Potent and Selective Non‐hydroxamate Histone Deacetylase 8 Inhibitors

Specific inhibition of histone deacetylase 8 (HDAC8) has been suggested as a promising option for the treatment of neuroblastoma and T‐cell malignancies. A novel class of highly potent and selective HDAC8 inhibitors with a pyrimido[1,2‐c][1,3]benzothiazin‐6‐imine scaffold was studied that is completely different from the traditional concept of HDAC inhibitors comprising a zinc binding group (ZBG), in most cases a hydroxamate group, a spacer, and a capping group that may interact with the surface of the target protein. Although lacking a ZBG, some of the new compounds were shown to have outstanding potency against HDAC8 in the single‐digit nanomolar range. The pyrimido[1,2‐c][1,3]benzothiazin‐6‐imines also inhibited the growth of solid and hematological tumor cells. The small size and beneficial physicochemical properties of the novel HDAC inhibitor class underline the high degree of drug likeness. This and the broad structure–activity relationship suggest great potential for the further development of compounds with the pyrimido[1,2‐c][1,3]benzothiazin‐6‐imine scaffold into innovative and highly effective therapeutic drugs against cancer.     

Theoretical study on potency and selectivity of novel non-peptide inhibitors of matrix metalloproteinases MMP-1 and MMP-3

By means of molecular modeling and docking studies, two novel non-peptide inhibitors (pyrogallic acid and myricetin) with a new zinc binding group (ZBG) have been evaluated as inhibitors of MMP-1 and MMP-3. The differences in binding affinities for MMP-1 and MMP-3 have been rationalized, and the results are consistent with the experiments of Fang et al. The density functional theory (DFT) method B3LYP/6-31G^∗ has also been employed to characterize the interactions between ZBG of pyrogallic acid and the catalytic zinc ion in MMP-1. Our results may be useful for further research in the structure-based design of inhibitors with improved potency and selectivity.     

Synthesis of N‐Hydroxycinnamides Capped with a Naturally Occurring Moiety as Inhibitors of Histone Deacetylase

Histone deacetylase (HDAC) inhibitors are regarded as promising therapeutics for the treatment of cancer. All reported HDAC inhibitors contain three pharmacophoric features: a zinc‐chelating group, a hydrophobic linker, and a hydrophobic cap for surface recognition. In this study we investigated the effectiveness of osthole, a hydrophobic Chinese herbal compound, as the surface recognition cap in hydroxamate‐based compounds as inhibitors of HDAC. Nine novel osthole‐based N‐hydroxycinnamides were synthesized and screened for enzyme inhibition activity. Compounds 9 d , 9 e , 9 g exhibited inhibitory activities (IC₅₀=24.5, 20.0, 19.6 nM ) against nuclear HDACs in HeLa cells comparable to that of suberoylanilide hydroxamic acid (SAHA; IC₅₀=24.5 nM ), a potent inhibitor clinically used for the treatment of cutaneous T‐cell lymphoma (CTCL). While compounds 9 d and 9 e showed SAHA‐like activity towards HDAC1 and HDAC6, compound 9 g was more selective for HDAC1. Compound 9 d exhibited the best cellular effect, which was comparable to that of SAHA, of enhancing acetylation of either α‐tubulin or histone H3. Molecular docking analysis showed that the osthole moiety of compound 9 d may interact with the same hydrophobic surface pocket exploited by SAHA and it may be modified to provide class‐specific selectivity. These results suggest that osthole is an effective hydrophobic cap when incorporated into N‐hydroxycinnamide‐derived HDAC inhibitors.     

Why Hydroxamates May Not Be the Best Histone Deacetylase Inhibitors—What Some May Have Forgotten or Would Rather Forget?

Hydroxamate‐based histone deacetylase inhibitors (HDACIs) have been approved as therapeutic agents by the US Food and Drug Administration for use in oncology applications. While the potential utility of such HDACIs in other areas of medicinal chemistry is tremendous, there are significant concerns that “pan‐HDAC inhibitors” may be too broadly acting and/or toxic for clinical use beyond oncology. In addition to the isozyme selectivity challenge, the potential mutagenicity of hydroxamate‐containing HDAC inhibitors represents a major hindrance in their application to other therapeutic areas. Herein we report on the mutagenicity of known hydroxamates, discuss the mechanisms responsible for their genotoxicity, and review some of the current alternatives to hydroxamates. We conclude that the hydroxamate group, while providing high‐potency HDACIs, is not necessarily the best zinc‐binding group for HDACI drug discovery.     

