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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

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.

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.

Potent and selective S1P3 receptor (S1P3-R) agonists may represent important proof-of-principle tools used to clarify the receptor biological function and assess the therapeutic potential of the S1P3-R in cardiovascular, inflammatory and pulmonary diseases. N,N-Dicyclohexyl-5-propylisoxazole-3-carboxamide was identified by a high-throughput screening of MLSMR library as a promising S1P3-R agonist. Rational chemical modifications of the hit allowed the identification of N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide, a S1P3-R agonist endowed with submicromolar activity and exquisite selectivity over the remaining S1P1,2,4,5-R family members. A combination of ligand competition, site-directed mutagenesis and molecular modeling studies showed that the N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide is an allosteric agonist and binds to the S1P3-R in a manner that does not disrupt the S1P3-R–S1P binding. The lead molecule herein disclosed constitutes a valuable pharmacological tool to explore the molecular basis of the receptor function, and provides the bases for further rational design of more potent and drug-like S1P3-R allosteric agonists.

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.

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.

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

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.