Histone Deacetylase-like Protein Research Articles

Exploration of Novel Mono Hydroxamic Acid Derivatives as Inhibitors for Histone Deacetylase Like Protein (HDLP) by Molecular Dynamics Studies

The acetylation modification process of histone has an essential role in the epigenetic regulation of gene expression. This process is controlled by the balance between histone deacetylases (HDAC) and histone acetyltransferases (HAT). HDACs are thought to be vital for cell function. Particularly, higher HDAC expression is frequent in various cancers, resulting in the dysregulation of several target genes involved in cell proliferation, differentiation, and survival. In this study, the inhibitory feasibility of several HDAC inhibitors was investigated, including vorinostat (SAHA), N-hydroxy-3-phenylprop-2-enamide (CPD1), N-hydroxy-3-(pyridine-4-yl)prop-2-enamide (CPD2), N-hydroxy-3-(pyridine-2-yl)prop-2-enamide (CPD3), 4-(diphenylamino)-N-(5-(hydroxyamino)-5-oxopentyl)benzamide (CPD4), 2-(6-(((6-fluoronaphthalen-2-yl)methyl)amino)-3-azabicyclo[3.1.0]hex-3-yl)-N-hydroxypirimidine-5-carboxamide (CPD5), and N-(3-aminopropyl)-N-hydroxy-2-((naphthalene-1-yloxy)methyl)oct-2-enediamide (CPD6). By examining the stability of the enzyme, positional stability of the individual amino acids, and binding energies of HDLP-inhibitor complexes, the inhibitory feasibility was assessed. The complexes of the HDLP enzyme with SAHA, CPD4, CPD5, and CPD6 had higher stability than the other studied complexes, according to the results of trajectory analysis and the Ramachandran plot. Based on the calculated MM-PBSA binding free energies, the stability of the HDLP enzyme followed this order CPD4 > CPD5 > SAHA > CPD6 > CPD2 > CPD3 > CPD1. The drugability values followed the same trend as the previous ones. Based on the obtained in silico results, CPD4, CPD5, and CPD6 were discovered to be possible lead compounds as reference inhibitors of SAHA.

Driving the new generation histone deacetylase inhibitors in cancer therapy; manipulation of the histone abbreviation at the epigenetic level: an in-silico approach

Histone deacetylase (HDAC) is an enzyme that deacetylates the histone protein by removing the acetyl group from the lysine residues. The overexpression of the HDAC enzyme alters the gene expressions and causes cancer development in the human body. The inhibition of HDAC is an excellent therapeutic way in current cancer therapy. In this regard, various inhibitors were selected, and the inhibitory potential of these inhibitors was examined by molecular dynamics (MD) simulation followed by trajectory analysis and binding energy calculations. The selected clinical trial II and III phase inhibitors are TSA (trichostatin-A), TFMK (trifluoromethyl-ketone-9,9,9-trifluoro-8-oxo- N-phenylnonanamide), AKA (alpha-ketoamide- N-cyclohexyl- N-methyl-2-oxononanediamide), ITF2357 (givinostat), MS275 (entinostat), CI994 (tacedinaline), and SAHA (suberoylanilide hydroxamic acid). This computational study examines the atomic level description of the drug binding site on the HDLP enzyme and investigates the interaction of the HDAC inhibitors with the amino acid residues attached to the active site of the histone deacetylase-like protein (HDLP). Root-mean-square deviation, radius of gyration, hydrogen bond analysis, MM-PBSA, linear interaction energy (LIE), and semi-LIE calculations have revealed that the HDLP enzyme is more stabilized when bound to TSA, ITF2357, and reference inhibitor SAHA. It was observed that the hydroxamic acid family inhibitors have more potent in inhibiting the HDLP enzyme than the benzamide and ketone families. The inhibitory efficacy of TSA and ITF2357 is much similar to that of SAHA. Therefore, these HDAC inhibitors have the potential to be used in future clinical practices for cancer-related treatments. The knowledge gathered from this study could also lead to discovering new HDAC inhibitors for clinical research.

