Identification and optimisation of a series of substituted 5-(1H-pyrazol-3-yl)-thiophene-2-hydroxamic acids as potent histone deacetylase (HDAC) inhibitors

Optimisation of ADS100380, a sub-micromolar HDAC inhibitor identified using a virtual screening approach, led to a series of substituted 5-(1H-pyrazol-3-yl)-thiophene-2-hydroxamic acids (6a-i), that possessed significant HDAC inhibitory activity. Subsequent functionalisation of the pendent phenyl group of compounds 6f and 6g provided analogues 6j-w with further enhanced enzyme and anti-proliferative activity. Compound 6j demonstrated efficacy in a mouse xenograft experiment.     

Factors affecting the substrate specificity of histone deacetylases

Histone deacetylases (HDACs) catalyze the deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones and thereby mediate changes in the chromatin structure and regulate gene expression in eukaryotic cells. So far, surprisingly little is known about the substrate specificities of different HDACs. Here, we prepared a library of fluorogenic tripeptidic substrates of the general format Ac-P(-2)-P(-1)-Lys(Ac)-MCA (P(-1), P(-2)=all amino acids except cysteine) and measured their HDAC-dependent conversion in a standard fluorogenic HDAC assay. Different HDAC subtypes can be ranked according to their substrate selectivity: HDAH > HDAC8 > HDAC1 > HDAC3 > HDAC6. HDAC1, HDAC3, and HDAC6 exhibit a similar specificity profile, whereas both HDAC8 and HDAH have rather distinct profiles. Furthermore, it was shown that second-site modification (e.g., phosphorylation) of substrate sequences as well as corepressor binding can modulate the selectivity of enzymatic substrate conversion.     

Inhibitors of histone deacetylase 6 based on a novel 3-hydroxy-isoxazole zinc binding group

Histone deacetylase 6 (HDAC6) is an established drug target for cancer treatment. Inhibitors of HDAC6 based on a hydroxamic acid zinc binding group (ZBG) are often associated with undesirable side effects. Herein, we describe the identification of HDAC6 inhibitors based on a completely new 3-hydroxy-isoxazole ZBG. A series of derivatives decorated with different aromatic or heteroaromatic linkers, and various cap groups were synthesised and biologically tested. In vitro tests demonstrated that some compounds are able to inhibit HDAC6 with good potency, the best candidate reaching an IC₅₀ of 700 nM. Such good potency obtained with a completely new ZBG make these compounds particularly attractive. The effect of the most active inhibitors on the acetylation levels of histone H3 and α- tubulin and their anti-proliferative activity of DU145 cells were also investigated. Docking studies were performed to evaluate the binding mode of these new derivatives and discuss structure-activity relationships.     

Amide-based derivatives of β-alanine hydroxamic acid as histone deacetylase inhibitors: Attenuation of potency through resonance effects

A library of amide-linked derivatives of β-alanine hydroxamic acid were prepared (2-7) and the activity as inhibitors of Zn(II)-containing histone deacetylases (HDACs) determined in vitro against HDAC1 and the anti-proliferative activity determined in BE(2)-C neuroblastoma cells. The IC(50) values of the best-performing compounds (3-7) against HDAC1 ranged between 38 and 84μM. The least potent compound (2) inhibited a maximum of only 40% HDAC1 activity at 250μM. The anti-proliferative activity of 2-7 at 50μM against BE(2)-C neuroblastoma cells ranged between 57.0% and 88.6%. The structural similarity between the potent HDAC inhibitor trichostatin A (TSA, 1; HDAC1, IC(50) 12nM) and the present compounds (2-7) was high at the Zn(II) coordinating hydroxamic acid head group; and in selected compounds (2, 5), at the 4-(dimethylamino)phenyl tail. The significantly reduced potency of 2-7 relative to 1 underscores the rank importance of the linker region as part of the HDAC inhibitor pharmacophore. Molecular modeling of 1-7 using HDAC8 as the template suggested that the conformationally constrained 4'-methyl group of 1 may contribute to HDAC inhibitor potency through a sandwich-like interaction with a hydrophobic region containing F152 and F208; and that the absence of this group in 2-7 may reduce potency. The close proximity of the 5'-carbonyl oxygen atom in 2-7 to the sulfur atom of Met274 in HDAC8 or the corresponding isobutyl group of Leu274 in HDAC1 may attenuate potency through repulsive steric and dipole-dipole forces. In a unique resonance stabilized form of 2, this interaction could manifest as stronger ion-dipole repulsive forces, resulting in a further decrease in potency. This work suggests that resonance structures of HDAC inhibitors could modulate intermolecular interactions with HDAC targets, and potency.     

