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Mass Spectrometry & Proteomics for Covalent Lead Discovery

Covalent drugs and degraders, defined by their ability to form covalent bonds with their targets, represent a powerful therapeutic modality for the potent and selective inhibition, modulation, or degradation of clinically relevant proteins. Thanks to technological innovations facilitating the screening and optimization of small-molecule compounds, interest in targeted covalent inhibitors has markedly accelerated in recent decades. The successful development and FDA approval of rationally designed covalent inhibitors targeting disease-associated kinases (such as the BTK inhibitor ibrutinib and the EGFR inhibitor dacomitinib) has further validated the utility of this compound class. Ongoing research efforts continue to expand the covalent toolbox through the design of novel scaffolds, the development of reversible covalent binders, and the targeting of alternative (i.e., non-cysteine) residues, among other advances.

While the earliest covalent inhibitors (including such heavy hitters as aspirin and penicillin) were discovered serendipitously, modern drug discovery is more often facilitated by high-throughput screening technologies, as well as structure-guided design efforts. These approaches enable researchers to efficiently identify promising small-molecule lead compounds, which can then be further modified to optimize activity, specificity, and other pharmacologically relevant properties. Once an effective covalent inhibitor has been identified and characterized, it can progress into preclinical and clinical testing to assess its safety and efficacy in a therapeutic context. From the discovery of a preliminary candidate to the proteome-wide evaluation of a drug’s in vivo effects, mass spectrometry can be integrated throughout the process of covalent drug discovery. By leveraging the analytical power of mass spectrometry towards cutting-edge screening, proteomic, and chemoproteomic approaches, researchers can develop potent covalent inhibitors with maximum efficiency and success.

Recent advances in high-throughput technology have made it possible for researchers to screen thousands of ligands against a protein of interest, discovering promising binders more readily than ever before. While a broad range of screening platforms have been developed to facilitate this process, the impressive sensitivity and accuracy of mass spectrometry-based approaches make them particularly suitable for drug discovery research. To further enhance the utility of mass spectrometry, state-of-the-art automation can be integrated throughout screening workflows to improve assay speed and consistency. One common approach for the large-scale screening of covalent inhibitors is intact protein mass spectrometry (Intact-MS). In this “top-down” technique, protein molecules are incubated with reactive ligands, then subjected to mass spectrometry without proteolytic digestion. Obtained spectra are deconvoluted to determine the mass of the ‘intact’ protein, with the presence of an adduct indicating successful binding of a covalent ligand. The same Intact-MS assay can also be performed in lower throughput to validate covalent leads, determine binding stoichiometry, and characterize reaction kinetics. As a result, Intact-MS not only accelerates the initial stages of covalent lead identification but also avoids the delays and disruptions associated with auxiliary assay development during the optimization process.

For more information about covalent screening with Intact-MS, check out our previous blog post: Intact Mass Screening for Covalent Compound Libraries: What and Why?

An alternative strategy for the discovery of covalent leads takes as its starting point an existing non-covalent drug, which is then modified to introduce a functional group that confers covalent reactivity. Proteomic techniques can be invaluable to this approach, providing critical information about target structure and ligandability that accelerate the drug discovery process. Of particular note, cysteine reactivity profiling is a chemoproteomic approach that identifies ligandable cysteines throughout a protein of interest, providing a foundation for covalent inhibitor development. Samples are first treated with a pan-cysteine probe, then analyzed using mass spectrometry; adducted residues are concluded to be sufficiently accessible to reactive ligands. To profile cysteine reactivityacross complex biological samples, activity-based protein profiling (ABPP) can be performed using a pan-cysteine probe, providing valuable information about ligandability in an in vivo context. These assays enable researchers to evaluate target accessibility and druggability before initiating extensive screening and development efforts, saving time, money, and labor.

Additional mass spectrometry-based proteomic approaches can inform drug development even earlier, from the preliminary stages of target selection. Global proteomic profiling of normal and diseased cells is a powerful approach for the discovery of disease-specific biomarkers, allowing researchers to identify putative drug targets and assess routes for therapeutic intervention. Global proteomic assays also provide critical information regarding the highly specific protein expression patterns that characterize a cell type, tissue, or specific condition. This information can facilitate the selection of tissue-specific drug targets, which are generally associated with improved safety profiles. Proteomic profiling data can also be used to predict responses to existing drugs, guiding initial development efforts and providing a useful foothold when assessing a novel cell type or disease state. Proteomic approaches targeting post-translational modifications (PTMs) can provide further valuable information; for example, global characterization of PTMs can be used to identify novel therapeutic targets, as well as to determine the mechanism of action of a known drug.

As multiple recent articles have summarized, mass spectrometry-based proteomics can provide substantial benefit in the development of covalent PROTACs and other TPD-based therapeutics. Chemoproteomic approaches have also been successfully applied for the identification of novel ligand-E3 ligase pairs, while global proteomic profiling can be used to identify exploitable tissue-specific E3 ligases. More broadly, global proteomic analysis using mass spectrometry remains the gold-standard technique for unbiased, proteome-wide evaluation of target engagement and degradation.

To learn more about the how PROTACs and other TPD technologies have revolutionized the therapeutic landscape, check out our previous blog post:  TPD/PROTAC as a transformative drug discovery paradigm.   

Mass spectrometry technology underlies a range of screening, proteomic, and chemoproteomic approaches that can facilitate the identification and development of covalent ligands. From target selection to mechanistic elucidation, the analytical accuracy and precision of mass spectrometry-based assays enable informed decisions to be made rapidly and decisively throughout the research process. By thoughtfully integrating these techniques throughout drug development, potent covalent therapeutics can be taken from bench to bedside with precision and efficiency.

 

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