Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target.

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-ß-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.

High-Throughput Enzyme Assay for Screening Inhibitors of the ZDHHC3/7/20 Acyltransferases.

As enzymes that mediate the attachment of long-chain fatty acids to cysteine residues, ZDHHC proteins have been reported to be promising therapeutic targets for treating cancer and autoimmune diseases. Yet, due to the lack of potent selective inhibitors, scrutiny of the biological functions of ZDHHCs has been limited. The main hindrance for developing ZDHHC inhibitors is the lack of a facile high-throughput assay. Here, we developed a ZDHHC3/7/20 high-throughput assay based on the acylation-coupled lipophilic induction of polarization (Acyl-cLIP) method and screened several potential ZDHHC inhibitors. Furthermore, we demonstrated that in vitro results from the Acyl-cLIP assay are supported by the results from cell-based assays. We envision that this new ZDHHC3/7/20 Acyl-cLIP assay will accelerate the high-throughput screening of large compound libraries for improved ZDHHC inhibitors and provide therapeutic benefits for cancer and autoimmune diseases.

Substrate recruitment by zDHHC protein acyltransferases.

Protein palmitoylation is the post-translational attachment of fatty acids, most commonly palmitate (C16 : 0), onto a cysteine residue of a protein. This reaction is catalysed by a family of integral membrane proteins, the zDHHC protein acyltransferases (PATs), so-called due to the presence of an invariant Asp-His-His-Cys (DHHC) cysteine-rich domain harbouring the catalytic centre of the enzyme. Conserved throughout eukaryotes, the zDHHC PATs are encoded by multigene families and mediate palmitoylation of thousands of protein substrates. In humans, a number of zDHHC proteins are associated with human diseases, including intellectual disability, Huntington's disease, schizophrenia and cancer. Key to understanding the physiological and pathophysiological importance of individual zDHHC proteins is the identification of their protein substrates. Here, we will describe the approaches and challenges in assigning substrates for individual zDHHCs, highlighting key mechanisms that underlie substrate recruitment.

Metallo-ß-lactamase domain-containing protein 2 is S-palmitoylated and exhibits acyl-CoA hydrolase activity.

Members of the metallo-ß-lactamase (MBL) superfamily of enzymes harbor a highly conserved αßßα MBL-fold domain and were first described as inactivators of common ß-lactam antibiotics. In humans, these enzymes have been shown to exhibit diverse functions, including hydrolase activity toward amides, esters, and thioesters. An uncharacterized member of the human MBL family, MBLAC2, was detected in multiple palmitoylproteomes, identified as a zDHHC20 S-acyltransferase interactor, and annotated as a potential thioesterase. In this study, we confirmed that MBLAC2 is palmitoylated and identified the likely S-palmitoylation site as Cys254. S-palmitoylation of MBLAC2 is increased in cells when expressed with zDHHC20, and MBLAC2 is a substrate for purified zDHHC20 in vitro. To determine its biochemical function, we tested the ability of MBLAC2 to hydrolyze a variety of small molecules and acylprotein substrates. MBLAC2 has acyl-CoA thioesterase activity with kinetic parameters and acyl-CoA selectivity comparable with acyl-CoA thioesterase 1 (ACOT1). Two predicted zinc-binding residues, Asp87 and His88, are required for MBLAC2 hydrolase activity. Consistent with a role in fatty acid metabolism in cells, MBLAC2 was cross-linked to a photoactivatable fatty acid in a manner that was independent of its S-fatty acylation at Cys254. Our study adds to previous investigations demonstrating the versatility of the MBL-fold domain in supporting a variety of enzymatic reactions.

Purification of Recombinant DHHC Proteins Using an Insect Cell Expression System.

DHHC enzymes are a family of integral membrane proteins that catalyze the posttranslational addition of palmitate, a 16-carbon fatty acid, onto a cysteine residue of a protein. While the library of identified palmitoylated proteins has grown tremendously over the years, biochemical and mechanistic studies on DHHC proteins are challenged by the innate difficulty of purifying the enzyme in large amounts. Here we describe our protocol for preparing recombinant DHHC proteins tagged with a hexahistidine sequence and a FLAG epitope that aid in the purification. This procedure has been tested successfully in purifying several members of the enzyme family; DHHC3 and its catalytically inactive cysteine mutant, DHHS3 are used as examples. The recombinant protein is extracted from whole cell lysates using the detergent dodecylmaltoside (DDM) and is subjected to a two-column purification. Homogeneity and monodispersity of the purified protein are checked by size exclusion chromatography (SEC). A preparation from a 400-mL infection of Sf9 insect cell culture typically yields 0.5 mg of DHHC3 and 1.0 mg of catalytically inactive DHHS3. Both forms appear monodisperse up to a concentration of 1 mg/mL by SEC.