Exploration of the internal cavity of histone deacetylase (HDAC) with selective HDAC1/HDAC2 inhibitors (SHI-1:2)

We report herein the initial exploration of novel selective HDAC1/HDAC2 inhibitors (SHI-1:2). Optimized SHI-1:2 structures exhibit enhanced intrinsic activity against HDAC1 and HDAC2, and are greater than 100-fold selective versus other HDACs, including HDAC3. Based on the SAR of these agents and our current understanding of the HDAC active site, we postulate that the SHI-1:2 extend the existing HDAC inhibitor pharmacophore to include an internal binding domain.     

Optimization of biaryl Selective HDAC1&2 Inhibitors (SHI-1:2)

A class of biaryl benzamides was identified and optimized as selective HDAC1&2 inhibitors (SHI-1:2). These agents exhibit selectivity over class II HDACs 4-7, as well as class I HDACs 3 and 8; providing examples of selective HDAC inhibitors for the HDAC isoforms most closely associated with cancer. The hypothesis for the increased selectivity is the binding of a pendant aromatic group in the internal cavity of the HDAC1&2 enzymes. SAR development based on an initial lead led to a series of potent and selective inhibitors with reduced off-target activity and tumor growth inhibition activity in a HCT-116 xenograft model.     

SAR profiles of spirocyclic nicotinamide derived selective HDAC1/HDAC2 inhibitors (SHI-1:2)

A potent family of spirocyclic nicotinyl aminobenzamide selective HDAC1/HDAC2 inhibitors (SHI-1:2) is profiled. The incorporation of a biaryl zinc-binding motif into a nicotinyl scaffold resulted in enhanced potency and selectivity versus HDAC3, but also imparted hERG activity. It was discovered that increasing polar surface area about the spirocycle attenuates this liability. Compound 12 induced a 4-fold increase in acetylated histone H2B in an HCT-116 xenograft model study with acute exposure, and inhibited tumor growth in a 21-day efficacy study with qd dosing.     

Protein Kinase CK2 Regulates the Dimerization of Histone Deacetylase 1 (HDAC1) and HDAC2 during Mitosis

Histone deacetylase 1 (HDAC1) and HDAC2 are components of corepressor complexes that are involved in chromatin remodeling and regulation of gene expression by regulating dynamic protein acetylation. HDAC1 and -2 form homo- and heterodimers, and their activity is dependent upon dimer formation. Phosphorylation of HDAC1 and/or HDAC2 in interphase cells is required for the formation of HDAC corepressor complexes. In this study, we show that during mitosis, HDAC2 and, to a lesser extent, HDAC1 phosphorylation levels dramatically increase. When HDAC1 and -2 are displaced from the chromosome during metaphase, they dissociate from each other, but each enzyme remains in association with components of the HDAC corepressor complexes Sin3, NuRD, and CoREST as homodimers. Enzyme inhibition studies and mutational analyses demonstrated that protein kinase CK2-catalyzed phosphorylation of HDAC1 and -2 is crucial for the dissociation of these two enzymes. These results suggest that corepressor complexes, including HDAC1 or HDAC2 homodimers, might target different cellular proteins during mitosis.     

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.     

Distinct and redundant functions of histone deacetylases HDAC1 and HDAC2 in proliferation and tumorigenesis

Histone deacetylases (HDACs) are negative regulators of gene expression and have been implicated in tumorigenesis and tumor progression. Therefore, HDACs are promising targets for antitumor drugs. However, the relevant isoforms of the 18 members encompassing HDAC family have not been identified. Studies utilizing either gene targeting or knockdown approaches reveal both specific and redundant functions of the closely related class I deacetylases HDAC1 and HDAC2 in the control of proliferation and differentiation. Combined ablation of HDAC1 and HDAC2 in different cell types led to a severe proliferation defects or enhanced apoptosis supporting the idea that both enzymes are relevant targets for tumor therapy. In a recent study on the role of HDAC1 in teratoma formation we have reported a novel and surprising function of HDAC1 in tumorigenesis. In this tumor model HDAC1 attenuates proliferation during teratoma formation. In the present work we discuss new findings on redundant and unique functions of HDAC1 and HDAC2 as regulators of proliferation and tumorigenesis and potential implications for applications of HDAC inhibitors as therapeutic drugs.Key words: tumor therapy, HDAC inhibitor, teratoma, chromatin, epigenetics, proliferation, histone acetylation, tumorigenesis     

