David House

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.

This file contain information on the Cell lines and organoids used in this study

This file contains details of the BE sgRNA library

Supplementary Figure 1. Generating base editor-expressing MSI cell lines for screening. Supplementary Figure 2. Quality control for base-editor screens in MSI cell lines. Supplementary Figure 3. Discovery and characterization of novel inhibitors of WRN helicase. Supplementary Figure 4. Correlation of WRN inhibitor sensitivity with genetic and molecular markers in MSI models. Supplementary Figure 5. Comparative analysis of DNA damage in MSI and MSS cells treated with WRN inhibitors and genetic inactivation. Supplementary Figure 6: Effects of GSK_WRN4 on MSI and MSS colorectal cancer xenografts.

This table lists the results from Base editing screen

Sulfonyl fluorides (SFs) are promising electrophiles for covalent drug discovery, enabling the targeting of diverse amino acids beyond cysteine. However, their widespread application has been hindered by limitations in stability and selectivity, often restricting their utility to early-stage tool development. We introduce heteroaryl sulfonyl fluorides as a next-generation class of tuneable electrophiles designed to overcome these challenges. These electrophiles demonstrate optimized intrinsic reactivity, enhanced solubility and lipophilicity, whilst exhibiting markedly improved stability in whole blood and plasma compared to traditional phenyl-based SFs. Employing a high-throughput chemistry direct-to-biology workflow, we rapidly explored structure-activity relationships, yielding potent covalent inhibitors of carbonic anhydrase II (CAII) with exceptional cellular selectivity. These findings establish heteroaryl SFs as versatile, tuneable electrophiles possessing superior drug-like properties and outstanding target specificity. This platform offers a powerful new avenue for developing highly selective covalent ligands that are suitable for drug discovery.

Identification of active site inhibitors of bacterial E3 ligases by covalent fragment screening coupled with high-throughput chemistry direct-to-biology (HTC-D2B).

O-GalNAc (N-acetylgalactosaminyl) glycosylation is an abundant posttranslational modification in mammalian cells. Dysregulation of O-GalNAc glycosylation is implicated in cancer metastasis and immune evasion; however, our mechanistic understanding remains limited due to the lack of small molecule tools. O-GalNAc biosynthesis depends heavily on the availability of UDP-GalNAc that is biosynthesised by the cytosolic enzyme UDP-galactose-4-epimerase (GalE). Knockout studies have demonstrated that loss of GalE severely impairs O-GalNAc glycosylation, positioning GalE as a promising enzymatic therapeutic target in oncology. Here, we present an efficient, convergent workflow that combines both covalent and high-throughput crystallographic non-covalent fragment screening with structure-based design to identify GalE inhibitors. Using these strategies, we discovered a ligandable pocket adjacent to a reactive tyrosine, enabling the development of a potent, “beyond cysteine” covalent inhibitor based on a sulfonyl fluoride warhead. Structurally-enabled fragment screening methodologies yielded nanomolar non-covalent as well as covalent binders within no more than 22 elaborated compounds. Our work demonstrates efficient synergism in next-generation delivery of chemical matter for GalE inhibition, with the broader potential for targeting non-cysteine residues in chemical biology and therapeutic applications.