Determining Cysteines Available for Covalent Inhibition Across the Human Kinome

Abstract

Covalently bound protein kinase inhibitors have been frequently designed to target non-catalytic cysteines at the ATP binding site. Thus, it is important to know if a given cysteine can form a covalent bond. Here we combine a function-site interaction fingerprint method and DFT calculations to determine the potential of cysteines to form a covalent interaction with an inhibitor. By harnessing the human structural kinome, a comprehensive structure-based binding site cysteine dataset was assembled. The orientation of the cysteine thiol group indicates which cysteines can potentially form covalent bonds. These covalent inhibitor accessible cysteines are located within five regions: P-loop, roof of pocket, front pocket, catalytic-2 of the catalytic loop and DFG-3 close to the DFG peptide. In an independent test set, these cysteines covered 95% of covalent kinase inhibitors. This study provides new insights into cysteine reactivity and preference which is important for the prospective development of covalent kinase inhibitors.

The Cysteinome of Protein Kinases as a Target in Drug Development

“…[18c] Amore recent report analysed the likelihood of covalent bond formation with cysteines present in the ATP binding pocket with an ATP-competitive inhibitor. This study analysed 2774 high-resolution structures using the functionsite interaction fingerprint (Fs-IFP) method and density functional theory (DFT), [62] identifying five regions with accessible cysteines that covered 95 %o fcovalent kinase inhibitors.H owever, the current landscape of covalent inhibitor development is based towards the large number of inhibitors that have been developed against major drug targets such as EGFR, BTK, ITK, and JAK3, all of which share ac ysteine residue located at the solvent-exposed front region. Considering available cysteines and the exposure of the thiol group,w ea dapted the nomenclature introduced previously to consider only cysteines that are likely to be targeted based on their side-chain orientation (Figures 7a nd 8).…”

Advances of small molecule targeting of kinases

“…Many kinases have an exposed cysteine side chain in the ATP site that could be targeted for covalent reaction with compounds harboring an electrophilic Michael Acceptor in the right position [33–35]. Such covalent inhibitors are designed as affinity labels that interact with the target kinase in a two-step reaction, in which the formation of an irreversible enzyme-inhibitor complex is preceded by a rapidly reversible collision complex.…”

DUckCov: a Dynamic Undocking‐Based Virtual Screening Protocol for Covalent Binders

“…According to these guidelines, the ligand's selectivity toward its protein target is still to be achievedb yo ptimizing the noncovalent interactions (hydrogen bonding, van der Waals, electrostatic, etc.) [7] Ligands that bind through ac ovalent mechanism are not subject to classical equilibrium kinetics, as their residence time in the binding pocket can last up to days.A saconsequence, the potency of these drugs is capable of surpassing the theoreticall imits of potency/ligand efficiency. Furthermore, increased specificity can be obtained by targeting ap oorly conserved reactiver esidue within the protein family.…”

“…Also, Liu and colleagues have coined the term "kinase cysteinome" to refer to the collection of targetable cysteine residues in the human kinome [6] and published ac omputational methodology to identify such cysteines. [7] Ligands that bind through ac ovalent mechanism are not subject to classical equilibrium kinetics, as their residence time in the binding pocket can last up to days.A saconsequence, the potency of these drugs is capable of surpassing the theoreticall imits of potency/ligand efficiency. [2] Another advantage is the prolonged duration of action, which can persist even when the ligand has already been cleared from the body.T his can be beneficial for alleviating the drug burden of ap atient due to less frequent drug dosing (depending on the turnover rate of the protein) and therefore ap ossibly lower risk of idiosyncratic toxicity, which has been linked to daily drug dosage.…”