2-Benzazolyl-4-Piperazin-1-Ylsulfonylbenzenecarbohydroxamic Acids as Novel Selective Histone Deacetylase-6 Inhibitors with Antiproliferative Activity

We have screened our compound collection in an established cell based assay that measures the derepression of an epigenetically silenced transgene, the locus derepression assay. The screen led to the identification of 4-[4-(1-methylbenzimidazol-2-yl)piperazin-1-yl]sulfonylbenzenecarbohydroxamic acid (9b) as an active which was found to inhibit HDAC1. In initial structure activity relationships study, the 1-methylbenzimidazole ring was replaced by the isosteric heterocycles benzimidazole, benzoxazole, and benzothiazole and the position of the hydroxamic acid substituent on the phenyl ring was varied. Whereas compounds bearing a para substituted hydroxamic acid (9a-d) were active HDAC inhibitors, the meta substituted analogues (8a-d) were appreciably inactive. Compounds 9a-d selectively inhibited HDAC6 (IC₅₀ = 0.1–1.0μM) over HDAC1 (IC₅₀ = 0.9–6μM) and moreover, also selectively inhibited the growth of lung cancer cells vs. patient matched normal cells. The compounds induce a cell cycle arrest in the S-phase while induction of apoptosis is neglible as compared to controls. Molecular modeling studies uncovered that the MM-GBSA energy for interaction of 9a-d with HDAC6 was higher than for HDAC1 providing structural rationale for the HDAC6 selectivity.     

The discovery of novel HDAC3 inhibitors via virtual screening and in vitro bioassay

Histone deacetylase 3 (HDAC3) is a potential target for the treatment of human diseases such as cancers, diabetes, chronic inflammation and neurodegenerative diseases. Previously, we proposed a virtual screening (VS) pipeline named “Hypo1_FRED_SAHA-3” for the discovery of HDAC3 inhibitors (HDAC3Is) and had thoroughly validated it by theoretical calculations. In this study, we attempted to explore its practical utility in a large-scale VS campaign. To this end, we used the VS pipeline to hierarchically screen the Specs chemical library. In order to facilitate compound cherry-picking, we then developed a knowledge-based pose filter (PF) by using our in-house quantitative structure activity relationship- (QSAR-) modelling approach and coupled it with FRED and Autodock Vina. Afterward, we purchased and tested 11 diverse compounds for their HDAC3 inhibitory activity in vitro. The bioassay has identified compound 2 (Specs ID: AN-979/41971160) as a HDAC3I (IC₅₀?=?6.1?μM), which proved the efficacy of our workflow. As a medicinal chemistry study, we performed a follow-up substructure search and identified two more hit compounds of the same chemical type, i.e. 2–1 (AQ-390/42122119, IC₅₀?=?1.3?μM) and 2–2 (AN-329/43450111, IC₅₀?=?12.5?μM). Based on the chemical structures and activities, we have demonstrated the essential role of the capping group in maintaining the activity for this class of HDAC3Is. In addition, we tested the hit compounds for their in vitro activities on other HDACs, including HDAC1, HDAC2, HDAC8, HDAC4 and HDAC6. We have identified these compounds are HDAC1/2/3 selective inhibitors, of which compound 2 show the best selectivity profile. Taken together, the present study is an experimental validation and an update to our earlier VS strategy. The identified hits could be used as starting structures for the development of highly potent and selective HDAC3Is.     

