at the fatty acid site and covalent bond formation with Cys43 were also demonstrated by crystallography. Furthermore, AUR104 showed a high degree of selectivity against a broad safety pharmacology panel of enzymes and receptors. AUR104 exhibited potent anti-proliferative activity in a large panel of cell lines derived from both hematological and solid cancers with a high degree of selectivity over normal cells. Anti-proliferative activity in lymphoma cell lines correlated with inhibition of MALT1 pathway activity, cleavage of RelB/Bcl10 and secretion of cytokines, IL-10 and IL-6. AUR104 displayed desirable drug-like properties and dose-dependent oral exposure in pharmacokinetic studies. Oral dosing with AUR104 resulted in dose-dependent anti-tumor activity in DLBCL (OCI-LY10) and NSCLC (NCI-H1975) xenograft models. In a repeated dose MTD studies in rodents and non-rodents, AUR104 showed good tolerability with an exposure multiple of >500 over cellular EC50 for up to 8 hours. In summary, we have identified a novel covalent FABP5 inhibitor with optimized properties that showed anti-tumor activity in in vitro and in vivo models with acceptable safety profile. The data presented here strongly support clinical development of AUR104. Citation Format: Dinesh Chikkanna, Leena Khare Satyam, Sunil Kumar Pnaigrahi, Vinayak Khairnar, Manoj Pothuganti, Lakshmi Narayan Kaza, Narasimha Raju Kalidindi, Vijaya Shankar Nataraj, Aditya Kiran Gatta, Narasimha Rao Krishnamurthy, Sandeep Patil, DS Samiulla, Kiran Aithal, Vijay Kamal Ahuja, Nirbhay Kumar Tiwari, KB Charamannna, Pravin Pise, Thomas Anthony, Kavitha Nellore, Sanjeev Giri, Shekar Chelur, Susanta Samajdar, Murali Ramachandra. Discovery and preclinical evaluation of a novel covalent inhibitor of FABP5 for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1266.

CD73 is a cell surface ectoenzyme, which is overexpressed in many types of human and mouse cancers. CD73 is the primary enzymatic producer of immunosuppressive adenosine in the tumor microenvironment, and high CD73 expression is associated with significantly poorer prognosis in several tumor types. Inhibition of either adenosine generation or signaling by inhibiting CD73 have been shown to be effective therapeutic approaches. Here we sought to discover and develop novel and orally bioavailable small molecule inhibitors that target CD73. Aurigene's non-nucleotide small molecule exhibit potent inhibition of CD73 in respective biochemical and cellular assays. High potency translated into rescue of AMP induced repression of IFN-Υ and IL-2 in human PBMCs. Lead compounds exhibited desirable drug-like properties including solubility, Caco2 permeability, lack of CYP inhibition and excellent oral pharmacokinetic exposure. In summary, we have identified small molecule inhibitors with good drug like properties, which showed good in vitro potency and excellent PK. Evaluation of these lead compounds in syngeneic tumor model along with in vivo toxicity studies are currently under way. Citation Format: Chandregowda Venkateshappa, Kishore Narayanan, Prasath Kothandaraman, Garima Priyadarshani, Rashmi Nair, Megha Goyal, Vijay Kamal Ahuja, Sagar Dadhania, AB Aravind, DS Samiulla, Girish Daginakkatte, Thomas Antony, Kavitha Nellore, Sunil Panigrahi, Dinesh Chikkanna, Susanta Samajdar, Murali Ramachandra. Novel, potent and orally bioavailable small molecule CD73 inhibitors for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1713.

Background: E1A binding protein (p300) and its paralog CREB binding protein (CBP or CREBBP) are ubiquitously expressed acetyl transferases (HAT) that also act as co-activators for number of transcription factors including HIF1a, BRCA-1, p53, c-Myc and androgen receptor (AR). Both CBP and p300 possess bromodomain (BRD) and a lysine acetyltransferase (KAT) domain. These two closely related epigenetic modulators are known to play oncogenic roles in a variety of cancers. Functional synthetic lethal screens have identified preferential killing in CBP-deficient and MYC-dependent hematological cancer cells by suppression of the paralogue p300. CBP/p300 BRD inhibitor could also prevent its coactivator function at AR, thereby potentially inhibit growth of AR-dependent prostate cancer cells. Thus, targeting CBP/p300 represents an attractive approach for developing personalized therapies. Experimental procedures and Results: Multiple potent and selective CBP/p300 BRD inhibitors that are structurally unrelated to known inhibitors have been identified by iterative medicinal chemistry and SAR based approaches. The lead compound, AU-18069 was optimized towards attaining good potency, physicochemical properties and DMPK profile. AU-18069 potently inhibited viability and proliferation of a wide range of cell lines derived from prostate cancer, CBP mutant and MYC-dependent hematological cancers and demonstrated improved PK profile in rodent models in comparison with a compound, currently in clinical trials. Excellent efficacy with significant tumor growth inhibition (TGI) was observed in MV4-11 xenograft model at well-tolerated doses along with downregulation of cMYC in a single dose PK-PD study. AU-18069 also showed modulation of different immune phenotypes including CD4+ T cell subsets. In summary, our lead candidate AU-18069 demonstrated that selective CBP/p300 bromodomain inhibitors are potent in models of hematologic malignancies and solid tumors in vitro and in vivo. Further evaluation of immune activation potential, efficacy studies in various xenograft models, long term toxicological evaluation in different species and other IND enabling studies are in progress. Citation Format: Chandrasekhar Abbineni, Saravanan Thiyagarajan, Ramesh S Senaiar, Subhendu Mukherjee, Mahaboobi Jaleel, Sivapriya Marappan, Raghavendra N R, Girish Aggunda Renukappa, Aravind A B, Naveen Kumar R, Venkata Siva Reddy, Asha Babu, Akhila Srinivas, Prasad Yadlapalli, Suraj T Gore, Pathange Hemasankar, Mamon Dey, Samiulla D S, Chandranath D Naik, Thomas Antony, Kavitha Nellore, Shekar Chelur, Girish Daginakatte, Mikko Myllymäki, Gerd Wohlfahrt, Murali Ramachandra, Susanta Samajdar. Evaluation of AU-18069, a novel small molecule CBP/p300 bromodomain inhibitor for the treatment of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1143.

