Targeting Non-Catalytic Cysteine Residues Through Structure-Guided Drug Discovery

Abstract

The targeting of non-catalytic cysteine residues with small molecules is drawing increased attention from drug discovery scientists and chemical biologists. From a biological perspective, genomic and proteomic studies have revealed the presence of cysteine mutations in several oncogenic proteins, suggesting both a functional role for these residues and also a strategy for targeting them in an ‘allele specific’ manner. For the medicinal chemist, the structure-guided design of cysteine-reactive molecules is an appealing strategy to realize improved selectivity and pharmacodynamic properties in drug leads. Finally, for chemical biologists, the modification of cysteine residues provides a unique means to probe protein structure and allosteric regulation. Here, we review three applications of cysteine-modifying small molecules: 1) the optimization of existing drug leads, 2) the discovery of new lead compounds, and 3) the use of cysteine-reactive molecules as probes of protein dynamics. In each case, structure-guided design plays a key role in determining which cysteine residue(s) to target and in designing compounds with the proper geometry to enable both covalent interaction with the targeted cysteine and productive non-covalent interactions with nearby protein residues.

Proteasome Inhibitors: Harnessing Proteostasis to Combat Disease

“…This has been exploited for finding inhibitors of the DUB Usp7, for example, which show efficacy in tumor models [209][210][211][212]. There is precedent for bringing thiol-reactive drugs to the clinic [213][214][215][216][217]. The recent development of AMG 510 by Amgen for treating solid tumors with a particular KRAS-G12C mutation further demonstrates that cysteine-reactive drugs can advance to clinical trials [218].…”

Cysteine Residues in Helicobacter pylori Adhesin HopQ are Required for CEACAM–HopQ Interaction and Subsequent CagA Translocation

“…Another approach is the use of antibodies to block the host-pathogen interaction [3], as well as nanobodies that overcome the limitations of traditional monoclonal antibodies and peptides as therapeutics [29,30]. An alternative approach that is prompted by identifying the importance of the CL1 disulfide bond in this study, and also avoids the limitations of other strategies, is to design small molecules that target and reduce the disulfide bond [31,32], thus preventing H. pylori binding; an approach that has previously been used to target HIV infection [33,34]. In this context, a previous study showed that binding of the H. pylori adhesin BabA to its cellular receptor Le b was impaired by the reduction in a conserved 8-residue cysteine-clasped loop in the adhesins glycan-binding site [8].…”

A Liquid Chromatography-Mass Spectrometry Method for Screening Disulfide Tethering Fragments

“…The last decade has seen an increase in the development of covalent inhibitors as potential therapeutic agents. This interest has been driven by an appreciation of the advantages of covalent mechanisms of inhibition [1][2] , including the ability to overcome resistance, such as in EGFR gatekeeping mutations 3 , the opportunity to increase affinity for otherwise 'undruggable' targets, and distinct pharmacokinetic properties due to very long target-residency times [4][5] . A barrier to the pursuit of such compounds has been the perception that electrophilic drugs present greater risk due to nonspecific binding to off-targets, formation of reactive metabolites, or rapid inactivation by reaction with glutathione or other endogenous nucleophiles [6][7][8] .…”

“…Interest in covalent drugs is driven by an appreciation of the advantages of covalent mechanisms of inhibition. These advantages are well reviewed 5,6 and include the ability to overcome resistance, such as in EGFR gatekeeping mutations 7 , a method to increase affinity for otherwise 'undruggable' targets, and distinct pharmacokinetic requirements owing to very long target residency times of covalent drugs 8, 9 . As interest in covalent drug discovery has grown, so have analytical techniques for the screening of covalent small molecules and the size of electrophilic compound libraries. Despite these improvements, the largest reported screen of a covalent library is of just 1000 compounds 10 .…”

“…However, if a screening compound is too reactive, the initial molecular recognition step becomes a minor component, leading to non-specific binding that is dominated by the energetics of covalent bond formation (some of these chemotypes are ubiquitous in HTS libraries, and are included in the category of 'pan assay interference compounds', or PAINs) 15,16 . The design of covalent compound libraries and the development of effective covalent screening conditions must therefore control for the differing reactivity of screening compounds, and/or include counter-screens to establish selectivity 6 . Finally, when identifying selective, covalent ligands is the goal, the ideal primary screen detects the formation of a covalent bond, with secondary screens for biochemical and cellular activity.…”