Target deconvolution of HDAC pharmacopoeia highlights MBLAC2 as common off-target

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 56 HDAC 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 identified novel interactome-dependent binding of drugs to HDAC1/2 epigenetic complexes. 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.