Background: SMARCA2 (BRM) and SMARCA4 (BRG1) are two mutually exclusive DNA-dependent ATPases of the SWI/SNF complex, which function in mobilizing nucleosomes to regulate transcription, DNA replication/repair and chromosome dynamics. SMARCA4 is known to be mutated in number of cancers lacking targetable oncogenes, with SMARCA4-mutant patient population representing 10%-20% of NSCLC, 100% small cell ovarian cancer (hypercalcemic type), 28% skin cancer, 16% glioma and 14% colon cancer. Genetic studies have established the necessity of SMARCA2 for survival of tumor cells lacking SMARCA4. Although genetic silencing of SMARCA2 leads to potent anti-proliferative activity in SMARCA4-deficient cancer cell lines, pharmacological studies with a probe capable of binding to SMARCA2 and SMARCA4 bromodomains have failed to recapitulate such anti-proliferative effects. This prompted us to evaluate targeted protein degradation as an alternate approach to target SMARCA4 altered cancers. Methods and Results: A variety of hetero bi-functional molecules were synthesized by conjugating selective SMARCA2/4 bromodomain inhibitors with either VHL or CRBN E3-ligase specific ligands. Rational design approach guided by our proprietary ternary complex modeling algorithm, ALMOND (ALgorithm for MOdeling Neosubstrate Degraders) resulted in the identification of highly selective SMARCA2 degraders. The lead compound, AU-19820 showed > 10000-fold selectivity for SMARCA2 degradation versus other homologous proteins in tested cell lines. AU-19820 demonstrated potent anti-proliferative activity in SMARCA4 mutant but not in SMARCA2/4 proficient cell lines. This compound displayed favorable IV PK profile in rodents along with clean CYP profile. Additionally, the lead compound exhibited significant tumor growth inhibition in RERF-LC-A1 (SMARCA4 mutant lung cancer) xenograft model when dosed via i.v. route. Efficacious exposures were well tolerated with excellent tumor penetration. The compound also demonstrated moderate oral bioavailability in mouse. Efforts are in progress to improve this further by SAR modifications and exploiting prodrug approach. Conclusions: Potent and extremely selective SMARCA2 protein degraders were identified by conjugating SMARCA2/4 inhibitors with known VHL or CRBN ligands. SMARCA2 vs SMARCA4 selectivity handles have been very well explored with expandable SAR. Lead compound also displayed a synthetic lethality phenotype in SMARCA4 mutant cancer models while sparing SMARCA2/4 proficient ones. Further optimization of the oral bioavailability and evaluation of efficacy through oral route as well as intermittent IV dosing are planned. Citation Format: Chandrasekhar Abbineni, Leena Khare Satyam, Bilash Kuila, Ashok Ettam, Khaji Abdul Rawoof, Sreevidya MR, Sandeep Vitthal Dukare, Suraj T. Gore, Rakesh P. Nankar, Vijay Kamal Ahuja, Charamanna KB, Megha Goyal, Kiran Aithal, Samiulla DS, Subhendu Mukherjee, Thomas Antony, Sanjeev Giri, Shekar Chelur, Kavitha Nellore, Girish Daginakatte, Murali Ramachandra, Susanta Samajdar. Orally bioavailable SMARCA2 degraders with exceptional selectivity and potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1144.

Background: Inhibition of Bromodomain and extra-terminal (BET) family proteins by small molecules is actively being pursued as a therapeutic strategy in the clinics. Targeted protein degradation is an emerging therapeutic modality that has shown initial promise in the clinic. BET protein degradation has inherent advantages over inhibition viz. expansion of indication scope and amenability to intermittent dosing schedules. While many BET degraders have been disclosed earlier, inferior pharmacokinetic properties limit their further development. Methods and Results: We have designed and synthesized various hetero bi-functional molecules by conjugating novel and selective BET BRD ligands with VHL or CRBN ligands. This exercise led to the identification of several potent and selective BRD4 protein degraders with activity in a wide range of hematological and solid tumor cell lines. We have profiled one of the lead compounds extensively in vitro to gain insights on the mechanism of action. The lead compound showed lasting effect on BET protein abundance post compound washout while leading to apoptosis. This compound has favorable IV PK profile in rodents had has clean CYP and hERG profiles. Additionally, the lead compound exhibited significant tumor growth inhibition in MV4-11 xenograft model when dosed via i.v. route. Both QD and Q48h dosing regimens were well tolerated and produced efficacy in mouse models. Further SAR in the linker portion resulted in compounds with lower iv clearance and oral bioavailability either as prodrugs or as such. The proteomics study revealed a high selectivity towards BET proteins for the lead compound. Conclusions: Potent and selective BET protein degraders were identified by conjugating novel BET BRD ligands with both VHL and CRBN ligands. Optimization of these first generation BET degraders led to improved metabolic stability, translating into low iv clearance in rodents. Further evaluation of these compounds as prodrugs resulted in good oral exposures. Lead compounds from both the series have low iv clearance and are orally bioavailable in a simple formulation. We believe these compounds serve as valuable tools to fully understand the clinical scope of BET degraders. Citation Format: Chandrasekhar Abbineni, Mahaboobi Jaleel, Subhendu Mukherjee, Sivapriya Marappan, Nirbhay Kumar Tiwari, DS Samiulla, AB Aravind, Naveen R Kumar, Indu Bansal, Raghurami B Reddy, NVM Rao Bandaru, Akhila Srinivas, Janith Mary Maben, Suraj Tgore, Avainash Kumar, Rakesh P. Nankar, Chandranath D. Naik, Thomas Antony, Kavitha Nellore, Sanjeev Giri, Girish Daginakatte, Shekar Chelur, Olli Törmäkangas, Gerd Wohlfahrt, Mari Björkman, Elina Mattila, Laura Ravanti, Anu Moilanen, Murali Ramachandra, Susanta Samajdar. First in class orally bioavailable BETBRD degraders [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 1754.

Alterations in the gene encoding for the FGFR and upregulation of the VEGFR are found often in cancer, which correlate with disease progression and unfavorable survival. In addition, FGFR and VEGFR signaling synergistically promote tumor angiogenesis, and activation of FGFR signaling has been described as functional compensatory angiogenic signal following development of resistance to VEGFR inhibition. Several selective small-molecule FGFR kinase inhibitors are currently in clinical development. ODM-203 is a novel, selective, and equipotent inhibitor of the FGFR and VEGFR families. In this report we show that ODM-203 inhibits FGFR and VEGFR family kinases selectively and with equal potency in the low nanomolar range (IC50 6–35 nmol/L) in biochemical assays. In cellular assays, ODM-203 inhibits VEGFR-induced tube formation (IC50 33 nmol/L) with similar potency as it inhibits proliferation in FGFR-dependent cell lines (IC50 50–150 nmol/L). In vivo, ODM-203 shows strong antitumor activity in both FGFR-dependent xenograft models and in an angiogenic xenograft model at similar well-tolerated doses. In addition, ODM-203 inhibits metastatic tumor growth in a highly angiogenesis-dependent kidney capsule syngenic model. Interestingly, potent antitumor activity in the subcutaneous syngenic model correlated well with immune modulation in the tumor microenvironment as indicated by marked decrease in the expression of immune check points PD-1 and PD-L1 on CD8 T cells and NK cells, and increased activation of CD8 T cells. In summary, ODM-203 shows equipotent activity for both FGFR and VEGFR kinase families and antitumor activity in both FGFR and angigogenesis models.

