Difluoromethyl-1,3,4-oxadiazoles are slow-binding substrate analog inhibitors of histone deacetylase 6 with unprecedented isotype selectivity.

Histone deacetylase 6 (HDAC6) is an attractive drug development target because of its role in the immune response, neuropathy, and cancer. Knockout mice develop normally and have no apparent phenotype, suggesting that selective inhibitors should have an excellent therapeutic window. Unfortunately, current HDAC6 inhibitors have only moderate selectivity and may inhibit other HDAC subtypes at high concentrations, potentially leading to side effects. Recently, substituted oxadiazoles have attracted attention as a promising novel HDAC inhibitor chemotype, but their mechanism of action is unknown. Here, we show that compounds containing a difluoromethyl-1,3,4-oxadiazole (DFMO) moiety are potent and single-digit nanomolar inhibitors with an unprecedented greater than 104-fold selectivity for HDAC6 over all other HDAC subtypes. By combining kinetics, X-ray crystallography, and mass spectrometry, we found that DFMO derivatives are slow-binding substrate analogs of HDAC6 that undergo an enzyme-catalyzed ring opening reaction, forming a tight and long-lived enzyme-inhibitor complex. The elucidation of the mechanism of action of DFMO derivatives paves the way for the rational design of highly selective inhibitors of HDAC6 and possibly of other HDAC subtypes as well with potentially important therapeutic implications.

Mechanistic and Structural Insights on Difluoromethyl-1,3,4-oxadiazole Inhibitors of HDAC6.

Histone deacetylase 6 (HDAC6) is increasingly recognized for its potential in targeted disease therapy. This study delves into the mechanistic and structural nuances of HDAC6 inhibition by difluoromethyl-1,3,4-oxadiazole (DFMO) derivatives, a class of non-hydroxamic inhibitors with remarkable selectivity and potency. Employing a combination of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) kinetic experiments, comprehensive enzymatic characterizations, and X-ray crystallography, we dissect the intricate details of the DFMO-HDAC6 interaction dynamics. More specifically, we find that the chemical structure of a DMFO and the binding mode of its difluoroacetylhydrazide derivative are crucial in determining the predominant hydrolysis mechanism. Our findings provide additional insights into two different mechanisms of DFMO hydrolysis, thus contributing to a better understanding of the HDAC6 inhibition by oxadiazoles in disease modulation and therapeutic intervention.

Pharmacophore-based virtual screening of ZINC database, molecular modeling and designing new derivatives as potential HDAC6 inhibitors.

To date, many HDAC6 inhibitors have been identified and developed but none is clinically approved as of now. Through this study, we aim to obtain novel HDAC6 selective inhibitors and provide new insights into the detailed structural design of potential HDAC6 inhibitors. A HypoGen-based 3D QSAR HDAC6 pharmacophore was built and used as a query model to screen approximately 8 million ZINC database compounds. First, the ZINC Database was filtered using ADMET, followed by pharmacophore-based library screening. Using fit value and estimated activity cutoffs, a final set of 54 ZINC hits was obtained that were further investigated using molecular docking with the crystal structure of human histone deacetylase 6 catalytic domain 2 in complex with Trichostatin A (PDB ID: 5EDU). Through detailed in silico screening of the ZINC database, we shortlisted three hits as the lead molecules for designing novel HDAC6 inhibitors with better efficacy. Docking with 5EDU, followed by ADMET and TOPKAT analysis of modified ZINC hits provided 9 novel potential HDAC6 inhibitors that possess better docking scores and 2D interactions as compared to the control ZINC hit molecules. Finally, a 50 ns MD analysis run followed by Protein-Ligand Interaction Energy (PLIE) analysis of the top scored hits provided a novel molecule N1 that showed promisingly similar results to that of Ricolinostat (a known HDAC6 inhibitor). The comparable result of the designed hits to established HDAC6 inhibitors suggests that these compounds might prove to be successful HDAC6 inhibitors in future. Designed novel hits that might act as good HDAC6 inhibitors derived from ZINC database using combined molecular docking and modeling approaches.

