Identification of Novel Histone Deacetylase 6-Selective Inhibitors Bearing 3,3,3-Trifluorolactic Amide (TFLAM) Motif as a Zinc Binding Group

Pharmacological inhibition of histone deacetylase 6 (HDAC6) is an effective therapeutic strategy for cancer and immunological diseases. Most of the previously reported HDAC6 inhibitors have a hydroxamate group as a zinc binding group (ZBG), which coordinates to the catalytic zinc ion of HDAC6. The hydroxamate group is liable to metabolically generate mutagenetic hydroxylamine; therefore, non-hydroxamate HDAC6 inhibitors would be advantageous. In this study, to identify novel non-hydroxamate HDAC6-selective inhibitors, screening of a chemical library and the subsequent structural optimization were performed, which led to the identification of HDAC6-selective inhibitors with 3,3,3-trifluorolactic amide (TFLAM) as a novel ZBG. The identified inhibitor showed potent and selective HDAC6-inhibitory activity in cells and induced regulatory T (Treg) cell differentiation.     

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.     

Natural Products as Zinc‐Dependent Histone Deacetylase Inhibitors

Zinc‐dependent histone deacetylases (HDACs), a family of hydrolases that remove acetyl groups from lysine residues, play an important role in the regulation of multiple processes, from gene expression to protein activity. The dysregulation of HDACs is associated with many diseases including cancer, neurological disorders, cellular metabolism disorders, and inflammation. Molecules that act as HDAC inhibitors (HDACi) exhibit a variety of related bioactivities. In particular, HDACi have been applied clinically for the treatment of cancers. Inhibition through competitive binding of the catalytic domain of these enzymes has been achieved by a diverse array of small‐molecule chemotypes, including a number of natural products. This review provides a systematic introduction of natural HDACi, with an emphasis on their enzyme inhibitory potency, selectivity, and biological activities, highlighting their various binding modes with HDACs.     

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.     

Structure-Based Inhibitor Discovery of Class I Histone Deacetylases (HDACs)

Class I histone deacetylases (HDACs) are promising targets for epigenetic therapies for a range of diseases such as cancers, inflammations, infections and neurological diseases. Although six HDAC inhibitors are now licensed for clinical treatments, they are all pan-inhibitors with little or no HDAC isoform selectivity, exhibiting undesirable side effects. A major issue with the currently available HDAC inhibitors is that they have limited specificity and target multiple deacetylases. Except for HDAC8, Class I HDACs (1, 2 and 3) are recruited to large multiprotein complexes to function. Therefore, there are rising needs to develop new, hopefully, therapeutically efficacious HDAC inhibitors with isoform or complex selectivity. Here, upon the introduction of the structures of Class I HDACs and their complexes, we provide an up-to-date overview of the structure-based discovery of Class I HDAC inhibitors, including pan-, isoform-selective and complex-specific inhibitors, aiming to provide an insight into the discovery of additional HDAC inhibitors with greater selectivity, specificity and therapeutic utility.     

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.     

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.     

A Structure‐Based Virtual Screening Approach toward the Discovery of Histone Deacetylase Inhibitors: Identification of Promising Zinc‐Chelating Groups

The inhibitors of histone deacetylases (HDACs) have drawn a great deal of attention due to their promising potential as small‐molecule therapeutics for the treatment of cancer. By means of virtual screening with docking simulations under consideration of the effects of ligand solvation, we were able to identify six novel HDAC inhibitors with IC₅₀ values ranging from 1 to 100 μM . These newly identified inhibitors are structurally diverse and have various chelating groups for the active site zinc ion, including N‐[1,3,4]thiadiazol‐2‐yl sulfonamide, N‐thiazol‐2‐yl sulfonamide, and hydroxamic acid moieties. The former two groups are included in many drugs in current clinical use and have not yet been reported as HDAC inhibitors. Therefore, they can be considered as new inhibitor scaffolds for the development of anticancer drugs by structure–activity relationship studies to improve the inhibitory activities against HDACs. Interactions with the HDAC1 active site residues responsible for stabilizing these new inhibitors are addressed in detail.     

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.     

Design and Synthesis of Dihydroxamic Acids as HDAC6/8/10 Inhibitors

We report the synthesis and evaluation of a class of selective multitarget agents for the inhibition of HDAC6, HDAC8, and HDAC10. The concept for this study grew out of a structural analysis of the two selective inhibitors Tubastatin A (HDAC6/10) and PCI-34051 (HDAC8), which we recognized share the same N-benzylindole core. Hybridization of the two inhibitor structures resulted in dihydroxamic acids with benzyl-indole and -indazole core motifs. These substances exhibit potent activity against HDAC6, HDAC8, and HDAC10, while retaining selectivity over HDAC1, HDAC2, and HDAC3. The best substance inhibited the viability of the SK-N-BE(2)C neuroblastoma cell line with an IC₅₀ value similar to a combination treatment with Tubastatin A and PCI-34051. This compound class establishes a proof of concept for such hybrid molecules and could serve as a starting point for the further development of enhanced HDAC6/8/10 inhibitors.     

Double click reaction for the acquisition of a highly potent and selective mPTPB inhibitor

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is a major worldwide threat to public health. Mycobacterium protein tyrosine phosphatase B (mPTPB) is a virulent phosphatase secreted by Mtb, which is essential for the survival and persistence of the bacterium in the host. Consequently, small‐molecule inhibitors of mPTPB are expected to serve as anti‐TB agents with a novel mode of action. Herein, we report the discovery of highly potent and selective mPTPB inhibitors using a novel, double Click chemistry strategy. The most potent mPTPB inhibitor from this approach possesses a K_i value of 160 nM and a >25‐fold selectivity for mPTPB over 19 other protein tyrosine phosphatases (PTBs). Molecular docking study of the enzyme–inhibitor complex provides a rationale for the high potency and selectivity of the lead compound and reveals an unusual binding mode, which may guide further optimization effort.     

