Assessing Lysine and Cysteine Reactivities for Designing Targeted Covalent Kinase Inhibitors

Abstract

Targeted covalent inhibitor design is gaining increasing interest and acceptance. A typical covalent kinase inhibitor design targets a reactive cysteine; however, this strategy is limited due to the low abundance of cysteine and acquired drug resistance from point mutations. Inspired by the recent development of lysine-targeted chemical probes, we asked if nucleophilic (reactive) catalytic lysines are common based on the published crystal structures of the human kinome. Using a newly developed pK a prediction tool based on continuous constant pH molecular dynamics, the catalytic lysines of 8 unique kinases from various human kinase groups were retro-and prospectively predicted to be nucleophilic, when kinase is in the rare DFG-out/αC-out type of conformation. Importantly, other reactive lysines as well as cysteines at various locations were also identified. Based on the finding, we proposed a new strategy in which selective type II reversible kinase inhibitors are modified to design highly selective, lysine-targeted covalent inhibitors. Traditional covalent drugs were discovered serendipitously; the presented tool, which can assess the reactivities of any potentially targetable residues, may accelerate the rational discovery of new covalent inhibitors. Another significant finding of the work is that lysines and cysteines in kinases may adopt neutral and charged states at physiological pH, respectively. This finding may shift the current paradigm of computational studies of kinases, which assume standard protonation states.

GPU-Accelerated Implementation of Continuous Constant pH Molecular Dynamics in Amber: pKa Predictions with Single-pH Simulations

“…A most recent study 53 demonstrated the capability of replica-exchange GBNeck2-CpHMD for accurate prediction of downshifted Cys and Lys pK a 's. Work is underway to benchmark the performance using single-pH simulations.…”

“…Although there remains much room for improvement, our present data are encouraging and demonstrate that single-pH simulations may be routinely performed on a desktop computer equipped with a single GPU card for a variety of applications, from assisting MD stimulations with protonation state assignment to offering pH-dependent corrections of binding free energies 10 and nucleophilic hot spots for covalent drug design. 53…”

Assessment of Proton-Coupled Conformational Dynamics of SARS and MERS Coronavirus Papain-like Proteases: Implication for Designing Broad-Spectrum Antiviral Inhibitors

“…All sidechains of Asp, Glu, His, Cys, and Lys were allowed to titrate, with their titration model parameters taken from our previous work. [28][29][30] An ionic strength of 0.15 M was used to represent the physiological salt condition. Simulations were run at a temperature of 300 K and an effectively infinite cutoff (999 Å) for nonbonded interactions.…”

Avoiding or Co-Opting ATP Inhibition: Overview of Type III, IV, V, and VI Kinase Inhibitors

“…However, surface-exposed lysines are generally thought to be poor nucleophiles because they are protonated (p K a ~10.5) at physiological pH [ 100 ]. Recent studies using computational predictions suggest that localized p K a values may shift several units, allowing lysines to be amenable to reacting with electrophilic warheads [ 124 ]. Given the success of current covalent inhibitors targeting EGFR and BTK protein kinases, the future will likely see the development of new type VI inhibitors for treating disease.…”