Deactylase inhibitors disrupt cellular complexes containing protein phosphatases and deacetylases

Affinity isolation of protein serine/threonine phosphatases on the immobilized phosphatase inhibitor microcystin-LR identified histone deacetylase 1(HDAC1), HDAC6, and HDAC10 as novel components of cellular phosphatase complexes. Other HDACs, specifically HDAC2, -3, -4, and -5, were excluded from such complexes. In vitro biochemical studies showed that recombinant HDAC6, but not HDAC4, bound directly to the protein phosphatase (PP)1 catalytic subunit. No association was observed between HDAC6 and PP2A, another major protein phosphatase. PP1 binding was mapped to the second catalytic domain and adjacent C-terminal sequences in HDAC6, and treatment of cells with trichostatin A (TSA) disrupted endogenous HDAC6.PP1 complexes. Consistent with the inhibition of tubulin deactylase activity of HDAC6, TSA enhanced cellular tubulin acetylation, and acetylated tubulin was present in the PP1 complexes from TSA-treated cells. Trapoxin B, a weak HDAC6 inhibitor, and calyculin A, a cell-permeable phosphatase inhibitor, had no effect on the stability of the HDAC6.PP1 complexes or on tubulin acetylation. Mutations that inactivated HDAC6 prevented its incorporation into cellular PP1 complexes and suggested that when bound together both enzymes were active. Interestingly, TSA disrupted all the cellular HDAC.phosphatase complexes analyzed. This study provided new insight into the mechanism by which HDAC inhibitors elicited coordinate changes in cellular protein phosphorylation and acetylation and suggested that changes in these protein modifications at multiple subcellular sites may contribute to the known ability of HDAC inhibitors to suppress cell growth and transformation.     

Novel Interaction of Class IIb Histone Deacetylase 6 (HDAC6) with Class IIa HDAC9 Controls Gonadotropin Releasing Hormone (GnRH) Neuronal Cell Survival and Movement

The impact of histone deacetylases (HDACs) in the control of gonadotropin releasing hormone (GnRH) neuronal development is unknown. We identified an increase in many HDACs in GT1-7 (differentiated) compared with NLT (undifferentiated) GnRH neuronal cell lines. Increased HDAC9 mRNA and protein and specific deacetylase activity in GT1-7 cells suggested a functional role. Introduction of HDAC9 in NLT cells protected from serum withdrawal induced apoptosis and impaired basal neuronal cell movement. Conversely, silencing of endogenous HDAC9 in GT1-7 cells increased apoptosis and cell movement. Comparison of WT and mutant HDAC9 constructs demonstrated that the HDAC9 pro-survival effects required combined cytoplasmic and nuclear localization, whereas the effects on cell movement required a cytoplasmic site of action. Co-immunoprecipitation demonstrated a novel interaction of HDAC9 selectively with the Class IIb HDAC6. HDAC6 was also up-regulated at the mRNA and protein levels, and HDAC6 catalytic activity was significantly increased in GT1-7 compared with NLT cells. HDAC9 interacted with HDAC6 through its second catalytic domain. Silencing of HDAC6, HDAC9, or both, in GT1-7 cells augmented apoptosis compared with controls. HDAC6 and -9 had additive effects to promote cell survival via modulating the BAX/BCL2 pathway. Silencing of HDAC6 resulted in an activation of movement of GT1-7 cells with induction in acetylation of α-tubulin. Inhibition of HDAC6 and HDAC9 together resulted in an additive effect to increase cell movement but did not alter the acetylation of αtubulin. Together, these studies identify a novel interaction of Class IIa HDAC9 with Class IIb HDAC6 to modulate cell movement and survival in GnRH neurons.     

