The present study explores the potential of pyridine-based synthetic amphiphiles C1 and C2 having 4-carbon and 12-carbon hydrophobic tails, respectively, as staphylococcal nuclease inhibitors. UV-visible titration with calf-thymus DNA (CT-DNA) revealed a hypochromic shift in the absorbance bands of C1 and C2, whereas fluorescence titration indicated a reduction in the emission intensity of the monomer bands of the amphiphiles. Interaction of deoxyribonuclease I (DNase 1) and micrococcal nuclease (MNase) with C1 or C2 led to a decrease in the emission intensity of tryptophan at λ=345 nm along with an increase in the monomer emission intensity of C1 and C2 at λ=375 nm for DNase I and excimer emission intensity at λ=470 nm for both DNase I and MNase. Scatchard's analysis indicated superior interaction of C2 with DNase I. Circular dichroism spectroscopy revealed major changes in the secondary structures of both DNase I and MNase upon interaction with the amphiphiles. A solution-based nuclease assay in conjunction with gel electrophoresis indicated amphiphile-mediated protection against nuclease-directed DNA cleavage. Interestingly, C2 could render inhibition of nuclease present in the culture supernatant of Staphylococcus aureus MTCC 96, which highlights the therapeutic prospect of the amphiphile against S. aureus.

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.