Divergent roles of HDAC1 and HDAC2 in the regulation of epidermal development and tumorigenesis

The histone deacetylases HDAC1 and HDAC2 remove acetyl moieties from lysine residues of histones and other proteins and are important regulators of gene expression. By deleting different combinations of Hdac1 and Hdac2 alleles in the epidermis, we reveal a dosage‐dependent effect of HDAC1/HDAC2 activity on epidermal proliferation and differentiation. Conditional ablation of either HDAC1 or HDAC2 in the epidermis leads to no obvious phenotype due to compensation by the upregulated paralogue. Strikingly, deletion of a single Hdac2 allele in HDAC1 knockout mice results in severe epidermal defects, including alopecia, hyperkeratosis, hyperproliferation and spontaneous tumour formation. These mice display impaired Sin3A co‐repressor complex function, increased levels of c‐Myc protein, p53 expression and apoptosis in hair follicles (HFs) and misregulation of HF bulge stem cells. Surprisingly, ablation of HDAC1 but not HDAC2 in a skin tumour model leads to accelerated tumour development. Our data reveal a crucial function of HDAC1/HDAC2 in the control of lineage specificity and a novel role of HDAC1 as a tumour suppressor in the epidermis.     

Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes

The development of selective histone deacetylase (HDAC) inhibitors with anti-cancer and anti-inflammatory properties remains challenging in large part owing to the difficulty of probing the interaction of small molecules with megadalton protein complexes. A combination of affinity capture and quantitative mass spectrometry revealed the selectivity with which 16 HDAC inhibitors target multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex. We identified several non-HDAC targets for hydroxamate inhibitors. HDAC inhibitors with distinct profiles have correspondingly different effects on downstream targets. We also identified the anti-inflammatory drug bufexamac as a class IIb (HDAC6, HDAC10) HDAC inhibitor. Our approach enables the discovery of novel targets and inhibitors and suggests that the selectivity of HDAC inhibitors should be evaluated in the context of HDAC complexes and not purified catalytic subunits.     

Distinct and redundant functions of histone deacetylases HDAC1 and HDAC2 in proliferation and tumorigenesis

Histone deacetylases (HDACs) are negative regulators of gene expression and have been implicated in tumorigenesis and tumor progression. Therefore, HDACs are promising targets for anti-tumor drugs. However, the relevant isoforms of the 18 members encompassing HDAC family have not been identified. Studies utilizing either gene targeting or knockdown approaches reveal both specific and redundant functions of the closely related class I deacetylases HDAC1 and HDAC2 in the control of proliferation and differentiation. Combined ablation of HDAC1 and HDAC2 in different cell types led to a severe proliferation defects or enhanced apoptosis supporting the idea that both enzymes are relevant targets for tumor therapy. In a recent study on the role of HDAC1 in teratoma formation we have reported a novel and surprising function of HDAC1 in tumorigenesis. In this tumor model HDAC1 attenuates proliferation during teratoma formation. In the present work we discuss new findings on redundant and unique functions of HDAC1 and HDAC2 as regulators of proliferation and tumorigenesis and potential implications for applications of HDAC inhibitors as therapeutic drugs.     

E3 ligase RCHY1 negatively regulates HDAC2

HDAC2, one of the class I histone deacetylase regulates epigenetic landscape through histone modification. Because HDAC2 is overexpressed in many cancers, cancer therapeutics against HDAC2 have been developed. Here we show novel mechanism of HDAC2 regulation by E3 ligase RCHY1. We found inverse correlation RCHY1 and HDAC2 levels in tumor tissue from six independent dataset using meta-analysis. Ectopic expression of RCHY1 decreased the level of HDAC2 from cancer cells including p53 wildtype, mutant and null cells. In addition, HDAC2 was increased by RCHY1 knockdown. RCHY1 directly interacts with HDAC2. Ectopic expression of wild type but not RING mutant RCHY1 increased HDAC2 levels. These data provide an evidence that RCHY1 negatively regulates HDAC2.     

