Fig. 4. Design of lysine-targeted acrylamide-derived electrophilic inhibitors of PD1. (A) Pembrolizumab (orange) and GY-14 (green) bulged CDR-H3 hairpin loops in contact with PD1 (PDB codes: 5GGS and 6J14). Sequence of ADAPTins2c and 2p showing substitutions of M12 and Y13 respectively with Dap/Dab residues attached to acrylamide-derived electrophiles. M12 and Y13 were selected for the introduction of electrophilic warheads based on distances between the corresponding M105 and Y106 to the targeted PD1K131, see ESI,† Fig. S15. (B) CD spectra for the electrophilic ADAPTins2ca–cd/2pa,b and TM values obtained from a global fit of CD-melts representing the temperature at which each peptide retain 50% folding. (C) Inhibitory activity of the covalent inhibitors against the PD1:PDL1 interaction, n = 3 (mean ± SD). (D) Kinetic curves of competitive inhibition targeting the PD 1: PDL1 interaction (1 : 12 ratio) showing the irreversible covalent nature of electrophilic inhibitors 2ca and 2cb. Error bars indicate the mean ± SD, n = 4.