AnIn-SilicoApproach to Evaluate the Inhibitory Potency of Selected Hydroxamic Acid Derivatives on Zinc-Dependent Histone Deacetylase Enzyme

Histone deacetylase (HDAC) enzymes modify the histone by removing the acetyl group from the lysine residues, known as histone deacetylation. HDACs have been involved in altering gene expressions, resulting in cancer cells in the body. This study focuses on HDAC inhibitors’ impact on histone deacetylase-like protein (HDLP) stability through computational techniques. Molecular dynamics (MD) analyses were used to examine the atomic-level description of drug binding sites and how the HDAC inhibitors change the HDLP enzyme environment. In this study, two hydroxamic acid-derived inhibitors, such as [Formula: see text]-Carboxycinnamic acid bis-hydroxamide (CBHA) and scriptaid (GCK1026), were selected to examine the inhibition ability in terms with suberanilohydroxamic acid (SAHA) as a reference drug. The crystal structure of the HDLP was downloaded from the Protein Data Bank. The structures of inhibitors were optimized using the G09W package. Docking studies were done by AutoDock-Vina, and the resultant complex was used to initiate MD studies. The trajectories obtained from MD simulation were used to perform the structural analysis. Root-mean-square deviation (RMSD), radius of gyration, hydrogen bond, binding free energy and interaction energy studies revealed that the stability of HDLP-SAHA and HDLP-CBHA is higher than the free HDLP enzyme. The HDLP-CBHA complex shows an increased number of hydrogen bonds (5), high MM-PBSA binding free energy ([Formula: see text][Formula: see text]kJ/mol), high interaction energy ([Formula: see text][Formula: see text]kJ/mol), and an increased number of alpha-helical amino acids (130) compared with HDLP-SAHA. It concluded that the CBHA has the relatively same potential as SAHA to inhibit the HDLP. Consequently, the use of CBHA in clinical application is recommended through this in-silico method.

Design, synthesis and biological evaluation of N-phenylquinazolin-4-amine hybrids as dual inhibitors of VEGFR-2 and HDAC

A single agent that simultaneously inhibits multiple targets may offer greater therapeutic benefits in cancer than single-acting agents through interference with multiple pathways and potential synergistic action. In this work, a series of hybrids bearing N-phenylquinazolin-4-amine and hydroxamic acid moieties were designed and identified as dual VEGFR-2/HDAC inhibitors. Compound 6fd exhibited the most potent inhibitory activity against HDAC with IC50 of 2.2 nM and strong inhibitory effect against VEGFR-2 with IC50 of 74 nM. It also showed the most potent inhibitory activity against a human breast cancer cell line MCF-7 with IC50 of 0.85 μM. Docking simulation supported the initial pharmacophoric hypothesis and suggested a common mode of interaction at the active binding sites of VEGFR-2 and HDLP ((Histone Deacetylase-Like Protein), which demonstrates that compound 6fd is a potential agent for cancer therapy deserving further researching.

Design, Synthesis, and Biological Activity of Hydroxamic Tertiary Amines as Histone Deacetylase Inhibitors

Herein we describe the design, synthesis, and biological evaluation of new hydroxamic tertiary amines as histone deacetylase (HDAC) inhibitors. These compounds have allowed us to clarify the influence of cap group dimension and hydrophobicity on HDAC inhibitory activity. This report also reveals the recognition pattern between the linear compounds and the histone deacetylase-like protein (HDLP) model receptor, and discusses the synthesis and in vitro evaluation of HDAC inhibitory activity in HeLa cell nuclear extracts. We obtained good qualitative agreement between experimental results and theoretical predictions, confirming that appropriately substituted hydroxamic tertiary amines are potential active HDAC inhibitors.

Synthesis, antiproliferation, and docking studies of N-phenyl-lipoamide and 8-mercapto-N-phenyloctanamide derivatives: effects of C6 position thiol moiety

Some N-phenyl lipoamide and 8-mercapto-N-phenyloctanamide derivatives were synthesized and their in vitro antiproliferative activity was evaluated. The experimental results indicated that 8-mercapto-N-phenyloctanamides might be good histone deacetylase inhibitors rather than N-phenyl lipoamides, who had thiol moiety at C6 position. To verify the antiproliferation data on structural basis, in silico docking studies of the representative compounds into the crystal structure of histone deacetylase-like protein using AutoDock 4.0 program were performed. Furthermore, sulfur acetylated 8-mercapto-N-phenyloctanamide improved its in vitro antiproliferative activity, probably due to the increasing of its cell membrane permeability. While the identification of enzymatic target of N-phenyl lipoamides with dithiolane is still ongoing.

Synthesis of new mono and bis amides projected as potential histone deacetylase (HDAC) inhibitors

In our ongoing efforts to discover new potent histone deacetylase (HDAC) inhibitors as promising anticancer candidates, we designed and synthesized a small collection of 3-substituted amines possessing macro heterocyclic skeletons bearing variable-length tails. As a metal binder domain, all the compounds possess an amide function suitable for Zn2+ chelation in the enzyme active site. A combination of solution and solid phase techniques were employed to synthesize the compounds and, as the key synthetic step to obtain the rings, a ring closing metathesis (RCM) reaction was adopted. The putative affinity of the compounds for the histone deacetylase-like protein (HDLP) model receptor active site was explored through docking calculations, and we also report preliminary studies on their pharmacological properties.

