The Importance of the "Time Factor" for the Evaluation of Inhibition Mechanisms: The Case of Selected HDAC6 Inhibitors.

Histone deacetylases (HDACs) participate with histone acetyltransferases in the modulation of the biological activity of a broad array of proteins, besides histones. Histone deacetylase 6 is unique among HDAC as it contains two catalytic domains, an N-terminal microtubule binding region and a C-terminal ubiquitin binding domain. Most of its known biological roles are related to its protein lysine deacetylase activity in the cytoplasm. The design of specific inhibitors is the focus of a large number of medicinal chemistry programs in the academy and industry because lowering HDAC6 activity has been demonstrated to be beneficial for the treatment of several diseases, including cancer, and neurological and immunological disorders. Here, we show how re-evaluation of the mechanism of action of selected HDAC6 inhibitors, by monitoring the time-dependence of the onset and relief of the inhibition, revealed instances of slow-binding/slow-release inhibition. The same approach, in conjunction with X-ray crystallography, in silico modeling and mass spectrometry, helped to propose a model of inhibition of HDAC6 by a novel difluoromethyloxadiazole-based compound that was found to be a slow-binding substrate analog of HDAC6, giving rise to a tightly bound, long-lived inhibitory derivative.

Difluoromethyl-1,3,4-oxadiazoles are slow-binding substrate analog inhibitors of histone deacetylase 6 with unprecedented isotype selectivity.

Histone deacetylase 6 (HDAC6) is an attractive drug development target because of its role in the immune response, neuropathy, and cancer. Knockout mice develop normally and have no apparent phenotype, suggesting that selective inhibitors should have an excellent therapeutic window. Unfortunately, current HDAC6 inhibitors have only moderate selectivity and may inhibit other HDAC subtypes at high concentrations, potentially leading to side effects. Recently, substituted oxadiazoles have attracted attention as a promising novel HDAC inhibitor chemotype, but their mechanism of action is unknown. Here, we show that compounds containing a difluoromethyl-1,3,4-oxadiazole (DFMO) moiety are potent and single-digit nanomolar inhibitors with an unprecedented greater than 104-fold selectivity for HDAC6 over all other HDAC subtypes. By combining kinetics, X-ray crystallography, and mass spectrometry, we found that DFMO derivatives are slow-binding substrate analogs of HDAC6 that undergo an enzyme-catalyzed ring opening reaction, forming a tight and long-lived enzyme-inhibitor complex. The elucidation of the mechanism of action of DFMO derivatives paves the way for the rational design of highly selective inhibitors of HDAC6 and possibly of other HDAC subtypes as well with potentially important therapeutic implications.

HDAC Inhibition as Potential Therapeutic Strategy to Restore the Deregulated Immune Response in Severe COVID-19.

The COVID-19 pandemic has had a devastating impact worldwide and has been a great challenge for the scientific community. Vaccines against SARS-CoV-2 are now efficiently lessening COVID-19 mortality, although finding a cure for this infection is still a priority. An unbalanced immune response and the uncontrolled release of proinflammatory cytokines are features of COVID-19 pathophysiology and contribute to disease progression and worsening. Histone deacetylases (HDACs) have gained interest in immunology, as they regulate the innate and adaptative immune response at different levels. Inhibitors of these enzymes have already proven therapeutic potential in cancer and are currently being investigated for the treatment of autoimmune diseases. We thus tested the effects of different HDAC inhibitors, with a focus on a selective HDAC6 inhibitor, on immune and epithelial cells in in vitro models that mimic cells activation after viral infection. Our data indicate that HDAC inhibitors reduce cytokines release by airway epithelial cells, monocytes and macrophages. This anti-inflammatory effect occurs together with the reduction of monocytes activation and T cell exhaustion and with an increase of T cell differentiation towards a T central memory phenotype. Moreover, HDAC inhibitors hinder IFN-I expression and downstream effects in both airway epithelial cells and immune cells, thus potentially counteracting the negative effects promoted in critical COVID-19 patients by the late or persistent IFN-I pathway activation. All these data suggest that an epigenetic therapeutic approach based on HDAC inhibitors represents a promising pharmacological treatment for severe COVID-19 patients.

Role of Fluorination in the Histone Deacetylase 6 (HDAC6) Selectivity of Benzohydroxamate-Based Inhibitors.