Studies of Benzamide‐ and Thiol‐Based Histone Deacetylase Inhibitors in Models of Oxidative‐Stress‐Induced Neuronal Death: Identification of Some HDAC3‐Selective Inhibitors

Less stress : We compare three structurally different classes of histone deacetylase (HDAC) inhibitors that contain benzamide, hydroxamate, or thiol groups as the zinc binding group (ZBG) for their ability to protect cortical neurons in culture from cell death induced by oxidative stress. Novel benzamide‐based ligands selectively inhibit HDAC3 but provide no neuroprotection in the HCA–cortical neuron model of oxidative stress.

     

Chemistry, biology, and QSAR studies of substituted biaryl hydroxamates and mercaptoacetamides as HDAC inhibitors-nanomolar-potency inhibitors of pancreatic cancer cell growth

The histone deacetylases (HDACs) are able to regulate gene expression, and inhibitors of the HDACs (HDACIs) hold promise in the treatment of cancer as well as a variety of neurodegenerative diseases. To investigate the potential for isoform selectivity in the inhibition of HDACs, we prepared a small series of 2,4'-diaminobiphenyl ligands functionalized at the para-amino group with an appendage containing either a hydroxamate or a mercaptoacetamide group and coupled to an amino acid residue at the ortho-amino group. A smaller series of substituted phenylthiazoles was also explored. Some of these newly synthesized ligands show low-nanomolar potency in HDAC inhibition assays and display micromolar to low-nanomolar IC(50) values in tests against five pancreatic cancer cell lines. The isoform selectivity of these ligands for class I HDACs (HDAC1-3 and 8) and class IIb HDACs (HDAC6 and 10) together with QSAR studies of their correlation with lipophilicity are presented. Of particular interest is the selectivity of the mercaptoacetamides for HDAC6.     

Probing the S1′ Site for the Identification of Non‐Zinc‐Binding MMP‐2 Inhibitors

Matrix metalloproteinases (MMPs) are zinc‐dependent enzymes involved in several pathological states. Among them, MMP‐2 is a relevant therapeutic target because of its role in cancer development and progression. Many MMP inhibitors (MMPIs) have been discovered over the last 30 years, and the majority of them contain a functional group that binds the zinc ion (zinc‐binding group; ZBG). Unfortunately, no MMPIs have reached the market yet, owing to toxic effects due to unselective interactions of the ZBG. The new generation of MMPIs that do not bind the zinc ion could overcome problems of selectivity and toxicity, but have so far been developed only for MMP‐8, ‐12, and ‐13. In this work, a virtual screening protocol was established by combining ligand‐ and structure‐based methods to identify non‐zinc‐binding MMP‐2 inhibitors using a new‐generation MMP‐8 inhibitor as a probe to find unexplored interactions in the MMP‐2 S1′ site. The screening allowed the identification of micromolar MMP‐2 inhibitors that putatively avoid binding the zinc ion, as demonstrated by docking calculations. The LIA model, built to correlate predicted and experimental binding energies of the identified non‐zinc‐binding MMP‐2 hits, underpins the reliability of the predicted docking poses.     

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular Target Engagement**

Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e. g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of most other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl-putrescine or -spermidine. Hence, it is also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin-labelled acetyl-spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibitors of HDAC10-mediated spermidine deacetylation in vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10-inhibitor structure using X-ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.     

Hybrids from Farnesylthiosalicylic Acid and Hydroxamic Acid as Dual Ras‐Related Signaling and Histone Deacetylase (HDAC) Inhibitors: Design,Synthesis and Biological Evaluation

A novel series of hybrids was designed and synthesized by combining key elements from farnesylthiosalicylic acid (FTS) and hydroxamic acid. Several 3,7,11‐trimethyldodeca‐2,6,10‐trien‐1‐yl) thio)benzamide derivatives, particularly those with branched and linear aliphatic linkers between the hydroxamic zinc binding group (ZBG) and the benzamide core, not only displayed significant antitumor activities against six human cancer cells but also exhibited histone deacetylase (HDAC) inhibitory effects in vitro. Among them, N‐(4‐(hydroxyamino)‐4‐oxobutyl)‐2‐(((2E,6E)‐3,7,11‐trimethyldodeca‐2,6, 10‐trien‐1‐yl)thio)benzamide ( 8 d ) was the most potent, with IC₅₀ values of 4.9–7.6 μM ; these activities are eight‐ to sixteen‐fold more potent than FTS and comparable to that of suberoylanilide hydroxamic acid (SAHA). Derivative 8 d induced cell cycle arrest in the G0/G1 phase, inhibited the acetylation of histone H3 and α‐tubulin, and blocked Ras‐related signaling pathways in a dose‐dependent manner. The improved tumor growth inhibition and cell‐cycle arrest in vitro might result from the dual inhibition. These findings suggest dual inhibitors of Ras‐related signaling pathway and HDAC hold promise as therapeutic agents for the treatment of cancer.     

