Histone deacetylase (HDAC) targeting drugs have entered the pharmacopoeia in the 2000s. However, some enigmatic phenotypes suggest off-target engagement. Here, we developed a quantitative chemical proteomics assay using immobilized HDAC inhibitors and mass spectrometry that we deployed to establish the target landscape of 53 drugs. The assay covers 9 of the 11 human zinc-dependent HDACs, questions the reported selectivity of some widely-used molecules, notably for HDAC6, and delineates how the composition of HDAC complexes influences drug potency. Unexpectedly, metallo-beta-lactamase domain-containing protein 2 (MBLAC2) featured as a frequent off-target of hydroxamate drugs. This poorly characterized palmitoyl-CoA hydrolase is inhibited by 24 HDAC inhibitors at low nM potency. MBLAC2 enzymatic inhibition and knock down led to the accumulation of extracellular vesicles. Given the importance of extracellular vesicle biology in neurological diseases and cancer, this HDAC-independent drug effect may qualify MBLAC2 as a target for drug discovery.

Although most cancer drugs modulate the activities of cellular pathways by changing post-translational modifications (PTMs), surprisingly little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. Here, we introduce a proteomic assay termed decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action (MoA). Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B-cells by over-activating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer.

Finding the targets of natural products is of key importance in both chemical biology and drug discovery, and deconvolution of cofactor interactomes contributes to the functional annotation of the proteome. Identifying the proteins that underlie natural compound activity in phenotypic screens helps to validate the respective targets and, potentially, expand the druggable proteome. Here, we present a generally applicable protocol for the photoactivated immobilization of unmodified and microgram quantities of natural products on diazirine-decorated beads and their use for systematic affinity-based proteome profiling. We show that among 31 molecules of very diverse reported activity and biosynthetic origin, 25 could indeed be immobilized. Dose−response competition binding experiments using lysates of human or bacterial cells followed by quantitative mass spectrometry recapitulated targets of 9 molecules with <100 μM affinity. Among them, immobilization of coenzyme A produced a tool to interrogate proteins containing a HotDog domain. Surprisingly, immobilization of the cofactor flavin adenine dinucleotide (FAD) led to the identification of nanomolar interactions with dozens of RNA-binding proteins.

HDAC drugs have entered the pharmacopoeia in the 2000s. However, some enigmatic phenotypes suggest off-target engagement. Here, we developed a chemical proteomics assay using three promiscuous chemotypes and quantitative mass spectrometry that we deployed to establish the target landscape of 53 drugs. The results highlight 14 direct targets, including 9 out of the 11 human zinc-dependent HDACs, question the reported selectivity of widely-used molecules, notably for HDAC6, and delineate how the composition of HDAC complexes influences drug potency. Unexpectedly, metallo-beta-lactamase domain-containing protein 2 (MBLAC2) featured as a frequent target of hydroxamate drugs. This ill-annotated palmitoyl-CoA hydrolase is inhibited by 24 HDAC inhibitors at low nM potency. Both enzymatic inhibition and knocking down the protein led to the accumulation of extracellular vesicles. Given the importance of exosome biology in neurological diseases or cancer, this HDAC-independent drug effect creates the incentive for considering MBLAC2 as a target for drug discovery.