Novel, small molecule inhibitors of PD-1/PD-L1 pathway.
Authors: Rastelli Luca , Rajagopal Sridharan , Gajendran Chandru , Sadhu Naveen M. , Mohd Zainuddin , Gosu Ramachandraiah , Friedmann-Morvinski Dinorah , Kandan Saravanan , Birudukota Swarnakumari , S Srinivasan , V Krishnakumar , Wahid Saif , Siddiqui Amir , Viswakarma Santosh , Narayan Ashwini , G Rudresh , Mullurwar Sadanand R. , Sher Divsha , Mansur Shahar , Sivanandhan Dhanalakshmi 
Published: June 2022

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Background: Programmed Cell Death 1 (PD-1) protein plays a key role in inhibiting immune responses and enhancing self-tolerance via modulation of T-cell activity, inducing T-cell apoptosis and inhibiting apoptosis of regulatory T cells. PD-L1 also plays an important role in various malignancies where it can attenuate the host immune response to tumor cells thereby favouring tumor progression and metastasis. High expression of PD-L1 in glioblastoma tumor tissues is associated with poor survival of patients, and PD-L1 may act as a prognostic predictor and an effective therapeutic target for glioblastoma. A number of monoclonal antibodies targeting PD-1/PD-L1 have approved for various malignancies. Still, efficacy of these antibodies in glioblastoma and brain metastasis continues to be moderate potentially owing to lack of or poor brain penetrance of these agents. Therefore, there is still a need for potent, selective small molecule PD-1/PD-L1 inhibitors with enhanced brain penetration in the treatment of such cancers. 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 signalling reporter assays as well cell based PD-L1 dimerization assays were used to assess the mechanistic and functional effects. In vivo efficacy was assessed in orthotopic GBM as well as in syngeneic and humanized subcutaneous tumor models in mice. Results: Our lead PD-L1 inhibitor JBI-2174 showed strong in vitro IC50 of ̃1 nM in TR-FRET assay that measures interaction between hPD-1 and hPD-L1 and a picomolar IC50 against monkey PD-L1. In selectivity assays for immunooncology targets, JBI-2174 was highly selective for PD-L1. JBI-2174 also inhibited PD-L1/PD-1 mediated signalling essential for T-cell modulation. JBI-2174 induced dimerization of PD-L1 as observed by size exclusion chromatography, which was confirmed by co-crystal structure and recapitulated in cell based dimerization assay. Elucidation of the co-crystal structure, clearly demonstrated that JBI-2174 clearly interacts with multiple amino acids on PD-L1 that are critical for PD-1 binding. JBI-2174 showed excellent oral bioavailability across pre-clinical species and sustained brain exposure. In the in vivo efficacy studies, JBI-2174 showed comparable efficacy to the anti-PD-L1 antibody or Atezolizumab in syngeneic (4T1, CT-26) and in partially humanized models (MC-38/hPD-L1). Further, oral administration of JBI-2174 resulted in statistically significant increase in survival (Day 27 in control vs day 38 in treated, p < 0.05) in a mouse glioma orthotopic model. Conclusions: The oral bioavailability and brain exposure of this molecule will make it attractive for cancers with unmet medical needs such as GBM and brain metastasis. IND enabling studies are being initiated for this compound.