Nonselective histone deacetylase (HDAC) inhibitors show dose-limiting side effects due to the inhibition of multiple, essential HDAC subtypes that can be limited or prevented by restricting their selectivity. We herein report the crystal structures of zebrafish HDAC6 catalytic domain 2 (zHDAC6-CD2) in complex with the selective HDAC6 inhibitors ITF3756 and ITF3985 and shed light on the role of fluorination in the selectivity of benzohydroxamate-based structures over class I isoforms. The reason for the enhancement in the selectivity of the benzohydroxamate-based compounds is the presence of specific interactions between the fluorinated linker and the key residues Gly582, Ser531, and His614 of zHDAC6, which are hindered in class I HDAC isoforms by the presence of an Aspartate that replaces Ser531. These results can be used in the design and development of novel, highly selective HDAC6 inhibitors.

Novel Benzohydroxamate-Based Potent and Selective Histone Deacetylase 6 (HDAC6) Inhibitors Bearing a Pentaheterocyclic Scaffold: Design, Synthesis, and Biological Evaluation.

Histone deacetylase 6 (HDAC6) is a peculiar HDAC isoform whose expression and functional alterations have been correlated with a variety of pathologies such as autoimmune disorders, neurodegenerative diseases, and cancer. It is primarily a cytoplasmic protein, and its deacetylase activity is focused mainly on nonhistone substrates such as tubulin, heat shock protein (HSP)90, Foxp3, and cortactin, to name a few. Selective inhibition of HDAC6 does not show cytotoxic effects in healthy cells, normally associated with the inhibition of Class I HDAC isoforms. Here, we describe the design and synthesis of a new class of potent and selective HDAC6 inhibitors that bear a pentaheterocyclic central core. These compounds show a remarkably low toxicity both in vitro and in vivo and are able to increase the function of regulatory T cells (Tregs) at well-tolerated concentrations, suggesting a potential clinical use for the treatment of degenerative, autoimmune diseases and for organ transplantation.

Amino acidic scaffolds bearing unnatural side chains: an old idea generates new and versatile tools for the life sciences.

The unnatural amino acids (UAAs) are members of a class of molecules with relevant impacts in the life sciences. Due to the role of these molecules in the modulation of the chemical and physical properties of biological and inorganic materials, UAAs have attracted increasing interest in recent years. The aim of this review is to highlight (i) the most recent and innovative synthetic routes for the preparation of UAAs, (ii) the recently marketed UAA-based drugs, and (iii) the most promising technological applications involving novel UAA-containing molecular entities.

Cyclic RGD peptidomimetics containing bifunctional diketopiperazine scaffolds as new potent integrin ligands.

The synthesis of eight bifunctional diketopiperazine (DKP) scaffolds is described; these were formally derived from 2,3-diaminopropionic acid and aspartic acid (DKP-1-DKP-7) or glutamic acid (DKP-8) and feature an amine and a carboxylic acid functional group. The scaffolds differ in the configuration at the two stereocenters and the substitution at the diketopiperazinic nitrogen atoms. The bifunctional diketopiperazines were introduced into eight cyclic peptidomimetics containing the Arg-Gly-Asp (RGD) sequence. The resulting RGD peptidomimetics were screened for their ability to inhibit biotinylated vitronectin binding to the purified integrins α(v)ß(3) and α(v)ß(5), which are involved in tumor angiogenesis. Nanomolar IC(50) values were obtained for the RGD peptidomimetics derived from trans DKP scaffolds (DKP-2-DKP-8). Conformational studies of the cyclic RGD peptidomimetics by (1)H NMR spectroscopy experiments (VT-NMR and NOESY spectroscopy) in aqueous solution and Monte Carlo/Stochastic Dynamics (MC/SD) simulations revealed that the highest affinity ligands display well-defined preferred conformations featuring intramolecular hydrogen-bonded turn motifs and an extended arrangement of the RGD sequence [Cß(Arg)-Cß(Asp) average distance ≥8.8 Å]. Docking studies were performed, starting from the representative conformations obtained from the MC/SD simulations and taking as a reference model the crystal structure of the extracellular segment of integrin α(v)ß(3) complexed with the cyclic pentapeptide, Cilengitide. The highest affinity ligands produced top-ranked poses conserving all the important interactions of the X-ray complex.