Comparative molecular dynamics simulations of histone deacetylase‐like protein: Binding modes and free energy analysis to hydroxamic acid inhibitors

Histone deacetylases (HDACs) play an important role in gene transcription, and inhibitors of HDACs can induce cell differentiation and suppress cell proliferation in tumor cells. Histone deacetylase1 (HDAC1) binds suberanilohydroxamic acid (SAHA) and 7-phenyl-2, 4, 6-hepta-trienoyl hydroxamic acid (CG-1521) with moderately low affinity (DeltaG = -8.6 and -7.8 kcal mol(-1)). The structurally related (E)-2-(3-(3-(hydroxyamino)-3-oxoprop-1-enyl)phenyl)-N(1),N(3)-diphenylmalonamide (SK-683), a Trichostatin A (TSA)-like HDAC1 inhibitor, and TSA are bound to the HDAC1 with -12.3 and -10.3 kcal mol(-1) of DeltaG, higher binding free energies than SAHA and CG-1521. Histone deacetylase-like protein (HDLP), an HDAC homologue, shows a 35.2% sequence identity of HDLP and human HDAC1. Molecular dynamics simulation and the molecular mechanics/generalized-Born surface area (MM-GBSA) free energy calculations were applied to investigate the factors responsible for the relatively activity of these four inhibitors to HDLP. In addition, computational alanine scanning of the binding site residues was carried out to determine the contribution components from van der Waals, electrostatic interaction, nonpolar and polar energy of solvation as well as the effects of backbones and side-chains with the MM-GBSA method. MM-GBSA methods reproduced the experimental relative affinities of the four inhibitors in good agreement (R(2) = 0.996) between experimental and computed binding energies. The MM-GBSA calculations showed that, the number of hydrogen bonds formed between the HDLP and inhibitors, which varied in the system studied, and electrostatic interactions determined the magnitude of the free energies for HDLP-inhibitor interactions. The MM-GBSA calculations revealed that the binding of HDLP to these four hydroxamic acid inhibitors is mainly driven by van der Waals/nonpolar interactions. This study can be a guide for the optimization of HDAC inhibitors and future design of new therapeutic agents for the treatment of cancer.

Functional analysis of a histone deacetylase-like protein of Thermus caldophilus GK24 in mammalian cell

The function of eukaryotic histone deacetylase (HDAC) has been extensively studied for its critical role in transcriptional regulation and carcinogenesis. However that of the prokaryotic counterpart remains largely unknown. Recently, we cloned HDAC-like protein in Thermus caldophilus GK24 ( Tca HDAC) from a genomic library of the microorganism based on homology analysis with human HDAC1. To explore the function of Tca HDAC in mammalian cells, Tca HDAC gene expressing vector was transfected into a human fibrosarcoma cell line, HT1080. Tca HDAC was mainly localized in nuclei of the mammalian cells as a human HDAC1 was, due to an N-terminal HDAC association domain. We further generated histidine-substituted Tca HDAC mutants and investigated their role in biochemical and cellular activity of the enzyme. Tca HDAC mutants exhibited dramatic loss of enzymatic activity and conditioned media (CM) from HT1080 cells transfected with mutant Tca HDAC was unable to stimulate angiogenic phenotypes of endothelial cells in vitro whereas that of wild Tca HDAC did. Collectively, these results demonstrate that a prokaryotic histone deacetylase from T. caldophilus GK24 is functionally active in mammalian cells and its function in gene expression is conserved from prokaryotes to eukaryotes.

Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8–substrate complex

Histone deacetylases (HDACs)-an enzyme family that deacetylates histones and non-histone proteins-are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 A resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor-HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.

