This talk will describe examples of how chemical biology probes and bioorthogonal chemistry were used for target identification and engagement, selectivity profiling (off-targets), and mechanism of action studies in drug discovery. Several different types of chemical biology probes were utilized, including enzyme class-specific, activity-based probes, cysteine-specific reactivity-based probes, as well as target-specific clickable covalent inhibitor and photoaffinity probes. These probes proved to be invaluable for target discovery.

Photoreactive and electrophilic probes are valuable tools in chemical biology to identify small-molecule/protein interactions. This presentation will compare and evaluate different photoreactive groups and electrophilic compounds in the context of drug discovery programs, with emphasis on target deconvolution, off-target identification, and activity-based protein profiling. Additional focus on the advancement of new sulfur (VI) fluoride probes, the development of a chemical biology toolbox, and synthetic chemistry advancements will also be discussed.

Small molecules that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. We and others have demonstrated that efficacious degradation of kinases and other targets can be achieved in vitro and in vivo, however, many targets remain recalcitrant to degradation. I will discuss the use of large-scale chemical proteomics approaches to accelerate the development of degraders as novel chemical probes for kinases and other targets.

Current methods for proteome-wide cysteine profiling rely on iodoacetamide-linked probes. Progess will be presented evaluating diverse heteroaromatic sulfides with different oxidation states, heteroatom substitutions, and a series of electron donating and electron-withdrawing substituents. Select substitutions profoundly influence reactivity and stability compared to conventional cysteine conjugation reagents, increasing the reaction rate by >3-orders of magnitude. Direct addition of desthiobiotin-functionalized probes to cultured cells simplified enrichment and elution to enable mass spectrometry discovery of unique thiols labeled in their native cellular environments in a fraction of the standard analysis time. These probes offer advantages over existing cysteine alkylation reagents, including accelerated reaction rates, improved stability, and robust ionization for mass spectrometry applications. Overall, heteroaromatic sulfones provide modular tunability, shifted chromatographic elution times, and superior in-cell cysteine profiling for in-depth proteome-wide analysis and covalent ligand discovery.

We have redesigned the entire workflow for activity-based protein profiling (ABPP) of reactive cysteine residues towards electrophilic compounds with an eye towards screening large libraries containing hundreds to thousands of compounds directly in cells. Altogether, a 42-fold improvement in sample throughput was achieved corresponding to profiling library members at a depth of >8,000 reactive cysteine sites using only 18 min per compound. We applied the method to screen hundreds of electrophiles directly in cells.

Many cancers are driven by complex signaling networks. It is therefore attractive to target more than one disease-relevant pathway or target. Optimal design of multi-targeted strategies requires detailed knowledge of drug targets and how drugs engage oncogenic signaling networks. Here, we will present examples of how the integration of chemical and phosphoproteomics approaches can dissect drug mechanisms of action within complex signaling networks and inform the rational design of novel drug combination approaches.

Probe-based chemical proteomics technologies play an integral role in target identification and validation of the biological target(s) through which the drug is proposed to exert its mechanistic effects. A suite of chemical probes was evaluated for selectivity profiling and measuring target engagement for several kinase inhibitors. A sulfonyl fluoride XO-44 probe was used for target identification and selectivity profiling of kinase drug candidates under physiological conditions and Ibrutinib-based probes were utilized to measure BTK target occupancy both in vitro and in vivo.

Phenotypic screening is a powerful approach for identification of novel disease-relevant targets and pathways for drug discovery. Here, I present a case study to identify targets, involved in pro-inflammatory to anti-inflammatory macrophage repolarization, that can be utilized for autoimmune drug discovery.­­ Using a small-molecule phenotypic screening approach, we were able to identify known, as well as novel, targets that facilitated induction of anti-inflammatory phenotype in LPS/IFNγ-activated, human monocyte-derived macrophages by distinct mechanisms. The targets were validated using tool compounds, target-to-phenotype correlation, profiling approaches, followed by target knock down and in vivo POC studies. Together, our phenotypic screening platform allowed discovery of novel targets and mechanisms to support development of macrophage-targeted therapeutics for autoimmune disorders.

The properties of eukaryotic messenger RNA can be modulated by dynamic chemical modifications that occur post-transcriptionally (known as the “epitranscriptome”). We are developing chemical proteomic strategies based upon metabolic RNA labeling with artificial nucleosides and synthetic oligonucleotide probes to discover and characterize RNA modification writers, erasers, and readers, and understand their biological roles. This should provide powerful and general strategies for interrogating fundamental RNA regulatory mechanisms and lead to new insights for targeting epitranscriptomic protein factors for therapeutic benefit.