We coupled multiplexed cell line viability technology to functional genomics and chemical proteomics for identifying novel cancer targets. Here, a specific case study from the initial screen to target deconvolution and follow-up functional validation studies will be presented.

Cell-cell interaction environments are attractive therapeutic regions of interest, yet while many key interactions take place across these environments, our ability to unbiasedly characterize the proteins and cell types involved with high resolution remains challenging. Here we provide updates on a recently disclosed visible light-based photocatalytic platform for the targeted labeling of cell surface proteins and its successful pairing with downstream workflows to uncover protein microenvironments and cell-cell interactions.

Brown adipose tissue (BAT) regulates metabolic physiology and disease. However, nearly all studies of BAT occurred in one mouse strain, C57BL/6, limiting the translational potential. Here we measure genotypes, phenotypes, and BAT proteomes across 163 Diversity Outbred mice, defining the Outbred Proteome Architecture of BAT (OPABAT) to annotate the functions of thousands of proteins in thermogenesis and metabolic disease. OPABAT is a resource for understanding conserved mechanisms of BAT regulation over metabolic physiology and disease.

Recent chemoproteomics advances have enabled covalent ligand discovery across a broad range of new targets.  Here, we discuss the expanding role of chemical biology and chemoproteomics to support covalent lead discovery efforts, from early hit-finding to late lead optimization.

Genomic and functional genomic approaches have revolutionized our understanding of the role the immune system plays in human health and disease, however our knowledge of the post-translational drivers of immune dysregulation in diverse pathologies is still incomplete. This talk will describe an integrated chemical proteomic and phenotypic screening approach that enables interrogation of structural and functional changes in the proteomes of immune cells and identification of small-molecule covalent protein degraders.

The enzyme SARM1 is a key regulator of axon degeneration through its capacity to consume NAD+, which promotes cell autonomous axonal self-destruction. Here, we provide evidence for an alternative allosteric mode of controlling SARM1 function through the chemical proteomic discovery of a druggable cysteine in the autoregulatory ARM1 domain of the enzyme.  In this talk we describe a series of electrophilic small molecules that site-specifically and stereoselectively react with this cysteine residue to suppress SARM1 activity and protect axons from chemical toxin-induced axonal degeneration. 

The bioorthogonal reaction between tetrazines and trans-cyclooctenes (TCOs) has shown immense utility in chemical biology. This presentation will focus on the development of new methods to prepare tetrazine-containing probe molecules and their utility in a variety of chemical biology applications. Moreover, a new approach, utilizing unreactive, stable, and cell permeable dihydrotetrazines, which under photochemical conditions are transformed into highly reactive tetrazines in cells, will also be described. This process referred to as the catalytic activation of bioorthogonal chemistry with light (or CABL) has proved particularly effective in spatiotemporally controlled labeling and no-wash imaging in live cells.

Dimethyl Fumarate (DMF, marketed as TECFIDERA) has been an established oral therapy for multiple sclerosis worldwide. Under physiological conditions, DMF is readily hydrolyzed to generate monoester monomethylfumarate (MMF), which is considered the active metabolite of DMF. To better understand the mechanism of action (MoA) of DMF/MMF and potentially develop target engagement biomarkers, we designed and utilized clickable probes to visualize and enrich probe-modified proteins. To profile target proteins on a proteome-wide level with high confidence, we perform quantitative chemoproteomics analysis and validate several unique/shared targets of DMF and MMF, which provide insight into the reactivity and selectivity of fumarates.

We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase. Applying this approach in Drosophila and mice indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.

Reversible protein phosphorylation is a fundamental process that controls protein function and intracellular signalling and failure of phospho-control accounts for many human diseases. We have developed the affinity-directed phosphatase (AdPhosphatase) system for targeted dephosphorylation of specific phospho-proteins in cells. By deploying the PP1/2A catalytic subunits conjugated to an antigen-stabilised anti-GFP nanobody, we can promote the dephosphorylation of two independent phospho-proteins, FAM83D or ULK1, knocked in with GFP-tags, with exquisite specificity.

XPO1 is a therapeutic target in both oncology and amyotrophic lateral sclerosis (ALS). The development and application of clickable probes and a mass spectrometry-based chemoproteomic workflow to measure in vivo XPO1 target occupancy will be presented. Additionally, we will discuss the use of these tools in different biological matrices and in vivo models.

Epigenetic proteins represent key therapeutic targets for a wide range of diseases. Mapping target selectivity, however, is challenging as the targets span a wide range of enzyme families and often exist as subunits within large and dynamic complexes. Such target selectivity information is important from a safety perspective, but also key for understanding the driver(s) of efficacy. In vitro biochemical screens using purified domains can provide initial selectivity information, but critically are removed from the native cellular context. Here, we will describe recent work at AstraZeneca to evaluate target engagement and selectivity using full-length epigenetic proteins in endogenous complexes using a combination of affinity matrices, cellular thermal shift assays, and proximity labeling technologies.

Several groundbreaking medicines in cancer produce a therapeutic response through a covalent mechanism of action. Our group developed sulfonyl-triazoles as a covalent binder of tyrosines, and lysines to a lesser extent, to enable ligand discovery of catalytic and non-catalytic sites on proteins through sulfur-triazole exchange (SuTEx) chemistry. In my talk, I will describe our efforts to advance the capabilities of SuTEx for covalent probes and therapeutic discovery.