Aromatic Ring Fluorination Patterns Modulate Inhibitory Potency of Fluorophenylhydroxamates Complexed with Histone Deacetylase 6.

Bavarostat (EKZ-001) is a selective inhibitor of histone deacetylase 6 (HDAC6) that contains a meta-fluorophenylhydroxamate Zn2+-binding group. The recently determined crystal structure of its complex with HDAC6 from Danio rerio (zebrafish) revealed that the meta-fluoro substituent binds exclusively in an aromatic crevice defined by F583 and F643 rather than being oriented out toward solvent. To explore the binding of inhibitor C-F groups in this fluorophilic crevice, we now report a series of 10 simple fluorophenylhydroxamates bearing one or more fluorine atoms with different substitution patterns. Inhibitory potencies against human and zebrafish HDAC6 range widely from 121 to >30,000 nM. The best inhibitory potency is measured for meta-difluorophenylhydroxamate (5) with IC50 = 121 nM against human HDAC6; the worst inhibitory potencies are measured for ortho-fluorophenylhydroxamate (1) as well as fluorophenylhydroxamates 4, 7, 9, and 10, although there are some variations in activity trends against human and zebrafish HDAC6. These studies show that aromatic ring fluorination at the meta position(s) does not improve inhibitory activity against human HDAC6 relative to the nonfluorinated parent compound phenylhydroxamate (IC50 = 120 nM), but meta-fluorination does not seriously compromise inhibitory activity either. Crystal structures of selected zebrafish HDAC6-fluorophenylhydroxamate complexes reveal that the fluoroaromatic ring is uniformly accommodated in the F583-F643 aromatic crevice, so ring fluorination does not perturb the inhibitor binding conformation. However, hydroxamate-Zn2+ coordination is bidentate for some inhibitors and monodentate for others. These studies will inform design strategies underlying the design of 18F-labeled HDAC6 inhibitors intended for positron emission tomography.

Do biological activities of selective histone deacetylase 6 (HDAC6) inhibitors rely on the modification of cap group?

Nowadays, significant progress has been made in the development of selective histone deacetylase 6 (HDAC6) inhibitors, exerting great potential in the treatment of various malignant tumors and neurodegenerative diseases. Previously, selective inhibitory activities of HDAC inhibitors were generally considered sensitive to the interactions between the Cap group and the binding site of HDAC6, and a large number of selective HDAC6 inhibitors have been designed and synthesized based on the strategy. However, some inhibitors without Cap group could also exhibit excellent potency and selective inhibition towards HDAC6, and in this study, BRD9757 and compound 8, as capless selective HDAC6 inhibitors, were selected as molecular probes to explore the difference of their binding interactions in HDAC1&6. Through the analysis of binding-free energies and conformational rearrangements after 1 µs molecular dynamics simulation, it could be learned that although the residues in the binding site remained highly consistent, the binding mechanisms of BRD9757 and compound 8 in HDAC1&6 were different, which will provide valuable hints for the discovery of novel selective HDAC6 inhibitors.

The extracellular HDAC6 ZnF UBP domain modulates the actin network and post-translational modifications of Tau.

BACKGROUND: Microtubule-associated protein Tau undergoes aggregation in Alzheimer`s disease (AD) and a group of other related diseases collectively known as Tauopathies. In AD, Tau forms aggregates, which are deposited intracellularly as neurofibrillary tangles. Histone deacetylase-6 (HDAC6) plays an important role in aggresome formation, where it recruits polyubiquitinated aggregates to the motor protein dynein. METHODS: Here, we have studied the effects of HDAC6 ZnF UBP on Tau phosphorylation, ApoE localization, GSK-3ß regulation and cytoskeletal organization in neuronal cells by immunocytochemical analysis. This analysis reveals that the cell exposure to the UBP-type zinc finger domain of HDAC6 (HDAC6 ZnF UBP) can modulate Tau phosphorylation and actin cytoskeleton organization. RESULTS: HDAC6 ZnF UBP treatment to cells did not affect their viability and resulted in enhanced neurite extension and formation of structures similar to podosomes, lamellipodia and podonuts suggesting the role of this domain in actin re-organization. Also, HDAC6 ZnF UBP treatment caused increase in nuclear localization of ApoE and tubulin localization in microtubule organizing centre (MTOC). Therefore, our studies suggest the regulatory role of this domain in different aspects of neurodegenerative diseases. Upon HDAC6 ZnF UBP treatment, inactive phosphorylated form of GSK-3ß increases without any change in total GSK-3ß level. CONCLUSIONS: HDAC6 ZnF UBP was found to be involved in cytoskeletal re-organization by modulating actin dynamics and tubulin localization. Overall, our study suggests that ZnF domain of HDAC6 performs various regulatory functions apart from its classical function in aggresome formation in protein misfolding diseases. Video abstract.