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

Cyclin-dependent kinase 12 (CDK12) is a transcription-associated protein that plays a critical role in DNA damage response, splicing, pre-mRNA processing and is crucial for maintaining genomic stability. CDK12 associated with Cyclin K (CycK) regulates transcription elongation by phosphorylating RNA polymerase II (RNAP II) at Serine 2 (pS2) in the C-terminal domain (CTD). Overexpression of CDK12 in various tumor types suggests the possibility that CDK12 has oncogenic properties, similarly to other transcription-associated kinases. Considering its critical role in transcription and RNA processing, CDK12 is emerging as a potential therapeutic target for cancer. Multiple series of potent and selective CDK12 covalent inhibitors were identified by structure-guided and iterative medicinal chemistry approaches. Early lead compounds were optimized towards achieving high on target potency with good selectivity and desirable drug like properties including pharmacokinetic profile to achieve anti-tumour activity. Optimization of early lead compounds from two distinct chemical series resulted in very potent and highly selective CDK12 covalent inhibitors with desirable oral bioavailability. The covalent mode of action for these biochemically potent compounds has been confirmed by CDK12 target engagement assay in the cellular context. These selective inhibitors showed significant anti-proliferative activity in TNBC and other cancer cell lines including those harbouring ETS fusion. Importantly, cell killing is observed in cancer cells but not in normal cells (RWPE1) with short time (2h) and long-time (72h) exposure of these compounds. Anti-proliferative activity is well correlated with the inhibition of pS2 and down-regulation of a number of DNA damage response genes including BRCA1, RAD51, ATM and FANCI. Consistent with the inhibition of genes involved in DNA damage repair, a highly synergistic anti-proliferative activity was observed when treated in combination with cisplatin and PARP inhibitors. Based on the robust efficacy as a single agent in a TNBC mouse xenograft model with one of the optimized leads, the preclinical candidate exhibiting a greater degree of selectivity is being evaluated for efficacy and tolerability in relevant preclinical models. Citation Format: Ramulu Poddutoori, Sujatha Rajagopalan, Subhendu Mukherjee, Sivapriya Marappan, Samiulla D. S., Sasirekha Sivakumar, Shilpa S Nayak, Ravindra M V, Devaraja TS, Srinivas Kondela, Suraj Tgore, Amit A Dhudashiya, Charamanna K. B, Aravind A B, Amith A, Pavithra S, Hema Sankar Pathange, Thomas Antony, Mahaboobi Jaleel, Sanjeev Giri, Girish Daginakatte, Kavitha Nellore, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Pharmacological characterization of a preclinical candidate covalently inhibiting CDK12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4418.

SMARCA2/BRM and SMARCA4/BRG1 are the mutually exclusive DNA-dependent ATPases within the SWI/SNF complexes, which function in mobilizing nucleosomes to regulate transcription, DNA replication and repair, and higher-order chromosome dynamics. SMARCA4 is mutated in a number of cancers, which generally lack targetable oncogenes. Genetic silencing studies have established a requirement of SMARCA2 for survival of tumor cells lacking SMARCA4. SMARCA4-deficient patient population represents 10%-20% of NSCLC cases, ∼5% pancreatic cancer patients and ∼10% ovarian cancer patients where SMARCA2 is overexpressed. Interestingly, SMARCA4 is highly expressed without mutation in certain tumor types, where overexpression contributes to increased proliferation and survival. SMARCA4 knockdown in these tumors leads to inhibition of proliferation and also increase sensitivity to known chemotherapeutic agents, supporting the validity of targeting SMARCA4. Although genetic silencing of SMARCA2 leads to potent anti-proliferative activity in SMARCA4-deficient cancer cell lines, pharmacological studies with a probe capable of binding to SMARCA2 and SMARCA4 bromodomain have failed to show such an anti-proliferative phenotype. These findings triggered us to evaluate chemical degradation as an alternate approach to target SMARCA2/4 altered cancers. Optimization of bifunctional molecules with binding moieties for SMARCA2/4 and E3 ligase to induce proteasome-mediated degradation resulted in the identification of selective SMARCA2 and SMARCA4 degraders. These degraders showed selectivity against other bromodomain containing proteins such as BRD4, CBP and p300 in Western blot analysis. Functional analysis of a preferential SMARCA2 degrader in a panel of cell lines indicated a potent anti-proliferative activity in the context of SMARCA4 mutation. Additionally, these compounds displayed acceptable drug-like properties including solubility, metabolic stability and pharmacokinetics in mice. Dose-dependent tumor growth inhibition was observed in a SMARCA4-deficient lung cancer xenograft model and a syngeneic model of lymphoma at well-tolerated doses. Observed efficacy was correlated with the target degradation in the tumor supporting the potential to further develop them for cancer therapy. Based on the reported vulnerability of SMARCA4-deficient cell lines of diverse tumor origin to agents targeting PARP, PI3K/AKT and EZH2, combination effects with SMARC2 degrader are being interrogated. Citation Format: Leena Khare Satyam, Sanjita Sasmal, Manoj K. Pothuganti, Sreevidya M.R., Ashokk Ettam, Sireesha Nunna, Marla Roshaiah, Shankaraiah Chithaluru, Rangarao Pallepati, Amitkumar A. Pawar, Leela P. Narukulla, Raghunadh A. Sripathi, Suraj Tgore, Rakesh P. Nankar, Charamanna KB, Nagesh Gowda S, Kiran Aithal, Samiulla DS, Subhendu Mukherjee, Shekar Chelur, Kavitha Nellore, Girish Daginakatte, Murali Ramachandra, Susanta Samajdar. Anti-tumor efficacy of SMARCA degraders in pre-clinical models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3844.

As a potent immunosuppressor adenosine is essential for maintaining tissue homeostasis and preventing an overzealous immune response during inflammation and infection. However, adenosine generated within the tumor microenvironment by the action of ecto-nucleotidases including CD73 hinders the immune reaction towards cancer cells by signaling through adenosine receptors such as high affinity A2AR expressed on immune cells. Inhibitions of either adenosine generation or signaling by inhibiting CD73 or A2AR have been shown to be effective therapeutic approaches. Interestingly, the co-blockade of CD73 and A2AR results in a more pronounced anti-tumor activity than blockade of either, likely due to increased CD73 expression upon A2AR inhibition and compensatory activity of other adenosine receptors such as A2BR. In view of this, we sought to discover and develop small molecule inhibitors that dually target CD73 and A2AR with oral bioavailability for ease of administration and use in combination with other anti-cancer therapies. Aurigene’s dual inhibitors exhibit potent inhibition of both A2AR and CD73 in respective biochemical and cellular assays. High potency translated into rescue of NECA or AMP induced repression of IFNΥ and IL2 in human PBMCs. Lead compounds exhibited desirable drug-like properties including solubility, permeability, lack of CYP inhibition, and pharmacokinetic exposure. In Syngeneic tumour model, treatment with lead compounds resulted in significant tumour growth inhibition while correlating well with tumour drug levels and modulation of pharmacodynamic markers. In summary, we have identified first-in-class dual inhibitors with good drug like properties, which showed significant antitumor efficacy. Evaluation of these lead compounds in additional tumour models and in vivo toxicity studies is currently under way. Citation Format: Dinesh Chikkanna, Kishore Narayanan, Sunil Panigrahi, Garima Priyadarshini, Megha Goyal, Jiju Mani, DS Samiulla, Sagar dadhania, AB Aravind, Girish Daginakatte, Thomas Anthony, Kavitha Nellore, Susanta Samajdar, Murali Ramachandra. Preclinical evaluation of pharmacokinetics, pharmacodynamics and efficacy of the dual CD73-A2AR Inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4086.

