Continuous enzyme activity assay for high-throughput classification of histone deacetylase 8 inhibitors

Aim: Human histone deacetylase 8 (KDAC8) is a well-recognized pharmaceutical target in Cornelia de Lange syndrome and different types of cancer, particularly childhood neuroblastoma. Several classes of chemotypes have been identified, which interfere with the enzyme activity of KDAC8. These compounds have been identified under equilibrium or near equilibrium conditions for inhibitor binding to the target enzyme. This study aims for the classification of KDAC8 inhibitors according to the mode of action and identification of most promising lead compounds for drug development. Methods: A continuous enzyme activity assay is used to monitor inhibition kinetics. Results: A high-throughput continuous KDAC8 activity assay is developed that provides additional mechanistic information about enzyme inhibition enabling the classification of KDAC8 inhibitors according to their mode of action. Fast reversible inhibitors act as a molecular chaperone and are capable to rescue the enzyme activity of misfolded KDAC8, while covalent inactivators and slow dissociating inhibitors do not preserve KDAC8 activity. Conclusions: The application of continuous KDAC8 activity assay reveals additional information about the mode of interaction with inhibitors, which can be used to classify KDAC8 inhibitors according to their mode of action. The approach is compatible with the high-throughput screening of compound libraries. Fast reversible inhibitors of KDAC8 act as molecular chaperones and recover enzyme activity from misfolded protein conformations. In contrast, slow-binding inhibitors and covalent inactivators of KDAC8 are not capable to recover enzyme activity

3‐Chloro‐5‐Substituted‐1,2,4‐Thiadiazoles (TDZs) as Selective and Efficient Protein Thiol Modifiers

The study of cysteine modifications has gained much attention in recent years. This includes detailed investigations in the field of redox biology with focus on numerous redox derivatives like nitrosothiols, sulfenic acids, sulfinic acids and sulfonic acids resulting from increasing oxidation, S-lipidation, and perthiols. For these studies selective and rapid blocking of free protein thiols is required to prevent disulfide rearrangement. In our attempt to find new inhibitors of human histone deacetylase 8 (HDAC8) we discovered 5-sulfonyl and 5-sulfinyl substituted 1,2,4-thiadiazoles (TDZ), which surprisingly show an outstanding reactivity against thiols in aqueous solution. Encouraged by these observations we investigated the mechanism of action in detail and show that these compounds react more specifically and faster than commonly used N-ethyl maleimide, making them superior alternatives for efficient blocking of free thiols in proteins. We show that 5-sulfonyl-TDZ can be readily applied in commonly used biotin switch assays. Using the example of human HDAC8, we demonstrate that cysteine modification by a 5-sulfonyl-TDZ is easily measurable using quantitative HPLC/ESI-QTOF-MS/MS, and allows for the simultaneous measurement of the modification kinetics of seven solvent-accessible cysteines in HDAC8

Synthesis and HDAC inhibitory activity of pyrimidine-based hydroxamic acids

Histone deacetylases (HDACs) play an essential role in the transcriptional regulation of cells through the deacetylation of nuclear histone and non-histone proteins and are promising therapeutic targets for the treatment of various diseases. Here, the synthesis of new compounds in which a hydroxamic acid residue is attached to differently substituted pyrimidine rings via a methylene group bridge of varying length as potential HDAC inhibitors is described. The target compounds were obtained by alkylation of 2-(alkylthio)pyrimidin-4(3 H )-ones with ethyl 2-bromoethanoate, ethyl 4-bromobutanoate, or methyl 6-bromohexanoate followed by aminolysis of the obtained esters with hydroxylamine. Oxidation of the 2-methylthio group to the methylsulfonyl group and following treatment with amines resulted in the formation of the corresponding 2-amino-substituted derivatives, the ester group of which reacted with hydroxylamine to give the corresponding hydroxamic acids. The synthesized hydroxamic acids were tested as inhibitors of the HDAC4 and HDAC8 isoforms. Among the synthesized pyrimidine-based hydroxamic acids N -hydroxy-6-[6-methyl-2-(methylthio)-5-propylpyrimidin-4-yloxy]hexanamide was found to be the most potent inhibitor of both the HDAC4 and HDAC8 isoforms, with an IC 50 of 16.6 µM and 1.2 µM, respectively

Multi-target weapons: diaryl-pyrazoline thiazolidinediones simultaneously targeting VEGFR-2 and HDAC cancer hallmarks

10.1039/d1md00125fRSC MEDICINAL CHEMISTRY1291540-155

Mechanistic Insights into Binding of Ligands with Thiazolidinedione Warhead to Human Histone Deacetylase 4

Recently, we have reported that non-hydroxamate thiazolidinedione (TZD) analogs are capable of inhibiting human deacetylase 4 (HDAC4). This study aims at the dissection of the molecular determinants and kinetics of the molecular recognition of TZD ligands by HDAC4. For this purpose, a structure activity relationship analysis of 225 analogs was combined with a comprehensive study of the enzyme and binding kinetics of a variety of HDAC4 mutant variants. The experimental data were rationalized by docking to the two major conformations of HDAC4. TZD ligands are competitive inhibitors and bind via a two-step mechanism involving principal molecular recognition and induced fit. The residence time of 24 g is (34 ± 3) min and thus much larger than that of the canonical pan-HDAC inhibitor SAHA ((5 ± 2) min). Importantly, the binding kinetics can be tuned by varying the structure of the CAP group