Exploring hydroxamic acid inhibitors of HDAC1 and HDAC2 using small molecule tools and molecular or homology modelling

Hydroxamic acid compounds 1–10 containing a N-hydroxycinnamamide scaffold and a 4-(benzylamino)methyl cap group that was either unsubstituted (1) or substituted with one (2–4) or two (5–10) methoxy groups in variable positions were prepared as inhibitors of Zn(II)-containing histone deacetylases (HDACs). The 3,4- (9) and 3,5- (10) bis-methoxy-substituted compounds were the least potent against HeLa nuclear extract, HDAC1 and HDAC2. Molecular modelling showed methoxy groups in the 3-, 4- and 5-position, but not the 2-position, had unfavourable steric interactions with the G32-H33-P34 triad on a loop at the surface of the HDAC2 active site cavity. An HDAC1 homology model showed potential ionic (E243..K288) and cation-pi (K247..F292) interactions between helix 10 and helix 11 that were absent in HDAC2 ((G243..K288) and (K247..V292)). This surface-located interhelical constraint could inform the design of bitopic HDAC1 and HDAC2 selective ligands using an allosteric approach, and/or protein-protein interaction (PPI) inhibitors.     

Phenylalanine-containing hydroxamic acids as selective inhibitors of class IIb histone deacetylases (HDACs)

We synthesized biarylalanine-containing hydroxamic acids and tested them on immunoprecipitated HDAC1 and HDAC6 and show a subtype selectivity for HDAC6 that was confirmed in cells by Western blot (tubulin vs histones). We obtained an X-ray structure with a HDAC6-selective inhibitor with the bacterial deacetylase HDAH. Docking studies were carried out using HDAC1 and HDAC6 protein models. Antiproliferative activity was shown on cancer cells for selected compounds.     

Fluorescent analogs of peptoid-based HDAC inhibitors: Synthesis,biological activity and cellular uptake kinetics

Fluorescent tagging of bioactive molecules is a powerful tool to study cellular uptake kinetics and is considered as an attractive alternative to radioligands. In this study, we developed fluorescent histone deacetylase (HDAC) inhibitors and investigated their biological activity and cellular uptake kinetics. Our approach was to introduce a dansyl group as a fluorophore in the solvent-exposed cap region of the HDAC inhibitor pharmacophore model. Three novel fluorescent HDAC inhibitors were synthesized utilizing efficient submonomer protocols followed by the introduction of a hydroxamic acid or 2-aminoanilide moiety as zinc-binding group. All compounds were tested for their inhibition of selected HDAC isoforms, and docking studies were subsequently performed to rationalize the observed selectivity profiles. All HDAC inhibitors were further screened in proliferation assays in the esophageal adenocarcinoma cell lines OE33 and OE19. Compound 2, 6-((N-(2-(benzylamino)-2-oxoethyl)-5-(dimethylamino)naphthalene)-1-sulfonamido)-N-hydroxyhexanamide, displayed the highest HDAC inhibitory capacity as well as the strongest anti-proliferative activity. Fluorescence microscopy studies revealed that compound 2 showed the fastest uptake kinetic and reached the highest absolute fluorescence intensity of all compounds. Hence, the rapid and increased cellular uptake of 2 might contribute to its potent anti-proliferative properties.     

Novel N-hydroxyfurylacrylamide-based histone deacetylase (HDAC) inhibitors with branched CAP group (Part 2)

Histone deacetylases (HDACs) are significant enzymes involved in tumor genesis and development. Herein, we report a series of novel N-hydroxyfurylacryl-amide-based HDAC inhibitors, which are marked by introducing branched hydrophobic groups as the capping group. The inhibitory activity of the synthesized compounds against HDACs and several tumor cell lines are firstly determined. Fifteen compounds with promising activities are selected for further evaluation of target selectivity profile against recombinant human HDAC1, HDAC4 and HDAC6. Compounds 10a, 10b, 10d and 16a exhibit outstanding selectivity against HDAC6. Analysis of HDAC4 X-ray structure and HDAC1, HDAC6 homology model indicates that these enzyme differ significantly in the rim near the surface of the active site. Although TSA has been known as a pan-HDAC inhibitor, it exhibits outstanding selectivity for HDAC6 over HDAC4. For further physicochemical properties study, six compounds are chosen for determination of their physicochemical properties including log D_7.4 and aqueous solubility. The results suggest that compounds with a smaller framework and with hydrophilicgroups are likely to have better aqueous solubility.     