Cyclin-dependent kinase 12 (CDK12) is a transcription-associated kinase that participates in various cellular processes such as DNA damage response, splicing and pre-mRNA processing. In association with Cyclin K (CycK), CDK12 regulates transcription elongation by phosphorylating RNA polymerase II (RNAP II) at Serine 2 in the C-terminal domain (CTD). Overexpression of CDK12 in various tumor types suggests the possibility that CDK12 has oncogenic properties, similarly to other transcription-associated kinases. Considering its critical role in transcription and RNA processing CDK12 is emerging as a potential therapeutic target for cancer. Multiple series of potent and selective CDK12 covalent inhibitors were identified by iterative medicinal chemistry efforts and SAR-based approaches. Early compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile to achieve anti-tumour activity. Very potent and highly selective CDK12 inhibitors have been identified from two distinct chemical series. The covalent mode of action for these biochemically potent compounds has been confirmed by CDK12 target engagement assay in the cellular context. These selective inhibitors showed significant anti-proliferative activity in TNBC and other cancer cell lines, which correlated with inhibition of pS2 (RNAP II), a bonafide CDK12 substrate and target engagement. In vivo target engagement, PD and efficacy data for optimized compounds with good oral bioavailability in a TNBC (HCC-70) xenograft model along with will be presented. Citation Format: Ramulu Poddutoori, Sujatha Rajagopalan, Subhendu Mukherjee, Sivapriya Marappan, Samiulla D S, Sasirekha Sivakumar, Shilpa S. Nayak, Ravindra M. V, Hadianawala Murtuza, Devaraja T. S, Srinivas Kondela, Suraj Tgore, Amit A. Dhudashiya, Charamanna K. B, Thomas Antony, Girish Daginakatte, Sanjeev Giri, Shekar Chelur, Murali Ramachandra, Chetan Pandit, Susanta Samajdar. Preclinical evaluation of PD and efficacy of novel potent selective and orally bioavailable CDK12 covalent inhibitors in TNBC model [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 2384.

PRMT5 is a typical type II methyltransferase, transferring two methyl groups to arginine, leading to symmetric dimethylation of the substrate. It can symmetrically methylate histones H2AR3, H3R2, H3R8, and H4R3 and can also methylate many non-histone proteins contributing to tumorigenesis by regulating cell cycle progression, DNA repair, cell growth, apoptosis, and inflammation. Overexpression of PRMT5 is reported in several human malignancies including lymphoma, glioma, melanoma, lung, breast, ovarian, and prostate cancers. Elevated levels correlate with poor prognosis in NSCLC, ovarian cancers, and GBM. Therefore, PRMT5 is considered an attractive target for cancer therapy. We sought to discover and develop PRMT5 inhibitors with the “best-in-class” profile with an emphasis on improved permeability for their potential use in solid tumors. Utilizing structure-guided drug design and SAR-based approaches, we have optimized two chemical series of substrate competitive PRMT5 inhibitors. Determination of co-crystal structures with several de novo designed hits aided in the identification of lead compounds that exhibited potent inhibition of PRMT5. Lead compounds AU-574 and AU-755 were highly active in inhibiting proliferation of a number of cell lines derived from solid tumors that correlated well with cellular H4R3Me2s inhibition, confirming the mechanism. Lead compounds exhibited desirable drug-like properties including solubility, permeability, lack of CYP inhibition, and pharmacokinetic exposure. In xenograft models of Z-138 (lymphoma) and H-358 (lung cancer), treatment with lead compounds resulted in significant tumor growth inhibition while correlating with tumor drug levels and modulation of H4R3Me2s as the pharmacodynamic effect. In summary, we have identified PRMT5 inhibitors with “best-in-class" drug-like properties including optimized permeability and antitumor efficacy. Evaluation of these lead compounds in in vitro selectivity screening and in toxicity studies in higher species is currently under way. Citation Format: Dinesh Chikkanna, Sunil Kumar Panigrahi, Sujatha Rajagopalan, Srinivasa Raju Sammeta, Darshan Chawla, Pavithra S, Samiulla D.S, Angelene Prasanna, Priyabrata Chand, Kiran Aithal, Sai Sudheer Marri, Naveen Kumar, Srinivasa Rao Ganipisetty, Raju Mutyala, Kasieswara Rao, Thomas Antony, Girish Daginakatte, Anirudha Lakshminarasimhan, Mohan R, Narasihmarao K, Shekar Chelur, Chetan Pandit, Susanta Samajdar, Murali Ramachandra. Preclinical In vivo evaluation of efficacy, pharmacokinetics and pharmacodynamics of novel PRMT5 inhibitors in multiple tumor models [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 1392.

