George S. Biggs

Covalent chemoproteomics has become a powerful route to expanding the druggable proteome, enabling proteome-wide screening of compounds that covalently modify nucleophilic residues, most commonly cysteine. Yet typical experiments detect only 5–18% of the ∼250,000 cysteines encoded by the human proteome, and the determinants of this limited coverage remain poorly defined. Here we combine cysteine featurisation, explainable machine learning (ML) and robust statistical filtering to establish what governs cysteine detectability in iodoacetamide-desthiobiotin (IA-DTB) chemoproteomics. Using a scalable feature-generation pipeline, we computed physicochemical and structural properties for cysteines and the corresponding tryptic peptides and trained models that predict detectability with high accuracy. Shapley analysis identifies protein abundance and intrinsic peptide properties governing LC–MS behaviour as the dominant determinants, whereas cysteine structural descriptors contribute comparatively little, indicating that detection is constrained downstream of probe reactivity. We further introduce a distance-based statistical filter that improves reproducibility across replicates, and show that cell line selection, subcellular fractionation and alternative electrophiles together expand coverage by >30%. More broadly, this work moves optimisation of chemoproteomics workflows from empirical trial-and-error towards rational, data-driven design. Graphical

Metal complexes offer unique opportunities as scaffolds in chemical biology and drug discovery, with tunable geometries, modular coordination environments, and structural features not readily accessible with organic molecules. Here, we introduce reactive metallo‐scaffolds ( r ‐mS ) as a class of metal complexes designed to map ligandable cysteines across the mammalian proteome. These covalent warhead‐bearing metal complexes use the metal centre and ligand architecture to modulate cysteine engagement, with cysteine labelling occurring through the electrophilic chloroacetamide warhead. Using chemoproteomics, we profiled a focused r ‐mS series in HEK293T lysate, identifying novel cysteine ligandability and demonstrating how metal identity, arene substituents and overall molecular topography govern cysteine reactivity and proteome‐wide targeting. Among the series screened, r ‐mS‐2 emerged as the most productive scaffold, which engaged cysteine 119 within the functionally relevant SAM‐binding domain of PRMT1. This interaction was validated by intact protein LC‐MS and was determined to functionally inhibit the activity of PRMT1. Structural modelling and docking provided insights into the molecular basis of binding, which implied π‐stacking and electrostatic complementarity in driving covalent engagement. Together, these results position reactive metallo‐scaffolds ( r ‐mS ) as a versatile platform for proteome‐wide covalent ligand discovery and the rational development of next‐generation metallodrugs.

Metal complexes offer unique opportunities as scaffolds in chemical biology and drug discovery, with tunable geometries, modular coordination environments, and structural features not readily accessible with organic molecules. Here, we introduce reactive metallo‐scaffolds ( r ‐mS ) as a class of metal complexes designed to map ligandable cysteines across the mammalian proteome. These covalent warhead‐bearing metal complexes use the metal centre and ligand architecture to modulate cysteine engagement, with cysteine labelling occurring through the electrophilic chloroacetamide warhead. Using chemoproteomics, we profiled a focused r ‐mS series in HEK293T lysate, identifying novel cysteine ligandability and demonstrating how metal identity, arene substituents and overall molecular topography govern cysteine reactivity and proteome‐wide targeting. Among the series screened, r ‐mS‐2 emerged as the most productive scaffold, which engaged cysteine 119 within the functionally relevant SAM‐binding domain of PRMT1. This interaction was validated by intact protein LC‐MS and was determined to functionally inhibit the activity of PRMT1. Structural modelling and docking provided insights into the molecular basis of binding, which implied π‐stacking and electrostatic complementarity in driving covalent engagement. Together, these results position reactive metallo‐scaffolds ( r ‐mS ) as a versatile platform for proteome‐wide covalent ligand discovery and the rational development of next‐generation metallodrugs.

Metal complexes offer unique opportunities as scaffolds in chemical biology and drug discovery, with tuneable geometry, modular coordination environments, and structural features not readily accessible with organic molecules. Here, we introduce reactive metallo-scaffolds (r-mS) - a new class of covalent warhead-containing metal complexes - designed to map ligandable cysteines across the mammalian proteome. Using chemoproteomics, we profiled a focused r-mS series in HEK293T lysate, identifying novel cysteine ligandability and demonstrating how metal identity, arene substituents and overall molecular topography govern cysteine reactivity and proteome-wide targeting. Among the series screened, r-mS-2 emerged as the most productive scaffold, which engaged cysteine 119 within the functionally relevant SAM binding domain of PRMT1. This interaction was validated via intact protein LC-MS and was determined to functionally inhibit the activity of PRMT1. Structural modelling and docking provided insights into the molecular basis of binding, which implied π-stacking and electrostatic complementarity in driving covalent engagement. Together, these results position reactive metallo-scaffolds as a versatile platform for proteome-wide covalent ligand discovery and the rational development of next-generation metallodrugs.

