HDAC11 is a class IV histone deacylase with no crystal structure reported so far. The catalytic domain of HDAC11 shares low sequence identity with other HDAC isoforms, which makes conventional homology modeling less reliable. AlphaFold is a machine learning approach that can predict the 3D structure of proteins with high accuracy even in absence of similar structures. However, the fact that AlphaFold models are predicted in the absence of small molecules and ions/cofactors complicates their utilization for drug design. Previously, we optimized an HDAC11 AlphaFold model by adding the catalytic zinc ion and minimization in the presence of reported HDAC11 inhibitors. In the current study, we implement a comparative structure-based virtual screening approach utilizing the previously optimized HDAC11 AlphaFold model to identify novel and selective HDAC11 inhibitors. The stepwise virtual screening approach was successful in identifying a hit that was subsequently tested using an in vitro enzymatic assay. The hit compound showed an IC50 value of 3.5 µM for HDAC11 and could selectively inhibit HDAC11 over other HDAC subtypes at 10 µM concentration. In addition, we carried out molecular dynamics simulations to further confirm the binding hypothesis obtained by the docking study. These results reinforce the previously presented AlphaFold optimization approach and confirm the applicability of AlphaFold models in the search for novel inhibitors for drug discovery.

• Keywords
• AlphaFold, HDAC11, docking, in vitro assay, modelling, molecular dynamics simulation, pharmacophore, virtual screening,
• MeSH
• Models, Chemical * MeSH
• Histone Deacetylase Inhibitors pharmacology   chemistry   MeSH
• Catalytic Domain MeSH
• Drug Design MeSH
• Molecular Dynamics Simulation * MeSH
• Molecular Docking Simulation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Histone Deacetylase Inhibitors MeSH

Histone deacetylase (HDAC) inhibitors used in the clinic typically contain a hydroxamate zinc-binding group (ZBG). However, more recent work has shown that the use of alternative ZBGs, and, in particular, the heterocyclic oxadiazoles, can confer higher isoenzyme selectivity and more favorable ADMET profiles. Herein, we report on the synthesis and biochemical, crystallographic, and computational characterization of a series of oxadiazole-based inhibitors selectively targeting the HDAC6 isoform. Surprisingly, but in line with a very recent finding reported in the literature, a crystal structure of the HDAC6/inhibitor complex revealed that hydrolysis of the oxadiazole ring transforms the parent oxadiazole into an acylhydrazide through a sequence of two hydrolytic steps. An identical cleavage pattern was also observed both in vitro using the purified HDAC6 enzyme as well as in cellular systems. By employing advanced quantum and molecular mechanics (QM/MM) and QM calculations, we elucidated the mechanistic details of the two hydrolytic steps to obtain a comprehensive mechanistic view of the double hydrolysis of the oxadiazole ring. This was achieved by fully characterizing the reaction coordinate, including identification of the structures of all intermediates and transition states, together with calculations of their respective activation (free) energies. In addition, we ruled out several (intuitively) competing pathways. The computed data (ΔG‡ ≈ 21 kcal·mol-1 for the rate-determining step of the overall dual hydrolysis) are in very good agreement with the experimentally determined rate constants, which a posteriori supports the proposed reaction mechanism. We also clearly (and quantitatively) explain the role of the -CF3 or -CHF2 substituent on the oxadiazole ring, which is a prerequisite for hydrolysis to occur. Overall, our data provide compelling evidence that the oxadiazole warheads can be efficiently transformed within the active sites of target metallohydrolases to afford reaction products possessing distinct selectivity and inhibition profiles.

• MeSH
• Histone Deacetylase 6 chemistry   MeSH
• Hydrolysis MeSH
• Histone Deacetylase Inhibitors * pharmacology   MeSH
• Hydroxamic Acids chemistry   MeSH
• Oxadiazoles * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Histone Deacetylase 6 MeSH
• Histone Deacetylase Inhibitors * MeSH
• Hydroxamic Acids MeSH
• Oxadiazoles * MeSH