The SWI/SNF complexes consist of one of two mutually exclusive DNA-dependent ATPases, BRG1/SMARCA4 or BRM/SMARCA2, together with core and accessory subunits that function in mobilizing nucleosomes to regulate transcription, DNA replication and repair, and higher-order chromosome dynamics. SMARCA2 (BRM) and SMARCA4 (BRG1), contain a bromodomain and an ATPase domain. The mammalian SWI/SNF complex functions as a tumor suppressor in many human malignancies. In addition to an essential role in pluripotency and development, genetic lesions of SWI/SNF complexes have been strongly linked to cancer development as components are mutated in many cancers. In some tumor types, mutations within the SWI/SNF complex lead to context specific vulnerabilities such as the requirement of SMARCA2 for survival of tumour cells lacking SMARCA4. This finding of SMARCA2/4 synthetic lethal relationship translates in vivo which emphasizes SMARCA2 as a promising therapeutic target for the treatment SMARCA4-mutant cancers. Moreover, the SMARCA4-deficient patient population generally lacks targetable oncogenes (such as mutant EGFR or ALK translocations), which further emphasizes the potential of developing SMARCA2 inhibitors. Previously, contrary to genetic silencing of SMARCA2 leading to potent anti-proliferative activity in SMARCA4-deficient cancer cell lines, pharmacological studies with a cell permeable probe capable of binding to SMARCA2 and SMARCA4 have failed to show such an anti-proliferative phenotype. These findings support that the ATPase domain, but not the bromodomain of SMARCA2, is the tractable therapeutic target for SMARCA4-deficient cancer. Significant hurdle in developing potent and selective ATPase inhibitors that must compete with intracellular concentrations of ATP (2-10 mM) triggered us to evaluate chemical degrader as an alternate approach. Herein we report the identification and characterization of potent SMARCA2/4 degraders. Design and SAR-based optimization of bifunctional molecules with binding moities for SMARCA2/4 and E3 ligase to induce proteosome-mediated degradation yielded compounds that potently degraded SMARCA2 alone or both SMARCA2 and SMARCA4. Selective binding to SMARCA2/4 was confirmed in biochemical assays followed by assessement of their cellular degradation potency in Western blot analysis. Selective degradation of SMARCA2/4 over other bromodomain containing protein such as BRD4 and CBP/p300 was also observed. Functional analysis of these compounds in a panel of cell lines indicated that the degradation of SMARCA2 or SMARCA2/4 resulted in potent anti-proliferative activity in selected cell lines that was not strictly dependent upon SMARCA4 status. Additionally, these compounds displayed reasonable drug-like properties including solubility, metabolic stability and pharmacokinetics in mice supporting their potential to fully evaluate the impact of SMARCA2/4 degradation and to develop them as a novel therapeutic approach. Citation Format: Sanjita Sasmal, Leena K. Satyam, Manoj K. Pothuganti, Ashokk Ettam, Sireesha Nunna, Mohammed A. Shareef, Sreevalsam Gopinath, Subhendu Mukherjee, Murali Ramachandra, Susanta Samajdar. Identification of SMARCA2/4 degraders for the treatment of SMARCA4-mutant and other cancers [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 LB-258.

Background: Cyclin-dependent kinase 12 (CDK12) coordinately regulates the transcription, splicing, and alternative splicing of several large pre-mRNAs. Defective CDK12 kinase activity has been associated with genomic instability and downregulation of genes in the DNA damage response (DDR) pathway. In addition, CDK12 depletion impairs alternate splicing, a process being increasingly implicated in cancer progression. Associated with Cyclin K (CycK), CDK12 regulates transcription elongation by phosphorylating RNA polymerase II (RNAP II) at S2 in the C-terminal domain (CTD). Considering its role in transcription and RNA processing to maintain genomic stability/integrity in cancer, CDK12 is emerging as a potential therapeutic target to treat cancer. Experimental Procedures: Potent and selective CDK12 inhibitors were identified from multiple series by iterative medicinal chemistry efforts and SAR-based approaches. Early compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity, and desirable pharmacokinetic profile to achieve antitumor activity.Summary: Very potent and highly selective compounds were identified from two distinct chemical series that are highly potent in inhibiting CDK12 in biochemical assays. Proving their covalent mode of action, these orally exposed compounds demonstrated robust engagement of CDK12 in a cellular context. Several of these potent and selective CDK12 inhibitors showed potent antiproliferative activity in various cancer cell lines derived from different origin, accompanied by robust CDK12 engagement and inhibition of pS2 (RNAP II). Further optimization of potency and ADME properties of initial lead compounds is in progress. Tolerability and efficacy studies are ongoing with selected early leads to test their impact on tumor growth inhibition in xenograft models. Conclusion: We identified novel, selective, and orally bioavailable covalent inhibitors of CDK12 from multiple distinct series with desirable drug-like properties, which are being evaluated for antitumor activity in xenograft models. Citation Format: Ramulu Poddutoori, Sujatha Rajagopalan, Subhendu Mukherjee, Sivapriya Marappan, Samiulla D S, Venkateswarlu Kasturi, Sasirekha Sivakumar, Shilpa Nayak, Ravindra M V, Suraj Tgore, Amit Dhudashiya, Charamanna K B, Thomas Antony, Mahaboobi M, Sanjeev Giri, Girish C. Daginakatte, Shekar Chelur, Murali Ramachandra, Chetan Pandit, Susanta Samajdar. Potent selective and orally bioavailable inhibition of CDK12 by novel covalent inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B165.

Background: Cyclin-dependent kinase 7 (CDK7) is an important constituent of the cellular transcriptional machinery, where it phosphorylates the C-terminal domain (CTD) of RNAP polymerase II (RNAPII). Because many tumor types are critically dependent on transcription for maintenance of their oncogenic state, pharmacologic modulation of CDK7 kinase activity is considered as an approach to treat cancer. Experimental Procedures: Multiple series of covalent CDK7 inhibitors were identified by iterative medicinal chemistry efforts and SAR-based approach. These compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity, and desirable pharmacokinetic profile to achieve antitumor activity. Summary: We have now identified a highly selective and orally bioavailable covalent CDK7 inhibitor AU-002 as a preclinical candidate. AU-002 is highly potent in inhibiting CDK7 in biochemical assays with robust engagement of CDK7 in cellular context. In a broad panel of 372 kinases, AU-002 shows excellent selectivity for CDK7. AU-002 exhibits excellent drug-like characteristics including solubility, permeability, metabolic stability, and good oral bioavailability. Oral treatment of this compound in a xenograft model resulted in dose-dependent tumor growth inhibition in AML xenograft model and with complete tumor regression at well-tolerated doses. Potent inhibition of tumor growth was accompanied by complete target engagement and suppression of pS5RNAPII in a parallel PK-PD study. Efficacy studies in additional xenograft models, advanced DMPK, and toxicity studies are ongoing for this compound. Conclusion: We have identified a novel and highly selective CDK7 covalent inhibitor candidate with good oral bioavailability that shows excellent efficacy in an AML xenograft model. Findings presented here support further development of AU-002 for the treatment of cancer. Citation Format: Ramulu Poddutoori, Leena Khare Satyam, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Charamanna KB, Lakshmi Narayana Kaza, Manoj Kumar Pothuganti, Sujatha Rajagopalan, Sasirekha Sivakumar, Bharath E N, Aravind A B, Amith A, Ravindra M V, Suraj Tgore, Thomas Antony, Chetan Pandit, Shekar Chelur, Girish Daginakatte, Murali Ramachandra, Susanta Samajdar. Identification of a novel highly selective and orally bioavailable preclinical candidate for CDK7 covalent inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B164.

During 2004-2006, group A rotavirus P[8] strains were the major VP4 genotype (43.2%, n = 317) among diarrhoeic children in Eastern India. Phylogenetic analysis of VP8* amino acid sequences of 16 of these strains with other P[8] strains revealed four distinct lineages. P[8] strains from Eastern India clustered within rare OP354-like and Hun9-like lineages, pointing towards co-prevalence of divergent P[8] strains. Although it is unclear whether the observed genetic diversity might affect to some extent the efficacy of vaccines, the present study emphasized further efforts to address the much lacking information on diversity of P[8] strains across the Indian subcontinent.