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.     

Identification of HDAC6‐Selective Inhibitors of Low Cancer Cell Cytotoxicity

The histone deacetylases (HDACs) occur in 11 different isoforms, and these enzymes regulate the activity of a large number of proteins involved in cancer initiation and progression. The discovery of isoform‐selective HDAC inhibitors (HDACIs) is desirable, as it is likely that such compounds would avoid some of the undesirable side effects found with the first‐generation inhibitors. A series of HDACIs previously reported by us were found to display some selectivity for HDAC6 and to induce cell‐cycle arrest and apoptosis in pancreatic cancer cells. In the present work, we show that structural modification of these isoxazole‐based inhibitors leads to high potency and selectivity for HDAC6 over HDAC1–3 and HDAC10, while unexpectedly abolishing their ability to block cell growth. Three inhibitors with lower HDAC6 selectivity inhibit the growth of cell lines BxPC3 and L3.6pl, and they only induce apoptosis in L3.6pl cells. We conclude that HDAC6 inhibition alone is insufficient for disruption of cell growth, and that some degree of class 1 HDAC inhibition is required. Moreover, the highly selective HDAC6Is reported herein that are weakly cytotoxic may find use in cancer immune system reactivation.     

Development of Selective Bisubstrate‐Based Inhibitors Against Protein Kinase C (PKC) Isozymes By Using Dynamic Peptide Microarrays

Kinase inhibitors are increasingly important in drug development. Because the majority of current inhibitors target the conserved ATP‐binding site, selectivity might become an important issue. This could be particularly problematic for the potential drug target protein kinase C (PKC), of which twelve isoforms with high homology exist in humans. A strategy to increase selectivity is to prepare bisubstrate‐based inhibitors that target the more selective peptide‐binding site in addition to the ATP‐binding site. In this paper a generally applicable, rapid methodology is presented to discover such bisubstrate‐based leads. Dynamic peptide microarrays were used to find peptide‐binding site inhibitors. These were linked with chemoselective click chemistry to an ATP‐binding site inhibitor, and this led to novel bisubstrate structures. The peptide microarrays were used to evaluate the resulting inhibitors. Thus, novel bisubstrate‐based inhibitors were obtained that were both more potent and selective compared to their constituent parts. The most promising inhibitor has nanomolar affinity and selectivity towards PKCθ amongst three isozymes.     

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.     

Biological evaluation and structural determinants of p38α mitogen-activated-protein kinase and c-Jun-N-terminal kinase 3 inhibition by flavonoids

A series of 42 naturally occurring flavonoids and one flavonoid glucuronide were tested for their ability to inhibit p38α mitogen-activated protein kinase (p38α) and c-Jun-N-terminal kinase 3 (JNK3). Potent inhibitors with IC(50) values in the low micromolar range were identified. Structure-activity relationships were evaluated and the most promising compounds were docked into the ATP binding site of these kinases. Among the different classes of flavonoids, the flavonol group showed better inhibition of p38α. Of this class, kaempferol-7,4'-dimethylether was a potent p38α inhibitor, displaying 13-fold selectivity for p38α over JNK3. The flavone compounds without a 6-methoxy group preferentially inhibited JNK3. The flavone glycoside, luteolin-7-O-glycoside, was identified as a potent inhibitor with the greatest selectivity toward JNK3. In contrast, the flavanol compounds displayed similar inhibitory activities toward both kinases.     

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.     

Highly Ligand Efficient and Selective N‐2‐(Thioethyl)picolinamide Histone Deacetylase Inhibitors Inspired by the Natural Product Psammaplin A

Novel picolinamide‐based histone deacetylase (HDAC) inhibitors were developed, drawing inspiration from the natural product psammaplin A. We found that the HDAC potency and isoform selectivity provided by the oxime unit of psammaplin A could be reproduced by using carefully chosen heterocyclic frameworks. The resulting (hetero)aromatic amide based compounds displayed very high potency and isoform selectivity among the HDAC family, in addition to excellent ligand efficiency relative to previously reported HDAC inhibitors. In particular, the high HDAC1 isoform selectivity provided by the chloropyridine motif represents a valuable design criterion for the development of new lead compounds and chemical probes that target HDAC1.     

Highly potent and selective substrate analogue factor Xa inhibitors containing D-homophenylalanine analogues as P3 residue: part 2

A series of highly potent substrate-analogue factor Xa inhibitors containing D-homophenylalanine analogues as the P3 residue has been identified by systematic optimization of a previously described inhibitor structure. An initial lead, benzylsulfonyl-D-hPhe-Gly-4-amidinobenzylamide (3), inhibits fXa with an inhibition constant of 6.0 nM. Most modifications of the P2 amino acid and P4 benzylsulfonyl group did not improve the affinity and selectivity of the compounds as fXa inhibitors. In contrast, further variation at the P3 position led to inhibitors with significantly enhanced potency and selectivity. Inhibitor 27, benzylsulfonyl-D-homo-2-pyridylalanyl(N-oxide)-Gly-4-amidinobenzylamide, inhibits fXa with a K(i) value of 0.32 nM. The inhibitor has strong anticoagulant activity in plasma and doubles the activated partial thromboplastin time and prothrombin time at concentrations of 280 nM and 170 nM, respectively. Compound 27 inhibits the prothrombinase complex with an IC(50) value of 5 nM and is approximately 50 times more potent than the reference inhibitor DX-9065a in this assay.