Mechanistic Exploration of Methionine 274 Acting as a “Switch” of the Selective Pocket Involved in HDAC8 Inhibition: An in Silico Study

The overexpression of histone deacetylase 8 (HDAC8) causes several diseases, and the selective inhibition of HDAC8 has been touted as a promising therapeutic strategy due to its fewer side effects. However, the mechanism of HDAC8 selective inhibition remains unclear. In this study, flexible docking and in silico mutation were used to explore the structural change of methionine (M274) during HDAC8 binding to inhibitors, along with the reason for this change. Meanwhile, steered and conventional molecular dynamics simulations were employed to explore the stability of the structural change. The findings suggest that M274 acts as a “switch” to control the exposure of the HDAC8-selective pocket. The structure of M274 changes from flipped-out to flipped-in only when L-shaped inhibitors bind to HDAC8. This structural change forms a groove that allows these inhibitors to enter the selective pocket. In other HDACs, a leucine residue replaces M274 in situ, and the same structural change is not observed. The findings reveal the mechanism of selective HDAC8 inhibition and provide guidance for the development of novel selective inhibitors.     

Chiral mercaptoacetamides display enantioselective inhibition of histone deacetylase 6 and exhibit neuroprotection in cortical neuron models of oxidative stress

Mercaptoacetamide-based ligands have been designed as a new class of histone deacetylase (HDAC) inhibitors for possible use in the treatment of neurodegenerative diseases. The thiol group of these compounds provides a key binding element for interaction with the catalytic zinc ion, and thus differs from the more typically employed hydroxamic acid based zinc binding groups. Herein we disclose the chemistry and biology of some substituted mercaptoacetamides with the intention of increasing HDAC6 isoform selectivity while maintaining potency similar to their hydroxamic acid analogues. The introduction of a stereocenter α to the thiol group was found to have a considerable impact on HDAC inhibitor potency. These new compounds were also profiled for their therapeutic potential in an in?vitro model of stress-induced neuronal injury and were found to act as nontoxic neuroprotective agents.     

Oxa Analogues of Nexturastat A Demonstrate Improved HDAC6 Selectivity and Superior Antileukaemia Activity

The acetylome is important for maintaining the homeostasis of cells. Abnormal changes can result in the pathogenesis of immunological or neurological diseases, and degeneration can promote the manifestation of cancer. In particular, pharmacological intervention in the acetylome with pan-histone deacetylase (HDAC) inhibitors is clinically validated. However, these drugs exhibit an undesirable risk-benefit profile due to severe side effects. Selective HDAC inhibitors might promote patient compliance and represent a valuable opportunity in personalised medicine. Therefore, we envisioned the development of HDAC6-selective inhibitors. During our lead structure identification, we demonstrated that an alkoxyurea-based connecting unit proves to be beneficial for HDAC6 selectivity and established the synthesis of alkoxyurea-based hydroxamic acids. Herein, we report highly potent N-alkoxyurea-based hydroxamic acids with improved HDAC6 preference compared to nexturastat A. We further validated the biological activity of these oxa analogues of nexturastat A in a broad subset of leukaemia cell lines and demonstrated their superior anti-proliferative properties compared to nexturastat A.     

Stereoselective HDAC Inhibition from Cysteine‐Derived Zinc‐Binding Groups

A series of small‐molecule histone deacetylase (HDAC) inhibitors, which feature zinc binding groups derived from cysteine, were synthesized. These inhibitors were tested against multiple HDAC isoforms, and the most potent, compound 10 , was determined to have IC₅₀ values below 1 μM . The compounds were also tested in a cellular assay of oxidative stress‐induced neurodegeneration. Many of the inhibitors gave near‐complete protection against cell death at 10 μM without the neurotoxicity seen with hydroxamic acid‐based inhibitors, and were far more neuroprotective than HDAC inhibitors currently in clinical trials. Both enantiomers of cysteine were used in the synthesis of a variety of novel zinc‐binding groups (ZBGs). Derivatives of L ‐cysteine were active in the HDAC inhibition assays, while the derivatives of D ‐cysteine were inactive. Notably, the finding that both the D ‐ and L ‐cysteine derivatives were active in the neuroprotection assays suggests that multiple mechanisms are working to protect the neurons from cell death. Molecular modeling was employed to investigate the differences in inhibitory activity between the HDAC inhibitors generated from the two enantiomeric forms of cysteine.     

