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.

• Klíčová slova
• AlphaFold, HDAC11, docking, in vitro assay, modelling, molecular dynamics simulation, pharmacophore, virtual screening,
• MeSH
• chemické modely * MeSH
• inhibitory histondeacetylas farmakologie   chemie   MeSH
• katalytická doména MeSH
• racionální návrh léčiv MeSH
• simulace molekulární dynamiky * MeSH
• simulace molekulového dockingu MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inhibitory histondeacetylas 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.

• Klíčová slova
• bovine serum albumin effect, continuous activity assay, fluorescence quenching, histone deacetylases, myristoylated substrates, sirtuin inhibitors, sirtuins,
• MeSH
• barvicí látky MeSH
• lysin MeSH
• peptidy MeSH
• sirtuin 1 metabolismus   MeSH
• sirtuin 2 metabolismus   MeSH
• sirtuin 3 * metabolismus   MeSH
• sirtuiny * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• barvicí látky MeSH
• lysin MeSH
• peptidy MeSH
• sirtuin 1 MeSH
• sirtuin 2 MeSH
• sirtuin 3 * MeSH
• sirtuiny * 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.

• Klíčová slova
• 2-aminobenzamides, HDAC1, HDAC2, HDAC3, SAR studies, acute myeloid leukemia (AML), docking, histone deacetylases,
• MeSH
• benzamidy chemická syntéza   chemie   farmakologie   MeSH
• HEK293 buňky MeSH
• inhibitory histondeacetylas chemická syntéza   chemie   farmakokinetika   farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• ortoaminobenzoáty chemická syntéza   chemie   MeSH
• protinádorové látky chemická syntéza   chemie   farmakokinetika   farmakologie   MeSH
• protonová magnetická rezonanční spektroskopie MeSH
• pyraziny chemie   MeSH
• pyridiny chemická syntéza   chemie   farmakologie   MeSH
• simulace molekulového dockingu * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• anthranilamide MeSH Prohlížeč
• benzamidy MeSH
• entinostat MeSH Prohlížeč
• inhibitory histondeacetylas MeSH
• ortoaminobenzoáty MeSH
• protinádorové látky MeSH
• pyraziny MeSH
• pyridiny MeSH

Histone deacetylase 11 (HDAC11) preferentially removes fatty acid residues from lysine side chains in a peptide or protein environment. Here, we report the development and validation of a continuous fluorescence-based activity assay using an internally quenched TNFα-derived peptide derivative as a substrate. The threonine residue in the +1 position was replaced by the quencher amino acid 3'-nitro-l-tyrosine and the fatty acyl moiety substituted by 2-aminobenzoylated 11-aminoundecanoic acid. The resulting peptide substrate enables fluorescence-based direct and continuous readout of HDAC11-mediated amide bond cleavage fully compatible with high-throughput screening formats. The Z'-factor is higher than 0.85 for the 15 μM substrate concentration, and the signal-to-noise ratio exceeds 150 for 384-well plates. In the absence of NAD+, this substrate is specific for HDAC11. Reevaluation of inhibitory data using our novel assay revealed limited potency and selectivity of known HDAC inhibitors, including Elevenostat, a putative HDAC11-specific inhibitor.

• Publikační typ
• časopisecké články MeSH