Mass spectrometry-based proteome-wide cysteine profiling has evolved from a tool for single compound profiling into a robust platform for covalent ligand discovery. Recent advances in cysteine-directed ligand discovery will be presented, including new innovations in chemical probes, in-cell labeling, instrumentation, automation, and data analysis.

I will describe the synthesis of sulfonyl-triazoles as a new phenol-reactive group for chemoselective modification of tyrosine residues on proteins through sulfur-triazole exchange (SuTEx) chemistry. We demonstrated that sulfonyl-triazoles can serve as global detection probes and fragment competitors for surveying ligandability of protein sites. We further showcase the high tunability of sulfonyl-triazoles for developing inhibitors that disrupt protein function by liganding catalytic and non-catalytic tyrosine sites. I will conclude my talk by describing our efforts to apply sulfonyl-triazoles for targeting cryptic binding sites on lipid enzymes to block metabolic and signaling activity in cells.

While photoaffinity labeling strategies have become the benchmark for target deconvolution of small molecules owing to their reliance on external activation to induce covalent protein capture, the process of target identification remains one of the most technically challenging aspects of early drug discovery. Thus, there is a strong demand for new technologies that allow for controlled activation of chemical probes to covalently label their protein target. In this presentation we introduce an electroaffinity labeling (ECAL) platform which leverages the use of a small, redox-active diazetidinone (DZE) functional group and the chemistry behind its development.

The clinical application of JAK inhibitors is limited by side effects arising from poor subtype selectivity. This talk will describe the use of chemical proteomics to identify a ligandable allosteric cysteine in the pseudokinase domain of JAK1 that is not conserved in JAK2 or JAK3. Proteomics guided optimization of a chemical probe targeting this site yields a potent inhibitor JAK1 that has high selectivity over JAK2 in primary immune cells, and a unique mechanism of action to existing orthosteric JAK inhibitors.

Identifying the targets of small molecule protein interactions is valuable in understanding target engagement and selectivity profiles. This presentation will focus on the use of photoaffinity and electrophilic probes to understand and profile these interactions.

Tag-based targeted protein degradation technologies including the degradation tag (dTAG) system have emerged as powerful approaches to control protein abundance using small molecule degraders. This talk will describe the development and advances of the dTAG technology platform and highlight case studies demonstrating utility for preclinical target discovery and validation.

Inducing novel protein-protein interactions has the potential to rewire pathways, inhibit or degrade novel targets through induced proximity with E3 ligases. Targeted protein degradation is a rapidly developing therapeutic modality. We report an approach to evaluate the ability of recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for hit finding to identify specific E3 ligase binders, this approach makes use of simple maleimide-thiol chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). By applying COFFEE to SPSB2, a SOCS box and SPRY-domain E3 ligase that has not previously been redirected for targeted protein degradation.

Global expression proteomics can serve as a powerful tool for discovery of protein degraders within electrophilic small molecule libraries. This approach is agnostic to the mechanism of degradation, which provides unique opportunities for unbiased degrader discovery and mechanistic characterization. This talk will discuss unexpected pharmacology revealed in studies of putative electrophilic degraders identified by MS-based proteomics.  

Chemical probes are potent and selective small molecule inhibitors that can be used in cellular assays to induce a phenotype.  Screening a library of epigenetic chemical probes in cellular assay from patient tissue, we have discovered novel targets and lead compounds for developing treatments for two rare diseases: Dupuytren's contracture and Friedreich’s Ataxia.

Creatine Transporter Deficiency (CTD) is defined by loss-of-function mutations in the creatine transporter, SLC6A8. Many SLC6A8 variants have substantial defects in plasma membrane localization, suggesting small-molecule correctors as a therapeutic strategy. We developed translational assays to profile the activity of novel correctors and validate their mechanism of action. Our lead molecules restore surface localization of SLC6A8 variants and increase creatine transport, validating correctors as a novel therapeutic opportunity for CTD.

Gene silencing by Polycomb Repressive Complex 1 (PRC1) is frequently misregulated in human diseases. Aberrant PRC1 targeting to histone H3 lysine 27 trimethylation is directed by cell type-specific, paralogous chromobox (CBX) proteins. While CBX proteins are attractive targets, development of selective small molecules has been challenging. We created a quantitative and target-specific cellular assay to discover potent, selective chemical probes against CBX proteins capable of efficiently displacing PRC1 from chromatin.

Choosing the most efficient and reliable target deconvolution method earlier minimizes the risk of late-stage failure, improve translational models, and provide a richer understanding of a compound's MoA and pathway effects. The high-resolution mass-spectrometry coupled with chemical biology target deconvolution techniques have shown to be indispensable, physiologically relevant system for an unbiased, proteome-wide discovery. In this talk, label free and labeled techniques will be reviewed with case studies on mass spec-based Cellular Thermal Shift Method.

SPROX (Stability of Proteins from Rates of Oxidation) and TPP (Thermal Proteome Profiling) have emerged as exciting methods for drug target engagement that do not require drug modifications. However, a systematic comparison between SPROX and TPP is still lacking. Insights from method comparison, optimization to OnePot 2D format, and a strategy for hit-ranking will be shared, including a 10X improvement in throughput and unique SPOX hits among atypical kinases.

High-throughput CETSA (HT-CETSA) has emerged as a promising screening strategy for identifying compounds bindings to the target of interest in the native cellular environment. This talk will discuss learnings from our HT-CETSA screening efforts toward ligand discovery for a difficult-to-drug transcription factor target.