Introduction: The PD-1/PD-L1 molecular pathway is one of the primary mechanisms of immune evasion deployed by cancer cells. Induction of PD-L1 expression on cancer cells is associated with inhibition of immune responses against cancer, thus favouring cancer progression and metastasis. Activation of PD-1/PD-L1 pathway induces T-cell anergy and exhaustion and enhances the function of regulatory T-cells (Tregs) thereby leading to an immune suppressive environment. Therefore, blocking this pathway restores the proliferation and cytotoxicity of T- lymphocytes, inhibits the function of Tregs and results in decreased T-cell apoptosis. A number of monoclonal antibodies agents targeting PD-1/PD-L1 have approved for a number of malignancies. These approved therapies require bolus intravenous injections, are administered in high dose and have a long half-life. The long residence time of these mAbs could contribute to the well-documented drug-related adverse effects. Therefore, there is still a need for potent, selective small molecule inhibitors of the PD-1/PD-L1 pathway. Such small molecule inhibitors, can provide increased oral bioavailability, and shorter half-life activity for a more controllable treatment, and flexibility for combination strategies. Methods: Rational design approaches were used to design novel small molecule PD-1/PD-L1 pathway inhibitors; potency of these inhibitors was assessed in an in-vitro TR-FRET assay. Checkpoints signaling reporter assays as well as ex-vivo co-culture assays were used to assess the ability of the compounds to restore T-cell proliferation and function. In vivo efficacy was assessed by syngeneic and humanized models in mice. Results: Our lead molecule JTi showed strong in vitro IC50 of 0.8 nM in TR-FRET assay that measures interaction between PD-1 and PD-L1. This molecule also augmented T-cell response as measured by IFN-gamma activation in a cancer cell-PBMC based co-culture assay. This molecule induced dimerization of PD-L1 in U2OS cell based assay with an EC50 of ~300 nM. Competition study between anti-PD-L1 blocking antibody and this small molecule inhibitor clearly suggested that it binds at the same site as the antibody. In a cell based assay, measuring PD-1/PD-L1 interaction, JTi clearly demonstrated inhibition of PD-1 and PD-L1 binding in a dose-dependent manner. JTi showed comparable efficacy to the anti-PD-L1 antibody in syngeneic (4T1, CT-26) and humanized models (MC-38/hPD-L1) by oral administration. Conclusion: Advanced studies to further characterize and progress the molecule into pre-clinical development are in progress. The oral administration route of these small molecule PD-1/PD-L1 inhibitors would provide an attractive option to be used as a maintenance therapy followed by mAb based treatment to enhance patient compliance. The lead compound will be entering IND enabling studies in the 1st half of 2021. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Naveen Sadhu M, Chandru Gajendran, Chandregowda venkateshappa, Muralidhar Reddy, Pendyala Satya Kishore, Pratima Deshpande, Sundarajan kannan, Tabassum Sahareen, Santhosh Viswakarma, Amir Siddiqui, Mohammed Zainuddin, Rudresh G, Prashanthi Daram, Ramchandraiah Gosu, Rashmi Rekha Devi. Novel, small molecule inhibitors of PD-L1/PD-1 interaction [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 1630.

Introduction: Arginine methylation deregulation in cancer has been well studied and PRMT5 which modulates dimethylation of arginine has emerged as an attractive therapeutic strategy in various cancer types, including lung cancer, lymphoma, glioblastoma, pancreatic cancers, etc. PRMT5 is over-expressed in multiple cancers leading to repression of tumor suppressor genes and genetic studies have identified it is a validated target in lymphoma. Recently, dysregulation of the splicing machinery in cancers has been identified to be one of the therapeutic vulnerabilities for PRMT5 inhibition, especially in glioblastoma. Therefore, inhibitors selectively targeting PRMT5 could be of high clinical value, especially in cancers with defects in spliceosome machinery. Methods: Rational design and structure based drug design were used to identify novel PRMT5 inhibitors. To assess in vitro potency, flash plate based activity assay was used. Cell based activity of these inhibitors was assessed by measuring the symmetrical dimethylation of known cellular protein SmD3 by ELISA and Western blotting. Long term cell proliferation assays were used to assess the functional effect of PRMT5 inhibition. Tumor growth inhibition was measured in orthotopic glioblastoma model in mice. Results: One of the lead PRMT5 inhibitors had an in vitro potency of 3 nM in the biochemical assay which translated well in the cell based SDMA ELISA where the EC50 was <10 nM. Anti-proliferative activity of this molecule in lymphoma, leukaemia, SCLC, pancreatic, lung and glioblastoma cell lines ranged from ~16 nM to 1035 nM. This molecule showed acceptable in vitro ADME properties in terms of aqueous solubility and metabolic stability and excellent oral bioavailability in rodent pharmacokinetics. In Z-138 mantle cell lymphoma xenograft model, oral administration of the lead compound at 50 mg/kg resulted in strong and complete (~95 %) tumour growth inhibition and with a concomitant complete inhibition of SDMA. The lead compound was well tolerated with no reduction in body weight at the tested doses. Interestingly, this molecule showed excellent brain exposure sufficient to achieve target engagement for 10h and significant tumour growth inhibition of orthotopic brain tumors by oral dosing. Repeat dose non-GLP study in rodents clearly demonstrated the safety of this molecule. Conclusion: Given the therapeutic importance of PRMT5 in glioblastoma and other lymphomas, this molecule will be extremely valuable in treating these cancers both as a standalone therapy and in combination with other standard of care agents. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Naveen Sadhu M, Chandru Gajendran, Saravanan Vadivelu, Natarajan Tamizharasan, Indu N. Swamy, Santhosh Viswakarma, Amir Siddiqui, Saif wahid, Mohammed Zainuddin, Rudresh G, Prashanthi Daram, Ramchandraiah Gosu, Dinesh Tiagaraj, Shivani Garapaty, Sreekala Nair, Namratha Kapoor. Selective, novel, small molecule PRMT5 inhibitors for treatment of cancer [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 1128.