Cloning, expression, and biochemical characterization of a new histone deacetylase-like protein from Thermus caldophilus GK24

Histone deactylases (HDACs) are members of an ancient enzyme family found in eukaryotes as well as in prokaryotes such as archaebacteria and eubacteria. We here report a new histone deacetylase ( Tca HDAC) that was cloned from the genomic library of Thermus caldophilus GK24 based on homology analysis with human histone deacetylase1 (HDAC1). The gene contains an open reading frame encoding 375 amino acids with a calculated molecular mass of 42,188 Da and the deduced amino acid sequence of Tca HDAC showed a 31% homology to human HDAC1. The Tca HDAC gene was over-expressed in Escherichia coli using a Glutathione-S transferase (GST) fusion vector (pGEX-4T-1) and the purified protein showed a deacetylase activity toward the fluorogenic substrate for HDAC. Moreover, the enzyme activity was inhibited by trichostatin A, a specific HDAC inhibitor, in a dose-dependent manner. Optimum temperature and pH of the enzyme was found to be approximately 70 °C and 7.0, respectively. In addition, zinc ion is required for catalytic activity of the enzyme. Together, these data demonstrate that Tca HDAC is a new histone deacetylase-like enzyme from T. caldophilus GK24 and will be a useful tool for deciphering the role of HDAC in the prokaryote and development of new biochemical reactions.

Molecular Insights into Azumamide E Histone Deacetylases Inhibitory Activity

Azumamide E, a cyclotetrapeptide isolated from the sponge Mycale izuensis, is the most powerful carboxylic acid containing natural histone deacetylase (HDAC) inhibitor known to date. In this paper, we describe design and synthesis of two stereochemical variants of the natural product. These compounds have allowed us to clarify the influence of side chain topology on the HDAC-inhibitory activity. The present contribution also reveals the identity of the recognition pattern between azumamides and the histone deacetylase-like protein (HDLP) model receptor and reports the azumamide E unprecedented isoform selectivity on histone deacetylases class subtypes. From the present studies, a plausible model for the interaction of azumamides with the receptor binding pocket is derived, providing a framework for the rational design of new cyclotetrapeptide-based HDAC inhibitors as antitumor agents.

Unexpected Deacetylation Mechanism Suggested by a Density Functional Theory QM/MM Study of Histone-Deacetylase-Like Protein

To characterize the catalytic mechanism for zinc-dependent histone deacetylases (HDAC), we have carried out density functional theory QM/MM studies on the deacetylation reaction catalyzed by a histone-deacetylase-like protein (HDLP). The calculation results do not support the previous mechanistic hypothesis, but suggest a lower protonation state for the active site as well as a 4-fold zinc coordination during the reaction process. To characterize such mechanistic difference is not only significant for our fundamental understanding of its inner workings but also crucial for the design of HDAC inhibitors.

Structure-Based Optimization of Phenylbutyrate-Derived Histone Deacetylase Inhibitors

Previously, we developed a strategy to develop a novel class of histone deacetylase (HDAC) inhibitors by tethering short-chain fatty acids with Zn(2+)-chelating motifs, which led to N-hydroxy-4-(4-phenylbutyryl-amino)benzamide (HTPB), a hydroxamate-tethered phenylbutyrate derivative with sub-micromolar potency in inhibiting HDAC activity and cancer cell proliferation. In this study, we carried out structure-based optimization of HTPB by using the framework generated by the structure of histone deacetylase-like protein (HDLP)-trichostatin A (TSA) complexes. Docking of HTPB into the HDLP binding domain suggested that the hydrophobic microenvironment encompassed by Phe-198 and Phe-200 could be exploited for structural optimization. This premise was corroborated by the greater potency of (S)-(+)-N-hydroxy-4-(3-methyl-2-phenylbutyrylamino)-benzamide [(S)-11] (IC(50) in HDAC inhibition, 16 nM), of which the isopropyl moiety was favorable in interacting with this hydrophobic motif. (S)-11 at concentrations as low as 0.1 microM was effective in causing histone hyperacetylation and p21(WAF/CIP1) overexpression and suppressing proliferation in cancer cells.

On the function of the 14 A long internal cavity of histone deacetylase-like protein: implications for the design of histone deacetylase inhibitors.

Histone deacetylases (HDACs) play an important role in gene transcription. Inhibitors of HDACs induce cell differentiation and suppress cell proliferation in tumor cells. AutoDock calculations of known and novel HDAC inhibitors as well as of several probe molecules to histone deacetylase-like protein (HDLP), using a modified scoring function for metalloproteins, demonstrate excellent agreement (R = 0.92) between experimental and computed binding constants. Analysis of the docked structures allows a determination of the different binding motifs in known inhibitors. Such calculations are a useful tool for the prediction of binding constants for new HDAC inhibitors. Exploration of the 14 A long internal cavity adjacent to the active site by docking of small molecular probes suggest that it plays a crucial role by accepting the cleaved acetate and releasing it at the far side of the cavity. The importance of the findings for the design of new inhibitors is discussed.

