Drugs that target histone deacetylase (HDAC) 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-β-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 nanomolar potency. MBLAC2 enzymatic inhibition and knockdown 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.

靶向组蛋白脱乙酰酶 (HDAC) 的药物于 2000 年代进入药典。然而，一些神秘的表型表明脱靶参与。在这里，我们使用固定化 HDAC 抑制剂和质谱法开发了一种定量化学蛋白质组学测定法，我们利用该测定法来建立 53 种药物的靶点图谱。该检测涵盖了 11 种人类锌依赖性 HDAC 中的 9 种，对一些广泛使用的分子（特别是 HDAC6）的选择性提出了质疑，并描述了 HDAC 复合物的组成如何影响药物效力。出乎意料的是，含有金属-β-内酰胺酶结构域的蛋白 2 (MBLAC2) 是异羟肟酸药物频繁脱靶的特征。这种特征不明的棕榈酰辅酶 A 水解酶可被 24 种低纳摩尔效力的 HDAC 抑制剂抑制。 MBLAC2 酶抑制和敲低导致细胞外囊泡的积累。鉴于细胞外囊泡生物学在神经系统疾病和癌症中的重要性，这种不依赖于 HDAC 的药物作用可能使 MBLAC2 成为药物发现的靶点。