Selectivity in histone deacetylase inhibition: A biophysical approach

• Main Author: Meyners, Christian Stephan
• Format: Others
• Language: en
• Published: 2017
• Online Access: http://tuprints.ulb.tu-darmstadt.de/6589/1/D_Christian%20Meyners_B.pdf; Meyners, Christian Stephan <http://tuprints.ulb.tu-darmstadt.de/view/person/Meyners=3AChristian_Stephan=3A=3A.html> : Selectivity in histone deacetylase inhibition: A biophysical approach. Technische Universität, Darmstadt [Ph.D. Thesis], (2017)

The development of selective and save compounds is an important task in drug discovery and during the past 25 years biophysical methods for the characterization of protein-ligand interactions have been developed to a valuable tool. On the one hand these methods allow to validate screening hits and on the other hand they complement traditional approaches in drug discovery by providing deep insights into the thermodynamics, kinetics and the mechanism of molecular interactions. Based on these information, approaches were developed which allow to identify promising lead structures for further development. Histone deacetylases (HDACs) are a protein family which are investigated as therapeutic targets for the treatment of different diseases like cancer neurodegenerative disorders and parasitic infections. By catalyzing the deacetylation of the ε-amino function of lysines of histones and other proteins they fulfill an important function for the epigenetic regulation. With SAHA, FK228, PXD101 and LBH589 four HDAC inhibitors were approved for the treatment of different cancers. They cause several unwanted side effects. SAHA, PXD101 and LBH589 are unselective inhibitors which inhibit most HDAC isotypes. In order to minimize unwanted side effects and to provide a more specific therapy isotype selective inhibitors are developed. The present work deals with the question how information from an analysis of the thermodynamics, kinetics and binding mechanisms can be exploited to characterize the selectivity of inhibitors of the HDAC family. The generated results of this doctoral thesis are provided in the cumulative part which consists of five articles published in peer reviewed journals and one article submitted for peer review. One the one hand they extend the methodology on the identification and biophysical characterization of HDAC inhibitors and on the other hand they contribute to the deep comprehension of the molecular basis of the inhibition of HDACs and of protein-ligand interactions. The first part of this work covers methods developed for the identification and biophysical characterization of HDAC inhibitors. In a cooperation with the research group of Prof. Wessig at the University of Potsdam ligands were generated whose fluorescence lifetime change upon binding to histone deacetylases. By a displacement of the ligands from the active site the binding of HDAC inhibitors can be monitored. A developed competitive binding assay is outstandingly suited for high throughput screening applications and the determination of binding constants. Furthermore, the developed assay is also applicable for class IIa HDACs resulting in the first binding assay for this HDAC class. For the determination of kinetic parameters and the binding mechanism of the binding inhibitors to histone deacetylases an established fluorescence spectroscopic binding assay was modified, so that a time-resolved monitoring of the binding of nonfluorescent ligands is allowed. The determined kinetic traces were subjected to a global fit analysis. With this method the reaction mechanisms of the binding of four ligands to the human histone deacetylases 1, 6 and 8 as well as to a bacterial histone deacetylase-like amidohydrolase were evaluated. In the second part of this work it was evaluated on the basis of a histone deacetylase-like amidohydrolase from Pseudomonas aeruginosa, HDAHpa, which structural elements of HDAC inhibitors affect the selectivity and how mechanistic and thermodynamic parameters can be applied to assess the selectivity. Therefore, the reaction mechanisms and the thermodynamic signature of structurally related inhibitors of the known HDAC inhibitor N-hydroxy-N′-phenyloctanediamide (SAHA) were determined and evaluated for their selectivity against the human histone deacetylases 1–8. Generally, SAHA and other compounds with a hydroxamic acid as zinc binding moiety exhibit a good selectivity against class IIa HDACs. In contrast, the compound SATFMK, where the hydroxamic acid moiety is exchanged by a trifluoromethyl ketone moiety, exhibits a good to very good selectivity against the HDACs 1–3 and 6 but a lower selectivity against class IIa HDACs and HDAC8. With an at least 1000-fold selectivity over human HDACs the highest selectivity was determined for the compound PFSAHA. This compound exhibits, compared to SAHA and SATFMK, a sterically more demanding perflourinated spacer. Furthermore, it was determined in another study that upon an exchange of the cap group the high selectivity is preserved in most cases and can be improved by methylation and chlorination. Surprisingly, an analysis of the determined binding mechanisms suggests that PFSAHA and SATFMK bind each to other protein conformations as the other ligands. These results support the assumption that selective ligands can be identified on the basis of their binding mechanism. In order to deepen the knowledge about the molecular recognition of inhibitors by HDAHpa the binding reactions were studied by isothermal titration calorimetry. The determination of the thermodynamic parameters revealed that the binding of PFSAHA to HDAHpa occurs with unfavorable entropic contributions and is solely enthallpically driven, while for the binding of SATFMK the entropic and enthalpic contributions are balanced. This indicates indeed that for selective binders the enthalpic contributions are more pronounced. However, the enthalpic contributions to binding are of limited suitability for the prediction of the selectivity of HDAC inhibitors. Only a newly defined enthalpy weighted binding constant exhibits a good correlation to the determined selectivity of the HDAC inhibitors. Certainly, the applicability and transferability to other protein targets have yet to be evaluated. In a further study, the binding of PFSAHA and SATFMK to HDAHpa and a to HDAHpa homologous histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes were investigated in more detail by protein crystallography and isothermal titration calorimetry. It was shown, that the thermodynamic signature and the mechanism of the binding reaction is largely influenced by flexibility and accessibility of the active site. With a better accessibility and a higher flexibility the binding occurs in an apparent one-step reaction and with more favorable enthalpic contributions to binding. But these are balanced by enthalpy-entropy compensation resulting in an unchanged binding affinity. These findings seem to be exceptionally important for the development of HDAC inhibitors, since HDACs interact with several proteins and it is likely that these interactions alter the flexibility of the HDACs and with it the recognition of ligands. Taken together these studies indicate that generally a solely consideration of thermodynamic signatures is insufficient for the identification of selective compounds, but especially in combination with mechanistic investigations useful information about the potential selectivity of compounds are provided, which supplement existing parameters for the early identification of leads with a high probability of success.