Pembrolizumab Induces an Unexpected Conformational Change in the CC'-loop of PD-1.

Abstract

Simple SummaryCancer cells are normally destructed by killer T-cells. However, T-cells expose the PD-1 receptor on their surface, acting as a checkpoint: If it is activated through a special molecule, PD-L1, the T-cell kills itself, ending the attack. Cells often need to present PD-L1 to prevent T-cells from over-aggressive attacks which cause autoimmune disease. There are tumors which also present PD-L1, thereby evading natural clearing, allowing them to continue growing. New anticancer drugs (checkpoint inhibitors: nivolumab and pembrolizumab) disrupt this evasion: They competitively bind to PD-1, without activating it, and re-enable immune tumor destruction. We scrutinize the binding mechanisms via molecular dynamics simulation. We demonstrate that these drugs deform the CC′-loop of the PD-1 in ways differing from those seen with PD-L1 as a binding partner. Pembrolizumab induces a new conformation of the CC′-loop not known to date. These findings might pave the way for the development of new anti-cancer drugs.To improve cancer immunotherapy, a clearer understanding of key targets such as the immune checkpoint receptor PD-1 is essential. The PD-1 inhibitors nivolumab and pembrolizumab were recently approved by the FDA. The CC′-loop of PD-1 has been identified as a hotspot for drug targeting. Here, we investigate the influence of nivolumab and pembrolizumab on the molecular motion of the CC′-loop of PD-1. We performed molecular dynamics simulations on the complete extracellular domain of PD-1, in complex with PD-L1, and the blocking antibodies nivolumab and pembrolizumab. Conformations of the CC′-loop were analyzed unsupervised with the Daura et al. clustering algorithm and multidimensional scaling. Surprisingly, two conformations found were seen to correspond to the ‘open’ and ‘closed’ conformation of CC′-loop in apo-PD-1, already known from literature. Unsupervised clustering also surprisingly reproduced the natural ligand, PD-L1, exclusively stabilizing the ‘closed’ conformation, as also known from literature. Nivolumab, like PD-L1, was found to shift the equilibrium towards the ‘closed’ conformation, in accordance with the conformational selection model. Pembrolizumab, on the other hand, induced a third conformation of the CC′-loop which has not been described to date: Relative to the conformation ‘open’ the, CC′-loop turned 180° to form a new conformation which we called ‘overturned’. We show that the combination of clustering and multidimensional scaling is a fast, easy, and powerful method in analyzing structural changes in proteins. Possible refined antibodies or new small molecular compounds could utilize the flexibility of the CC′-loop to improve immunotherapy.