Simple synthesis of substituted pyrroles using iodine-catalyzed and montmorillonite KSF-clay-induced modified Paal-Knorr methods has been accomplished with excellent yields. N-Substituted carbazole has also been prepared by following this method. If one of the reactants is a liquid, the reaction proceeds exceedingly well without a solvent. This method gives pyrroles with less nucleophilic multicyclic aromatic amines at room temperature.

Bismuth nitrate in wet silica gel was found to be an excellent reagent for the regeneration of ketones from oximes.

Background: ODM-207 is a potent and selective BET inhibitor that is structurally unrelated to the benzodiazepine-based inhibitors including JQ1, I-BET762, and OTX015. Phase I clinical trials have now been initiated with this agent based on its potent anti-tumor activity in various in vitro and in vivo models of hematologic malignancies and solid tumors. In view of the recent publications implicating a role for BET protein BRD4 in the suppression of PD-L1 expression, an immune checkpoint ligand for PD-1, we sought to evaluate ODM-207 for its effect on immune-mediated anti-tumor efficacy in pre-clinical models. Methods and Results: Mouse splenocytes were stimulated with anti-CD3 and anti-CD28 in the presence or absence of ODM-207 for four days and changes in immune cell population were analyzed by FACS. Results revealed an increase in the level of activated cytotoxic CD8+ T cells as indicated by increased intracellular IFNγ and granzyme B with ODM-207 treatment. After confirming the lack of direct anti-proliferative activity on the mouse colon carcinoma cell line CT26, in vivo evaluation of ODM-207 was carried out in the syngeneic CT26 subcutaneous tumor model established in BALB/c mice. Daily oral administration of ODM-207 at 30 mg/kg was well tolerated in this model and resulted in a statistically significant inhibition of tumor growth. Interestingly, the tumor growth inhibition observed with ODM-207 was comparable to that with a commercially available anti-mouse PD1 antibody. Studies to characterize the immune changes in the tumor and anti-tumor activity of ODM-207 in combination with an anti-mouse PD1 antibody are currently underway and the results will be presented. Conclusions: In summary, these studies demonstrate the anti-tumor activity of BET inhibitor in a syngeneic model of colon carcinoma in the absence of a direct anti-proliferative activity on tumor cells. Observed tumor growth inhibition correlated with the in vitro activation of cytotoxic CD8+ T cells supporting the immune-mediated effect leading to tumor growth inhibition. In view of the remarkable success with the immune-based therapeutic approaches, these findings are relevant in devising appropriate strategies for the continued clinical development of ODM-207. Citation Format: Pratima Deshpande, Ravi Krishna Babu, Prashant Yallappa Vadnal, Mahaboobi Jaleel, Murali Ramachandra, Chandrasekhar Abbineni, Susanta Samajdar, Anu-Maarit Moilanen, Pekka Kallio. Immune-mediated anti-tumor activity with a clinical stage BET bromodomain inhibitor ODM-207 in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-113. doi:10.1158/1538-7445.AM2017-LB-113

Cyclin-dependent kinase 7 (CDK7) is an important constituent of the cellular transcriptional machinery, where it phosphorylates the C-terminal domain (CTD) of RNAP polymerase II (RNAPII). Because many tumor types are critically dependent on transcription for maintenance of their oncogenic state, pharmacological modulation of CDK7 kinase activity is considered as an approach to treat cancer. Multiple series of covalent CDK7 inhibitors were identified by iterative medicinal chemistry efforts and SAR based approach. These compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile to achieve anti-tumor activity. We have now identified a pre-clinical candidate AU-BGB-002 which is highly potent in inhibiting CDK7 in biochemical as well as cellular assays while fully efficiently engaging the target. In a panel of kinases, AU-BGB-002 shows selectivity for CDK7. A panel of cell lines derived from a diverse set of indications are sensitive to AU-BGB-002. AU-BGB-002 exhibits excellent drug-like characteristics including solubility, permeability, metabolic stability and good oral bioavailability. When tested in a xenograft model, AU-BGB-002 treatment resulted in dose dependent tumor growth inhibition in AML xenograft model with tumor stasis at a dose of 10 mg/kg. Potent inhibiton of tumor growth was accompanied by complete target engagement and suppression of pS5RNAPII RNAPolII Ser5 phosphorylation in a parallel PK-PD study. Efficacy studies in additional xenograft models, advanced DMPK and toxicity studies are ongoing for this compound. In summary, we have identified a novel and selective CDK7 covalent inhibitor candidate with desirable drug-like properties that shows excellent efficacy in an AML xenograft model. Findings presented here support further development of AU-BGB-002 for the treatment of cancer. Citation Format: Leena K. Satyam, Ramulu Poddutoori, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Aravind Basavaraju, Lakshmi Narayana Kaza, Manoj Kumar Pothuganti, Shilpa Nayak, Nandish C, Amith A, Ravindra MV, Dabbeeru Madhu Babu, Nagaraju A, Suraj Tgore, Thomas Antony, Chetan Pandit, Murali Ramachandra, Shekar Chelur, Girish Daginakatte, Susanta Samajdar. Identification of a novel preclinical candidate for CDK7 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-317. doi:10.1158/1538-7445.AM2017-LB-317

Background: Bromodomain and extra-terminal (BET) family proteins are dual bromodomain-containing epigenetic readers that bind to acetylated-lysine residues at gene promoter and enhancer elements in histones and recruit protein complexes to promote transcriptional elongation. Recent evidence demonstrates that BET bromodomain inhibition leads to anti-proliferative activity in pre-clinical models of many hematological malignancies and solid tumors. Selective inhibition of BET bromodomains by small molecule inhibitors has emerged as a promising therapeutic strategy for the treatment of cancer. In this study, we evaluated the antitumor activity of ODM-207, a novel, potent and highly selective BET bromodomain inhibitor. Methods and Results: ODM-207 is a potent and selective BET inhibitor that is structurally unrelated to the benzodiazepine like inhibitors such as JQ1, I-BET762, and OTX015. We tested the preclinical activity of ODM-207 across multiple tumor types in a 4-day growth inhibition in vitro assay. ODM-207 potently inhibits cell viability of a wide range of hematological and solid tumor cell lines. ODM-207 also shows potent antiproliferative effects in patient-derived cancer cells representing various tumor types. In VCaP prostate cancer cell lines, ODM-207 induced apoptosis consistent with increased expression of pro-apoptotic regulators, whereas potent antiproliferative effects associated with cell cycle arrest and cellular senescence were seen in e.g. LNCaP prostate cancer cell line. To gain insight on mechanisms of acquired BET inhibitor resistance, we generated a cell line with resistance to BET-inhibition by culturing LNCaP prostate cancer cells with increasing concentrations of OTX015, a potent and selective BET-inhibitor. Interestingly, ODM-207 is also able to inhibit proliferation and downregulate Myc levels in cells that have acquired resistance to BET-inhibitors OTX015 and I-BET762. In xenograft models, oral treatment with ODM-207 significantly inhibits tumor growth at a dose which is well tolerated. Conclusions: In summary, these studies demonstrate that ODM-207 is a potent inhibitor of BET proteins in models of hematologic malignancies and solid tumors in vitro and in vivo and support its clinical development for the treatment of cancer. Citation Format: Anu-Maarit Moilanen, Mari Björkman, Reetta Riikonen, Chandrasekhar Abbineni, Mahaboobi Jaleel, Sivapriya Marappan, Tarja Ikonen, Girish Daginakatte, Aravind A B, Elina Mattila, Juha Rantala, Susanta Samajdar, Murali Ramachandra, Pekka Kallio. Targeting cancer with a novel BET bromodomain inhibitor ODM-207 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 118. doi:10.1158/1538-7445.AM2017-118

Montmorillonite impregnated with bismuth nitrate has been found to be an excellent reagent for the synthesis of several nitro compounds of biological significance in high yield.