Phosphonate as a Stable Zinc-Binding Group for “Pathoblocker” Inhibitors of Clostridial Collagenase H (ColH)

Microbial infections are a significant threat to public health, and resistance is on the rise, so new antibiotics with novel modes of action are urgently needed. The extracellular zinc metalloprotease collagenase H (ColH) from Clostridium histolyticum is a virulence factor that catalyses tissue damage, leading to improved host invasion and colonisation. Besides the major role of ColH in pathogenicity, its extracellular localisation makes it a highly attractive target for the development of new antivirulence agents. Previously, we had found that a highly selective and potent thiol prodrug (with a hydrolytically cleavable thiocarbamate unit) provided efficient ColH inhibition. We now report the synthesis and biological evaluation of a range of zinc-binding group (ZBG) variants of this thiol-derived inhibitor, with the mercapto unit being replaced by other zinc ligands. Among these, an analogue with a phosphonate motif as ZBG showed promising activity against ColH, an improved selectivity profile, and significantly higher stability than the thiol reference compound, thus making it an attractive candidate for future drug development.     

Strategies To Design Selective Histone Deacetylase Inhibitors

This review classifies drug-design strategies successfully implemented in the development of histone deacetylase (HDAC) inhibitors, which have many applications including cancer treatment. Our focus is on especially demanded selective HDAC inhibitors and their structure-activity relationships in relation to corresponding protein structures. The main part of the paper is divided into six subsections each narrating how optimization of one of six structural features can influence inhibitor selectivity. It starts with the impact of the zinc binding group on selectivity, continues with the optimization of the linker placed in the substrate binding tunnel as well as the adjustment of the cap group interacting with the surface of the protein, and ends with the addition of groups targeting class-specific sub-pockets: the side-pocket-, lower-pocket- and foot-pocket-targeting groups. The review is rounded off with a conclusion and an outlook on the future of HDAC inhibitor design.     

Synthesis,Molecular Docking and Biological Characterization of Pyrazine Linked 2-Aminobenzamides as New Class I Selective Histone Deacetylase (HDAC) Inhibitors with Anti-Leukemic Activity

Class I histone deacetylases (HDACs) are key regulators of cell proliferation and they are frequently dysregulated in cancer cells. We report here the synthesis of a novel series of class-I selective HDAC inhibitors (HDACi) containing a 2-aminobenzamide moiety as a zinc-binding group connected with a central (piperazin-1-yl)pyrazine or (piperazin-1-yl)pyrimidine moiety. Some of the compounds were additionally substituted with an aromatic capping group. Compounds were tested in vitro against human HDAC1, 2, 3, and 8 enzymes and compared to reference class I HDACi (Entinostat (MS-275), Mocetinostat, CI994 and RGFP-966). The most promising compounds were found to be highly selective against HDAC1, 2 and 3 over the remaining HDAC subtypes from other classes. Molecular docking studies and MD simulations were performed to rationalize the in vitro data and to deduce a complete structure activity relationship (SAR) analysis of this novel series of class-I HDACi. The most potent compounds, including 19f, which blocks HDAC1, HDAC2, and HDAC3, as well as the selective HDAC1/HDAC2 inhibitors 21a and 29b, were selected for further cellular testing against human acute myeloid leukemia (AML) and erythroleukemic cancer (HEL) cells, taking into consideration their low toxicity against human embryonic HEK293 cells. We found that 19f is superior to the clinically tested class-I HDACi Entinostat (MS-275). Thus, 19f is a new and specific HDACi with the potential to eliminate blood cancer cells of various origins.     

Discovery of Pyridone‐Based Histone Deacetylase Inhibitors: Approaches for Metabolic Stability

Histone deacetylases (HDACs) are important enzymes in epigenetic regulation and are therapeutic targets for cancer. Most zinc‐dependent HDACs induce proliferation, dedifferentiation, and anti‐apoptotic effects in cancer cells. We designed and synthesized a new series of pyridone‐based HDAC inhibitors that have a pyridone ring in the core structure and a conjugated system with an olefin connecting the hydroxamic acid moiety. Consequently, most of the selected pyridone‐based HDAC inhibitors showed similar or higher inhibition profiles in addition to remarkable metabolic stability against hydrolysis relative to the corresponding lactam‐based HDAC inhibitors. Furthermore, the selectivity of the novel pyridine‐based compounds was evaluated across all of the HDAC isoforms. One of these compounds, (E)‐N‐hydroxy‐3‐{1‐[3‐(naphthalen‐2‐yl)propyl]‐2‐oxo‐1,2‐dihydropyridin‐3‐yl}acrylamide, exhibited the highest level of HDAC inhibition (IC₅₀=0.07 μM ), highly selective inhibition of class I HDAC1 and class II HDAC6 enzymes, metabolic stability in mouse liver microsomal studies, and effective growth inhibition of various cancer cell lines. Docking studies indicated that a long alkyl linker and bulky hydrophobic cap groups affect in vitro activities. Overall, the findings reported herein regarding pyridone‐based HDAC inhibitors can be used to guide future research efforts to develop new and effective anticancer therapeutics.     

