Small-molecule inhibitors of PD-1/PD-L1 immune checkpoint alleviate the PD-L1-induced exhaustion of T-cells Small-molecule inhibitors of PD-1/PD-L1 immune checkpoint alleviate the PD-L1-induced exhaustion of T-cells

The dimeric molecular chaperone Hsp90 is required for the activation and stabilization of hundreds of substrate proteins, many of which participate in signal transduction pathways. The activation process depends on the hydrolysis of ATP by Hsp90. Hsp90 consists of a C-terminal dimerization domain, a middle domain, which may interact with substrate protein, and an N-terminal ATP-binding domain. A complex cycle of conformational changes has been proposed for the ATPase cycle of yeast Hsp90, where a critical step during the reaction requires the transient N-terminal dimerization of the two protomers. The ATPase cycle of human Hsp90 is less well understood, and significant differences have been proposed regarding key mechanistic aspects. ATP hydrolysis by human Hsp90α and Hsp90β is 10-fold slower than that of yeast Hsp90. Despite these differences, our experiments suggest that the underlying enzymatic mechanisms are highly similar. In both cases, a concerted conformational rearrangement involving the N-terminal domains of both subunits is controlling the rate of ATP turnover, and N-terminal cross-talk determines the rate-limiting steps. Furthermore, similar to yeast Hsp90, the slow ATP hydrolysis by human Hsp90s can be stimulated up to over 100-fold by the addition of the co-chaperone Aha1 from either human or yeast origin. Together, our results show that the basic principles of the Hsp90 ATPase reaction are conserved between yeast and humans, including the dimerization of the N-terminal domains and its regulation by the repositioning of the ATP lid from its original position to a catalytically competent one.

CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins – important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 – a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57.

We describe a new class of potent PD-L1/PD-1 inhibitors based on a terphenyl scaffold that is derived from the rigidified biphenyl-inspired structure. Using in silico docking, we designed and then experimentally demonstrated the effectiveness of the terphenyl-based scaffolds in inhibiting PD-1/PD-L1 complex formation using various biophysical and biochemical techniques. We also present a high-resolution structure of the complex of PD-L1 with one of our most potent inhibitors to identify key PD-L1/inhibitor interactions at the molecular level. In addition, we show the efficacy of our most potent inhibitors in activating the antitumor response using primary human immune cells from healthy donors.