Total synthesis of the monoterpene β-necrodol has been accomplished. The key step involves an ortho -ester Claisen rearrangement in a highly sterically congested cyclopentane derivative resulting in a high level of 1,3- trans diastereoselection. A novel photo-induced decarboxylation of the obtained γ,δ-unsaturated acid afforded β-necrodol.

Montmorillonite impregnated with bismuth nitrate was found to be an excellent reagent for aromatic nitration in high yield.

Exhaustive methylation of 8,8a-diphenyl-1,2,3,5,6,8a-hexahydroimidazo[1,2-a]pyrazine by NaH and CH3I in dehydrated tetrahydrofuran yielded 4,5-diphenyl-3,6-diazaocta-1,3,5,7-tetraene (1). Using 1 as an N,N donor ligand, cationic copper(I) complexes of the type [Cu(1)2]X·nH2O (X− = ClO4−, n = 1.5; X− = PF6−, n = 0) and [Cu(1)(PPh3)2]X (X− = ClO4− or PF6−) have been synthesized. Compound 1 and its copper(I) complexes have been thoroughly characterised by 1H NMR (300 MHz). The CuII/I potential in [Cu(1)2]X·nH2O is rather high, 0.92 V vs. SCE in CH2Cl2 at a glassy carbon electrode. This indicates that 1 is a potential π acid which preferentially stabilises copper(I) much more than copper(II). It is photoluminescent in fluid solution, its emission being somewhat quenched in [Cu(1)2]X·nH2O.

A simple stereocontrolled route involving intramolecular [2+2] photocycloaddition followed by rearrangement of the resulting cyclobutane derivatives is described for the construction of cyclopentanones with substituents upto three contiguous chiral centres.

Introduction: BET (bromodomain and extraterminal) family proteins (BRD2, BRD3, BRD4, and BRDT) are epigenetic readers that bind to acetylated-lysine residues in histones and recruit protein complexes to promote transcription elongation. In many cancers, BET proteins have been shown to regulate expression of MYC and other oncogenic drivers that are important for cell proliferation and survival. Pharmacologic inhibition of the BET-histone interaction has been shown to result in transcriptional downregulation of a number of oncogenes and inhibition of tumor growth providing a novel strategy for treatment of cancer. Therefore, in this study, we evaluated the in vitro and in vivo antitumor activity of ODM-207, a novel, potent and highly selective BET bromodomain inhibitor using cell lines derived from prostate and breast cancer as well as patient-derived tumor cell cultures of breast cancer. Methods and Results: ODM-207 has antiproliferative effects on several hematological and solid tumor cell lines. In a panel of prostate cancer cell lines, ODM-207 attenuates cell growth of androgen receptor (AR)-positive cell lines such as VCaP and 22Rv1. RNA-sequencing and Western blot studies revealed that the exposure of sensitive prostate cancer cells to ODM-207 is associated with rapid down-regulation c-Myc expression levels while wtAR was not affected. In 22Rv1 prostate cancer xenograft, which expresses both the full-length androgen receptor and androgen receptor splice variant V7, oral administration of ODM-207 was very efficacious in suppressing tumor growth at well tolerated doses whereas enzalutamide had only a modest effect. Contrary to our findings in prostate cancer cells, BET-inhibitor treatment of estrogen-dependent MCF-7 breast cancer cell line inhibits tumor growth in cell proliferation assays but is associated with down-regulation of ERα while the c-Myc levels are very low, highlighting the context-dependent functional effects of BET inhibition. Interestingly, ODM-207 produces potent antiproliferative effects associated with cell-cycle arrest and cellular senescence in patient-derived breast cancer cells. Conclusions: In summary, ODM-207 is a new generation BET inhibitor found to possess excellent pharmacological properties and antitumor activity both in vitro and in vivo. Our data suggest the potential utilization of ODM-207 for the treatment of prostate and breast cancer. Citation Format: Mari Björkman, Elina Mattila, Reetta Riikonen, Chandra Sekhar, Mahaboobi Jaleel, Sivapriya Marappan, Tarja Ikonen, Daniel Nicorici Nicorici, Juha Rantala, Susanta Samajdar, Murali Ramachandra, Pekka Kallio, Anu-Maarit Moilanen. ODM-207, a novel BET-bromodomain inhibitor as a therapeutic approach for the treatment of prostate and breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4649.

Interleukin-1 receptor associated kinases (IRAKs) are serine/threonine protein kinases belonging to the tyrosine-like kinase (TLK) family. IRAKs function as mediators of Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling pathways and play an important role in innate immune signaling. TLR/IL-1R stimulation leads to recruitment of MYD88, an adaptor molecule, to the activated receptor complex, which then complexes with IRAK4 and activates IRAK1. TRAF6 is then activated by IRAK1 leading to NFkB activation. Recent studies have reported the occurrence of gain of function oncogenic mutation (L265P) in MYD88 in ∼30% of activated B cell diffuse large B-cell lymphoma(ABC DLBCL) and ∼90% of Waldenstrom's macroglobulinemia (WM) leading to constitutive activation of IRAK4 and NFkB pathway. Among the DLBCL subtypes (GCB, ABC DLBCL and PMBL), ABC DLBCL is the most refractory. Inhibition of constitutive IRAK4 signalling can be used as a therapeutic strategy to treat ABC DLBCL Small molecule inhibitors of IRAK4 were synthesized based on hits originating from Aurigene's compound library. Structure guided drug design approach was used to further improve the potency. Lead compounds demonstrated moderate to very high selectivity towardsIRAK4 (S35 score of 0.03) when screened against a large panel of 329 kinases. Aurigene's lead compounds have excellent PK profile and good oral bioavailability in mice, leading to good in-vivo activity in TLR4 induced cytokine release model. Selected lead compounds were tested in a OCI-Ly3 xenograft model, which has a MYD88(L265P) mutation leading to constitutive activation of IRAK4 signaling. An advanced lead compound has demonstrated excellent efficacy in OCI-Ly3 model, with tumor stasis at low doses and tumor regression at higher doses. The compound is well tolerated and has a good therapeutic window as determined in a 14 day rodent toxicity study. In summary, a selective IRAK4 inhibitor has been identified with excellent efficacy and good safety profile. Citation Format: Wesley Roy Balasubramanian, Venkateshwar Rao Gummadi, Kavitha Nellore, Subhendu Mukherjee, Sivapriya Marappan, Aravind Basavaraju, Bharathi Raja Ainan, Girish Daginakatte, Sreevalsam Gopinath, Sanjeev Giri, Thomas Antony, Shekar Chelur, Susanta Samajdar, Chetan Pandit, Murali Ramachandra. Efficacy and safety of highly selective novel IRAK4 inhibitors for treatment of ABC-DLBCL. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4798.

