Design and optimization of tetrahydroisoquinoline-based small molecule inhibitors of the Programmed Cell Death-1 (PD-1)/Programmed Cell Death-Ligand 1 (PD-L1) protein-protein interaction as potential novel cancer immunotherapeutics
Creators: name: Yang, Jeffrey
Publisher: name: No Publisher Supplied
Year: 2022

description: The PD-1/PD-L1 immune checkpoint pathway is a clinically validated drug target engaged in the suppression of tumor immunity. Tumor cells are known to overexpress the PD-L1 ligand, which in turn binds to the PD-1 receptor expressed on activated T lymphocytes to inactivate effector T cell function, evading immune detection and destruction. In recent years, several clinical trials assessing the efficacy of anti-PD-1/PD-L1 monoclonal antibodies as immune checkpoint inhibitors (ICIs) have demonstrated remarkable therapeutic successes—tumor regression and prolonged survival—in advanced malignancies. Despite these therapeutic advances, only a fraction of cancer patients respond to ICIs due to the evolution of multiple mechanisms of immune escape. Additionally, the antibody-based ICIs possess several drawbacks: intravenous administration, prolonged half-life, intractable immune-related toxicities, and unsustainable costs to patients, manufacturers, and healthcare institutions. It is therefore imperative to develop small molecule alternatives with greater oral bioavailability and tumor penetration, shorter half-lives for easier control of toxicities, and higher benefit-cost ratios, which can then be combined with other oral anti-cancer treatments.A structure-based, computer-assisted drug design approach was pursued to discover novel small molecule inhibitors of the PD-1/PD-L1 protein-protein interaction (PPI). Starting from the disclosed structure of BMS-202 (1) and structure-activity relationships reported in the patent literature, we derived a highly potent small molecule inhibitor LH1305 (24) by introducing a (5-cyanopyridin-3-yl)methoxy moiety on the central resorcinol ring. Examination of the crystal structure of 1 bound within the PD-L1 homodimer led to the realization of the C₂-symmetric nature of the homodimer, inspiring the design of C₂-symmetric analogs (e.g., LH1307/25b). The lead compound 25b was found to be a highly potent inhibitor of the PD-1/PD-L1 PPI (IC₅₀ = 3.0 nM vs. 2.5 nM for 24) in a TR-FRET PD-1/PD-L1 PPI inhibition assay and of the PD-1-mediated immune signaling pathway (EC₅₀ = 79 nM vs. 219 nM for 24) in a cellular co-culture assay. However, 25b did not possess drug-like properties amenable for oral administration. Two separate inhibitor series were subsequently explored to overcome the shortcomings of 25b. Based on the non-C₂-symmetric analog 24, conformational restriction of the N-acetylethylenediamine and (5-cyanopyridin-3-yl)methoxy substituents led to new inhibitors with the 1,2,3,4-tetrahydroisoquinoline (THIQ)-3-carboxylic acid scaffold. Within this series, substitution at the 1-position of the THIQ ring (e.g., LH1388/(1S,3S)-32e with IC₅₀ = 22 nM) led to greater inhibitory potency against PD-1/PD-L1 PPI than direct substitution on the THIQ nitrogen atom (e.g., LH1320/(S)-29a and LH1352/(S)-29e with IC₅₀ = ~6 µM and 329 nM, respectively). (1S,3S)-32e could be further optimized into more potent inhibitors. In the second inhibitor series, a molecular simplification strategy was employed to pare down one half of 25b to the minimum structure required to maintain comparable potency while improving physicochemical properties such as solubility and permeability. This lead optimization effort led to several inhibitors bearing the N-acyl-7-phenyl-1,2,3,4-THIQ core scaffold (LH1398/64b, LH1425/66c, LH1464/67g, and LH1465/67i) with comparable PPI inhibition activity as 25b (IC₅₀ values ranging 2.5 to 6.6 nM) and superior solubility (172 µM for 67g vs. 14 µM for 25b) and/or permeability (48 nm/s for 66c vs. 0.1 nm/s for 25b). The new inhibitors also displayed nanomolar inhibition constants (Ki values ranging 7.7 to 16 nM vs. 0.003 nM for 25b) as determined in a newly established TR-FRET PD-L1 competitive binding assay. These inhibitors have now advanced to in vitro cellular cytotoxicity studies and may soon be submitted for in vivo PK and efficacy studies. Further medicinal chemistry efforts will be needed to strike a better balance between solubility and permeability with this inhibitor series.