Lysine deacetylases, like histone deacetylases (HDACs) and sirtuins (SIRTs), are involved in many regulatory processes such as control of metabolic pathways, DNA repair, and stress responses. Besides robust deacetylase activity, sirtuin isoforms SIRT2 and SIRT3 also show demyristoylase activity. Interestingly, most of the inhibitors described so far for SIRT2 are not active if myristoylated substrates are used. Activity assays with myristoylated substrates are either complex because of coupling to enzymatic reactions or time-consuming because of discontinuous assay formats. Here we describe sirtuin substrates enabling direct recording of fluorescence changes in a continuous format. Fluorescence of the fatty acylated substrate is different when compared to the deacylated peptide product. Additionally, the dynamic range of the assay could be improved by the addition of bovine serum albumin, which binds the fatty acylated substrate and quenches its fluorescence. The main advantage of the developed activity assay is the native myristoyl residue at the lysine side chain avoiding artifacts resulting from the modified fatty acyl residues used so far for direct fluorescence-based assays. Due to the extraordinary kinetic constants of the new substrates (KM values in the low nM range, specificity constants between 175,000 and 697,000 M-1s-1) it was possible to reliably determine the IC50 and Ki values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats.

• Keywords
• bovine serum albumin effect, continuous activity assay, fluorescence quenching, histone deacetylases, myristoylated substrates, sirtuin inhibitors, sirtuins,
• MeSH
• Coloring Agents MeSH
• Lysine MeSH
• Peptides MeSH
• Sirtuin 1 metabolism   MeSH
• Sirtuin 2 metabolism   MeSH
• Sirtuin 3 * metabolism   MeSH
• Sirtuins * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Coloring Agents MeSH
• Lysine MeSH
• Peptides MeSH
• Sirtuin 1 MeSH
• Sirtuin 2 MeSH
• Sirtuin 3 * MeSH
• Sirtuins * MeSH

Class I histone deacetylases (HDACs) are key regulators of cell proliferation and they are frequently dysregulated in cancer cells. We report here the synthesis of a novel series of class-I selective HDAC inhibitors (HDACi) containing a 2-aminobenzamide moiety as a zinc-binding group connected with a central (piperazin-1-yl)pyrazine or (piperazin-1-yl)pyrimidine moiety. Some of the compounds were additionally substituted with an aromatic capping group. Compounds were tested in vitro against human HDAC1, 2, 3, and 8 enzymes and compared to reference class I HDACi (Entinostat (MS-275), Mocetinostat, CI994 and RGFP-966). The most promising compounds were found to be highly selective against HDAC1, 2 and 3 over the remaining HDAC subtypes from other classes. Molecular docking studies and MD simulations were performed to rationalize the in vitro data and to deduce a complete structure activity relationship (SAR) analysis of this novel series of class-I HDACi. The most potent compounds, including 19f, which blocks HDAC1, HDAC2, and HDAC3, as well as the selective HDAC1/HDAC2 inhibitors 21a and 29b, were selected for further cellular testing against human acute myeloid leukemia (AML) and erythroleukemic cancer (HEL) cells, taking into consideration their low toxicity against human embryonic HEK293 cells. We found that 19f is superior to the clinically tested class-I HDACi Entinostat (MS-275). Thus, 19f is a new and specific HDACi with the potential to eliminate blood cancer cells of various origins.

• Keywords
• 2-aminobenzamides, HDAC1, HDAC2, HDAC3, SAR studies, acute myeloid leukemia (AML), docking, histone deacetylases,
• MeSH
• Benzamides chemical synthesis   chemistry   pharmacology   MeSH
• HEK293 Cells MeSH
• Histone Deacetylase Inhibitors chemical synthesis   chemistry   pharmacokinetics   pharmacology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• ortho-Aminobenzoates chemical synthesis   chemistry   MeSH
• Antineoplastic Agents chemical synthesis   chemistry   pharmacokinetics   pharmacology   MeSH
• Proton Magnetic Resonance Spectroscopy MeSH
• Pyrazines chemistry   MeSH
• Pyridines chemical synthesis   chemistry   pharmacology   MeSH
• Molecular Docking Simulation * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• anthranilamide MeSH Browser
• Benzamides MeSH
• entinostat MeSH Browser
• Histone Deacetylase Inhibitors MeSH
• ortho-Aminobenzoates MeSH
• Antineoplastic Agents MeSH
• Pyrazines MeSH
• Pyridines MeSH