The nuclear hormone receptor RORγ controls the differentiation of Th17 cells that plays a key pro-inflammatory role in a variety of autoimmune diseases. RORγ inverse agonists from multiple structural classes were discovered and screened in a RORγ radio-ligand binding assay as well as in a cell based reporter assay and functional effect was evaluated using primary mouse/human CD4+ve T-cells differentiation to Th17 cells. Pharmacokinetic profile of potent compounds was determined to support evaluation of efficacy as well as safety profile in rodents. Lead compounds have demonstrated good activity (< 100 nM) in reporter assay. Crystal structure of RORγ in complex with known inverse agonists as well as novel compounds, were solved to support lead optimization. Selected compounds demonstrated >100 fold selectivity against RORα as well as other nuclear receptors with significant inhibition of IL-17 release in Th17 differentiation assay. Lead compounds have shown optimal physicochemical profile including good solubility and high free fraction, leading to excellent pharmacokinetic profile in mice and rats. Efficacy of lead compounds has been demonstrated in multiple disease models including asthma, psoriasis, Experimental auto immune encephalomyelitis and collagen induced arthritis. Potential lead candidates have been identified with excellent in-vitro safety profile including CEREP-44 safety panel, Ames test and cytotoxicity. One of the lead candidates has also demonstrated good therapeutic window as determined by a 7 day rat toxicity study. Conclusions We have identified structurally diverse small molecule inverse agonists of RORγ and have demonstrated efficacy in relevant disease models as well as good therapeutic window.

An indium-induced reduction-rearrangement reaction of nitro-substituted β-lactams has been used for facile synthesis of oxazines in aqueous ethanol.

Cyclin-dependent kinase 7 (CDK7) is an important constituent of the cellular transcriptional machinery, where it phosphorylates the C-terminal domain (CTD) of RNAP polymerase II (RNAPII). Because many tumor types are critically dependent on transcription for maintenance of their oncogenic state, pharmacological modulation of CDK7 kinase activity is considered as an approach to treat cancer. Multiple series of CDK7 inhibitors were identified by iterative medicinal chemistry efforts and SAR based approach. Early compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile to achieve anti-tumor activity. We have identified compounds from two distinct chemical series that are highly potent in inhibiting CDK7 in biochemical assays. These inhibitors demonstrate time-dependent inhibition of CDK7 indicating covalent nature of binding. The compounds showed potent anti-proliferative activity in cell lines derived from various tumor types and this was accompanied by CDK7 modulation in cells as monitored by pS5RNAPII levels. They have excellent drug-like characteristics including solubility, permeability, metabolic stability and good oral bioavailability. In a broad panel of kinases (332 kinase), selected compounds from both series showed good selectivity profile. Tolerability and efficacy studies are ongoing with selected early leads to test their impact on tumor growth inhibition in xenograft models. We have identified novel and selective CDK7 covalent inhibitors from two series with desirable drug-like properties, which are being evauated for anti-tumor activity in xenograft models. Citation Format: Ramulu Poddutoori, Leena K. Satyam, Girish Daginakatte, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Raghuveer Ramachandra, Anirudha Lakshminarasimhan, Manoj Pothuganti, Shilpa Nayak, Nandish C, Chandranath Naik, Ravindra MV, Madhu Dabbeeru, Thomas Antony, Chetan Pandit, Murali Ramachandra, Shekar Chelur, Susanta Samajdar. Potent and selective inhibition of CDK7 by novel covalent inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C190.

Interleukin-1 receptor associated kinases (IRAKs) are serine/threonine protein kinases belonging to tyrosine-like kinase (TLK) family. The IRAK family consists of IRAK1, IRAK2, IRAK3 and IRAK4 out of which only IRAK1 and IRAK4 exhibit kinase activity. IRAKs function as mediators of Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling pathways and play an important role in innate immune signaling. Recent studies have reported the occurrence of oncogenic mutations in MYD88 in 30% of activated B cell diffuse large B-cell lymphoma (ABC DLBCL) and 90% of Waldenstrom's macroglobulinemia (WM) leading to constitutive activation of the IRAK4 and NFkB pathway. Recent studies have also highlighted the association of dysregulated innate immune signaling with Myelodysplastic syndrome (MDS) and Acute Myeloid leukaemia (AML). TLRs and their associated signal transducers are frequently overexpressed and/or constitutively activated in MDS. Overexpression and activation of IRAK1 is observed in AML. Thus IRAKs are attractive therapeutic targets for treatment of tumors with altered innate immune signaling such as ABC DLBCL and AML. We have designed, synthesized and tested small molecule inhibitors of IRAK4 based on hits originating from Aurigene's compound library. We have identified a series of novel bicyclic heterocycles as potent inhibitors of IRAK-4 with moderate to very high selectivity (S35 score = 0.03) in a 329 kinase panel. Lead compounds were profiled in proliferation and mechanistic assays (p-IRAK1 and p-TAK1 inhibition) in appropriate ABC DLBCL/AML cell lines. Aurigene lead compounds demonstrate potent inhibition of cellular proliferation with a good correlation to inhibition of phosphorylation of signaling intermediates in mechanistic assays. Lead compounds exhibit excellent PK profile and good oral bioavailability in mice. Preliminary in-vitro toxicology studies indicate a clean safety profile. Selected compounds demonstrate excellent in-vivo efficacy in relevant tumor models with >90% tumor growth inhibition and good in-vivo PD modulation. In summary, a series of potent and selective IRAK4 inhibitors have been discovered and are being evaluated for treatment of cancers with dysregulated innate immune signaling. Citation Format: Venkateshwar Rao, Wesley Roy Balasubramanian, Kavitha Nellore, Sivapriya Marappan, Aravind Basavaraju, Bharathi Raja Ainan, Girish Daginakatte, Sreevalsam Gopinath, Sanjeev Giri, Thomas Antony, Shekar Chelur, Susanta Samajdar, Chetan Pandit, Murali Ramachandra. Efficacy of novel IRAK4 inhibitors in ABC-DLBCL and AML models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C191.