Balancing Histone Deacetylase (HDAC) Inhibition and Drug-likeness: Biological and Physicochemical Evaluation of Class I Selective HDAC Inhibitors

Herein we report the structure-activity and structure-physicochemical property relationships of a series of class I selective ortho-aminoanilides targeting the “foot-pocket” in HDAC1&2. To balance the structural benefits and the physicochemical disadvantages of these substances, we started with a set of HDACi related to tacedinaline (CI-994) and evaluated their solubility, lipophilicity (log D_7.4) and inhibition of selected HDAC isoforms. Subsequently, we selected the most promising “capless” HDACi and transferred its ZBG to our previously published scaffold featuring a peptoid-based cap group. The resulting hit compound 10 c ( LSH-A54) showed favorable physicochemical properties and is a potent, selective HDAC1/2 inhibitor. The following evaluation of its slow binding properties revealed that LSH-A54 binds tightly to HDAC1 in an induced-fit mechanism. The potent HDAC1/2 inhibitory properties were reflected by attenuated cell migration in a modified wound healing assay and reduced cell viability in a clonogenic survival assay in selected breast cancer cell lines.     

Natural and Synthetic Macrocyclic Inhibitors of the Histone Deacetylase Enzymes

Inhibition of histone deacetylase (HDAC) enzymes has emerged as a target for development of cancer chemotherapy. Four compounds have gained approval for clinical use by the Food and Drug Administration in the US, and several are currently in clinical trials. However, none of these compounds possesses particularly good isozyme selectivity, which would be a highly desirable feature in a tool compound. Whether selective inhibition of individual HDAC isozymes will provide improved drug candidates remains to be seen. Nevertheless, it has been speculated that using macrocyclic compounds to target HDAC enzymes might hold an advantage over the use of traditional hydroxamic‐acid‐containing inhibitors, which rely on chelation to the conserved active‐site zinc ion. Here we review the literature on macrocyclic HDAC inhibitors obtained from natural sources and on structure–activity relationship studies inspired by these molecules, as well as on efforts aimed at fully synthetic macrocyclic HDAC inhibitors.     

Novel Pyridine-Based Hydroxamates and 2′-Aminoanilides as Histone Deacetylase Inhibitors: Biochemical Profile and Anticancer Activity

Starting from the N-hydroxy-3-(4-(2-phenylbutanoyl)amino)phenyl)acrylamide ( 5 b ) previously described by us as a HDAC inhibitor, we prepared four aza-analogues, 6 – 8 , 9 b , as regioisomers containing the pyridine nucleus. Preliminary screening against mHDAC1 highlighted the N-hydroxy-5-(2-(2-phenylbutanoyl)amino)pyridyl)acrylamide ( 9 b ) as the most potent inhibitor. Thus, we further developed both pyridylacrylic- and nicotinic-based hydroxamates ( 9 a , 9 c – f , and 11 a – f ) and 2′-aminoanilides ( 10 a – f and 12 a – f ), related to 9 b , to be tested against HDACs. Among them, the nicotinic hydroxamate 11 d displayed sub-nanomolar potency (IC₅₀: 0.5 nM) and selectivity up to 34 000 times that of HDAC4 and from 100 to 1300 times that of all the other tested HDAC isoforms. The 2′-aminoanilides were class I-selective HDAC inhibitors, generally more potent against HDAC3, with the nicotinic anilide 12 d being the most effective (IC₅₀^HDAC3=0.113 μM). When tested in U937 leukemia cells, the hydroxamates 9 e , 11 c , and 11 d blocked over 80 % of cells in G2/M phase, whereas the anilides did not alter cell-cycle progress. In the same cell line, the hydroxamate 11 c and the anilide 10 b induced about 30 % apoptosis, and the anilide 12 c displayed about 40 % cytodifferentiation. Finally, the most potent compounds in leukemia cells 9 b , 11 c , 10 b , 10 e , and 12 c were also tested in K562, HCT116, and A549 cancer cells, displaying antiproliferative IC₅₀ values at single-digit to sub-micromolar level.