Design,synthesis and evaluation of novel HDAC inhibitors as potential antitumor agents

Phenyl imidazolidin-2-one was introduced as the linker for novel HDAC inhibitors. A focused library of 20 compounds was designed and synthesized, among which eight compounds showed equivalent or higher potencies against HDAC1 as compared to vorinostat. In vitro antitumor activity assays in HCT-116, PC-3 and HL-60 cancer cells revealed six compounds with potent antitumor activities, and compound 1o showed 6- to 9-fold higher potencies compared to vorinostat. In an HCT-116 nude mice xenograft model, compound 1o displayed significant antitumor activity in both continuous and intermittent dosing schedules.     

Novel pyrrole-containing histone deacetylase inhibitors endowed with cytodifferentiation activity

A novel series of aroyl-pyrrolyl-hydroxy-amides (APHAs) active as histone deacetylase (HDAC) inhibitors has been reported. The new derivatives were designed by replacing the benzene ring of the prototype 1 with both aromatic and aliphatic, monocyclic and polycyclic rings (compounds 3a-i), or by inserting a number of substituents on the methylene linker of 1 (compounds 4a-l). Compounds 3a-i and 4a-l were active at sub-micromolar level against the maize deacetylases HD1-B (class I), HD1-A (class II), and HD2. Tested at 5 microM against human HDAC1 and HDAC4, 3b, 4a, and 4j showed significant HDAC1 inhibition, whereas on HDAC4 only 4a was highly effective. On the human leukemia U937 cell line, the same compounds did not alter the cell cycle phases and failed in inducing apoptosis. However, they displayed granulocytic differentiation at 5 microM, with 3b being the most potent (76% CD11c positive cells). Tested to evaluate their effects on histone H3 and alpha-tubulin acetylation, 3b and 4a showed high H3 acetylation, whereas 4a and 4b were the most potent with alpha-tubulin as a substrate.     

New pyrrole-based histone deacetylase inhibitors: binding mode, enzyme- and cell-based investigations

Aroyl-pyrrolyl-hydroxy-amides (APHAs) are a class of synthetic HDAC inhibitors described by us since 2001. Through structure-based drug design, two isomers of the APHA lead compound 1, the 3-(2-benzoyl-1-methyl-1H-pyrrol-4-yl)-N-hydroxy-2-propenamide 2 and the 3-(2-benzoyl-1-methyl-1H-pyrrol-5-yl)-N-hydroxy-2-propenamide 3 (iso-APHAs) were designed, synthesized and tested in murine leukemia cells as antiproliferative and cytodifferentiating agents. To improve their HDAC activity and selectivity, chemical modifications at the benzoyl moieties were investigated and evaluated using three maize histone deacetylases: HD2, HD1-B (class I human HDAC homologue), and HD1-A (class II human HDAC homologue). Docking experiments on HD1-A and HD1-B homology models revealed that the different compounds selectivity profiles could be addressed to different binding modes as observed for the reference compound SAHA. Smaller hydrophobic cap groups improved class II HDAC selectivity through the interaction with HD1-A Asn89-Ser90-Ile91, while bulkier aromatic substituents increased class I HDAC selectivity. Taking into account the whole enzyme data and the functional test results, the described iso-APHAs showed a behaviour of class I/IIb HDACi, with 4b and 4i preferentially inhibiting class IIb and class I HDACs, respectively. When tested in the human leukaemia U937 cell line, 4i showed altered cell cycle (S phase arrest), joined to high (51%) apoptosis induction and significant (21%) differentiation activity.