Docking Study of HDAC Implication for Benzamide Inhibitors Binding Mode

The paper proposed a possible binding mode of MS-275, a benzamide historic deacetylase(HDAC) inhibitor, to HDAC by intensive docking study. This binding mode is different from those observed in the crystal structure of complexes formed by a histone deacetylase-like protein (HDLP) with trichostatin A(TSA) or suberoylanilide hydroxamic acid (SARA). The docking result implicates that the main target of MS-275 is the narrowest part of HDAC active pocket. It seems to be able to explain the low toxicity of MS-275 and provides new insights on the design of novel HDAC inhibitors.

Ab initio study of the binding of Trichostatin A (TSA) in the active site of histone deacetylase like protein (HDLP).

Histone deacetylase (HDAC) inhibitors have recently attracted considerable interest because of their therapeutic potential for the treatment of cell proliferative diseases. An X-ray structure of a very potent inhibitor, Trichostatin A (TSA), bound to HDLP (an HDAC analogue isolated from Aquifex aeolicus), is available. From this structure, an active site model (322 atoms), relevant for the binding of TSA and structural analogues, has been derived, and TSA has been minimized in this active site at HF 3-21G* level. The resulting conformation is in excellent accordance with the X-ray structure, and indicates a deprotonation of the hydroxamic acid in TSA by His 131. Also, a water molecule was minimized in the active site. In addition to a similar deprotonation, in accordance with a possible catalytic mechanism of HDAC as proposed by Finnin et al. (M. S. Finnin, J. R. Donigian, A. Cohen, V. M. Richon, R. A. Rifkind and P. A. Marks, Nature, 1999, 401, 188-193), a displacement of the resulting OH- ion in the active site was observed. Based on these results, the difference in energy of binding between TSA and water was calculated. The resulting value is realistic in respect to experimental binding affinities. Furthermore, the mechanism of action of the His 131-Asp 166 charge relay system was investigated. Although the Asp residue in this motif is known to substantially increase the basicity of the His residue, no proton transfer from His 131 to Asp 166 was observed on binding of TSA or water. However, in the empty protonated active site, this proton transfer does occur.

Binding mode analysis of 3-(4-benzoyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide: a new synthetic histone deacetylase inhibitor inducing histone hyperacetylation, growth inhibition, and terminal cell differentiation.

The binding mode of 3-(4-aroyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides 1a-c, belonging to a recently reported class of synthetic histone deacetylase (HDAC) inhibitors (Massa, S.; et al. J. Med. Chem. 2001, 44, 2069-2072), into the new modeled HDAC1 catalytic core is presented, and enzyme/inhibitor interactions are discussed. HDAC1 X-ray coordinates were obtained by virtual "mutation" of those of histone deacetylase-like protein, a bacterial HDAC homologue. In in vitro antimaize HD2 as well as antimouse HDAC1 assay, compounds 1a-c showed inhibitory activities in the low micromolar range. Similarly, 1a-c are endowed with anti-HDAC activity in vivo: on mouse A20 cells, 1a-c induced histone hyperacetylation leading to a highly increased acetylation level of H4 as compared to control histones. Results obtained with acid-urea-triton polyacrylamide gel electrophoresis have been confirmed by Western Blot experiments. Finally, compound 1a, chosen as a representative member of this class of HDAC inhibitors, resulted endowed with antiproliferative (45 and 85% cell growth inhibition at 40 and 80 microM, respectively) and cellular differentiation (18 and 21% of benzidine positive cells at the same concentrations) activities in murine erythroleukemic cells.

3-(4-aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamides, a new class of synthetic histone deacetylase inhibitors.

Novel 3-(4-aroyl-2-pyrrolyl)-N-hydroxy-2-propenamides are disclosed as a new class of histone deacetylase (HDAC) inhibitors. Three-dimensional structure-based drug design and conformational analyses into the histone deacetylase-like protein (HDLP) catalytic core suggested the synthesis and biological evaluation of compounds 7a-h. Experimental pK(i) values are in good agreement with VALIDATE predicted pK(i) values of new derivatives. All compounds 7a-h show HDAC inhibitory activity in the micromolar range, with 7e as the most potent derivative (IC(50) = 1.9 microM). The influence of the 4'-substituent in the aroyl moiety is not significant for the inhibitory activity, as all compounds 7a-g show IC(50) values between 1.9 and 3.9 microM. Otherwise, the unsaturated chain linking the pyrrole ring to the hydroxamic acid group is clearly important for the anti-HDAC activity, the saturated analogue 7h being 10-fold less active than the unsaturated counterpart 7a.