Identifying pharmacological probes for human proteins represents a key opportunity to accelerate the discovery of new therapeutics. High-content screening approaches to expand the ligandable proteome offer the potential to expedite the discovery of novel chemical probes to study protein function. Screening libraries of reactive fragments by chemoproteomics offers a compelling approach to ligand discovery, however, optimising sample throughput, proteomic depth, and data reproducibility remains a key challenge. We report a versatile, label-free quantification proteomics platform for competitive profiling of cysteine-reactive fragments against the native proteome. This high-throughput platform combines SP4 plate-based sample preparation with rapid chromatographic gradients. Data-independent acquisition performed on a Bruker timsTOF Pro 2 consistently identified ~23,000 cysteine sites per run, with a total of ~32,000 cysteine sites profiled in HEK293T and Jurkat lysate. Crucially, this depth in cysteinome coverage is met with high data completeness, enabling robust identification of liganded proteins. In this study, 80 reactive fragments were screened in two cell lines identifying >400 ligand-protein interactions. Hits were validated through concentration-response experiments and the platform was utilised for hit expansion and live cell experiments. This label-free platform represents a significant step forward in high-throughput proteomics to evaluate ligandability of cysteines across the human proteome.

Reactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target's perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low‐throughput, constrained by the need for synthesis and purification of target compounds. We report an efficient platform for ‘direct‐to‐biology’ (D2B) screening of cysteine‐targeting chloroacetamide RFs, wherein synthesis is performed in 384‐well plates allowing direct assessment in downstream biological assays without purification. Here, the developed platform was used to optimise inhibitors of SARS‐CoV‐2 main protease (M^Pro), an established drug target for the treatment of COVID‐19. An initial RF hit was developed into a series of potent inhibitors, and further exploration using D2B screening enabled a ‘switch’ to a reversible inhibitor series. This example of ligand discovery for M^Pro illustrates the acceleration that D2B chemistry can offer for optimising RFs towards covalent inhibitor candidates, as well as providing future impetus to explore the evolution of RFs into non‐covalent ligands.

Reactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target's perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low‐throughput, constrained by the need for synthesis and purification of target compounds. We report an efficient platform for ‘direct‐to‐biology’ (D2B) screening of cysteine‐targeting chloroacetamide RFs, wherein synthesis is performed in 384‐well plates allowing direct assessment in downstream biological assays without purification. Here, the developed platform was used to optimise inhibitors of SARS‐CoV‐2 main protease (M^Pro), an established drug target for the treatment of COVID‐19. An initial RF hit was developed into a series of potent inhibitors, and further exploration using D2B screening enabled a ‘switch’ to a reversible inhibitor series. This example of ligand discovery for M^Pro illustrates the acceleration that D2B chemistry can offer for optimising RFs towards covalent inhibitor candidates, as well as providing future impetus to explore the evolution of RFs into non‐covalent ligands.

Reactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target’s perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low-throughput, constrained by the need for synthesis and purification of target compounds. We report a high-throughput platform for ‘direct-to-biology’ (D2B) screening of cysteine-targeting chloroacetamide RFs, wherein synthesis is performed in 384-well plates allowing direct assessment in downstream biological assays without purification. Here, the developed platform was used to optimise inhibitors of SARS-CoV-2 main protease (MPro), an established drug target for the treatment of COVID-19. An initial RF hit was developed into a series of potent inhibitors, and further exploration using D2B screening enabled a ‘switch’ to a reversible inhibitor series. This example of ligand discovery for MPro illustrates the acceleration that D2B chemistry can offer for optimising RFs towards covalent inhibitor candidates, as well as providing future impetus to explore the evolution of RFs into non-covalent ligands.

Identifying pharmacological probes for human proteins represents a key opportunity to accelerate the discovery of new therapeutics. High-content screening approaches to expand the ligandable proteome offer the potential to expedite the discovery of novel chemical probes to study protein function. Screening libraries of reactive fragments by chemoproteomics offers a compelling approach to ligand discovery, however, optimising sample throughput, proteomic depth, and data reproducibility remains a key challenge. We report a versatile, label-free quantification proteomics platform for competitive profiling of cysteine-reactive fragments against the native proteome. This high-throughput platform combines SP4 plate-based sample preparation with rapid chromatographic gradients. Data-independent acquisition performed on a Bruker timsTOF Pro 2 consistently identified ∼23,000 cysteine sites per run, with a total of ∼32,000 cysteine sites profiled in HEK293T and Jurkat lysate. Crucially, this depth in cysteinome coverage is met with high data completeness, enabling robust identification of liganded proteins. In this study, 80 reactive fragments were screened in two cell lines identifying >400 ligand-protein interactions. Hits were validated through concentration-response experiments and the platform was utilised for hit expansion and live cell experiments. This label-free platform represents a significant step forward in high-throughput proteomics to evaluate ligandability of cysteines across the human proteome.

To survive, many pathogens extract heme from their host organism and break down the porphyrin scaffold to sequester the Fe²⁺ ion via a heme oxygenase. Recent studies have revealed that certain pathogens can anaerobically degrade heme. Our own research has shown that one such pathway proceeds via NADH-dependent heme degradation, which has been identified in a family of hemoproteins from a range of bacteria. HemS, from Yersinia enterocolitica, is the main focus of this work, along with HmuS (Yersinia pestis), ChuS (Escherichia coli) and ShuS (Shigella dysenteriae). We combine experiments, Energy Landscape Theory, and a bioinformatic investigation to place these homologues within a wider phylogenetic context. A subset of these hemoproteins are known to bind certain DNA promoter regions, suggesting not only that they can catalytically degrade heme, but that they are also involved in transcriptional modulation responding to heme flux. Many of the bacterial species responsible for these hemoproteins (including those that produce HemS, ChuS and ShuS) are known to specifically target oxygen-depleted regions of the gastrointestinal tract. A deeper understanding of anaerobic heme breakdown processes exploited by these pathogens could therefore prove useful in the development of future strategies for disease prevention.