The p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression

Regulation of NF-kappaB transactivation function is controlled at several levels, including interactions with coactivator proteins. Here we show that the transactivation function of NF-kappaB is also regulated through interaction of the p65 (RelA) subunit with histone deacetylase (HDAC) corepressor proteins. Our results show that inhibition of HDAC activity with trichostatin A (TSA) results in an increase in both basal and induced expression of an integrated NF-kappaB-dependent reporter gene. Chromatin immunoprecipitation (ChIP) assays show that TSA treatment causes hyperacetylation of the wild-type integrated NF-kappaB-dependent reporter but not of a mutant version in which the NF-kappaB binding sites were mutated. Expression of HDAC1 and HDAC2 repressed tumor necrosis factor (TNF)-induced NF-kappaB-dependent gene expression. Consistent with this, we show that HDAC1 and HDAC2 target NF-kappaB through a direct association of HDAC1 with the Rel homology domain of p65. HDAC2 does not interact with NF-kappaB directly but can regulate NF-kappaB activity through its association with HDAC1. Finally, we show that inhibition of HDAC activity with TSA causes an increase in both basal and TNF-induced expression of the NF-kappaB-regulated interleukin-8 (IL-8) gene. Similar to the wild-type integrated NF-kappaB-dependent reporter, ChIP assays showed that TSA treatment resulted in hyperacetylation of the IL-8 promoter. These data indicate that the transactivation function of NF-kappaB is regulated in part through its association with HDAC corepressor proteins. Moreover, it suggests that the association of NF-kappaB with the HDAC1 and HDAC2 corepressor proteins functions to repress expression of NF-kappaB-regulated genes as well as to control the induced level of expression of these genes.     

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.     

Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors

The structure–activity and structure–kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11 Å channel leading to the Zn²⁺ catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.     

Structural basis for the design and synthesis of selective HDAC inhibitors

Histone Deacetylases are considered promising targets for cancer epigenetic therapy, and small molecules able to modulate their biological function have recently gained an increasing interest as potential anticancer agents. In spite of their potential application in cancer therapy, most HDAC inhibitors unselectively bind the several HDAC isoforms, giving rise to different side-effects. In this context, we have traced out the structural elements responsible of selective binding for the therapeutically relevant different HDAC isoforms. The structural analysis has been carried out by molecular modeling, docking in the binding pockets of HDAC1–4 and HDAC6–8, 36 inhibitors presenting a well defined selectivity for the different isoforms. As quick proof of evidence, we have designed, synthesized and experimentally tested three selective ligands. The experimental data suggest that the obtained structural guidelines can be useful tools for the rational design of new potent inhibitors against selected HDAC isoforms.     

Combined HDAC1 and HDAC2 Depletion Promotes Retinal Ganglion Cell Survival After Injury Through Reduction of p53 Target Gene Expression

Histones deacetylases (HDACs), besides their function as epigenetic regulators, deacetylate and critically regulate the activity of nonhistone targets. In particular, HDACs control partially the proapoptotic activity of p53 by balancing its acetylation state. HDAC inhibitors have revealed neuroprotective properties in different models, but the exact mechanisms of action remain poorly understood. We have generated a conditional knockout mouse model targeting retinal ganglion cells (RGCs) to investigate specifically the functional role of HDAC1 and HDAC2 in an acute model of optic nerve injury. Our results demonstrate that combined HDAC1 and HDAC2 ablation promotes survival of axotomized RGCs. Based on global gene expression analyses, we identified the p53-PUMA apoptosis-inducing axis to be strongly activated in axotomized mouse RGCs. Specific HDAC1/2 ablation inhibited this apoptotic pathway by impairing the crucial acetylation status of p53 and reducing PUMA expression, thereby contributing to the ensuing enhanced neuroprotection due to HDAC1/2 depletion. HDAC1/2 inhibition and the affected downstream signaling components emerge as specific targets for developing therapeutic strategies in neuroprotection.