HDAC6 as privileged target in drug discovery: A perspective.

HDAC6, a class IIB HDAC isoenzyme, stands unique in its structural and physiological functions. Besides histone modification, largely due to its cytoplasmic localization, HDAC6 also targets several non-histone proteins including Hsp90, α-tubulin, cortactin, HSF1, etc. Thus, it is one of the key regulators of different physiological and pathological disease conditions. HDAC6 is involved in different signaling pathways associated with several neurological disorders, various cancers at early and advanced stage, rare diseases and immunological conditions. Therefore, targeting HDAC6 has been found to be effective for various therapeutic purposes in recent years. Though several HDAC6 inhibitors (HDAC6is) have been developed till date, only two ACY-1215 (ricolinostat) and ACY-241 (citarinostat) are in the clinical trials. A lot of work is still needed to pinpoint strictly selective as well as potent HDAC6i. Considering the recent crystal structure of HDAC6, novel HDAC6is of significant therapeutic value can be designed. Notably, the canonical pharmacophore features of HDAC6is consist of a zinc binding group (ZBG), a linker function and a cap group. Significant modifications of cap function may lead to achieve better selectivity of the inhibitors. This review details the study about the structural biology of HDAC6, the physiological and pathological role of HDAC6 in several disease states and the detailed structure-activity relationships (SARs) of the known HDAC6is. This detailed review will provide key insights to design novel and highly effective HDAC6i in the future.

Overcome the tumor immunotherapy resistance by combination of the HDAC6 inhibitors with antitumor immunomodulatory agents.

Tumor immunotherapy is currently subject of intense scientific and clinical developments. In previous decade, therapists used natural immune system from the human body to treat several diseases. Although tumor immune disease is a big challenge, combinatorial therapeutic strategy has been succeeded to show the clinical significance. In this context, we discuss the HDAC6 and tumor immune diseases relationship. Also, we summarized the current state of knowledge that based on the combination treatments of the HDAC6 inhibitors (HDAC6is) with antitumor immunomodulatory agents. We observed that, the combination therapies slow down the tumor immune diseases by blocking the aggresome and proteasome pathway. The combination therapy was able to reduce M2 macrophage and increasing PD-L1 blockade sensitivity. Most importantly, multiple combinations of HDAC6is with other agents may consider as potential strategies to treat tumor immune diseases, by reducing the side effects and improve efficacy for the future clinical development.

HDAC6 ZnF UBP as the Modifier of Tau Structure and Function.

Histone deacetylase 6 is a class II histone deacetylase primarily present in the cytoplasm and involved in the regulation of various cellular functions. It consists of two catalytic deacetylase domains and a unique zinc finger ubiquitin binding protein domain, which sets it apart from other HDACs. HDAC6 is known to regulate cellular activities by modifying the function of microtubules, HSP90, and cortactin through deacetylation. Apart from the catalytic activity of HDAC6, it interacts with other proteins through either the SE14 domain or the ZnF UBP domain to modulate their functions. Here, we have studied the role of the HDAC6 ZnF UBP domain as a modifier of Tau aggregation by its direct interaction with the polyproline region/repeat region of Tau. Interaction of HDAC6 ZnF UBP with Tau was found to reduce the propensity of Tau to self-aggregate and to disaggregate preformed aggregates in a concentration-dependent manner and also bring about the conformational changes in Tau protein. The interaction of HDAC6 ZnF UBP with Tau results in its degradation, suggesting either proteolytic activity of HDAC6 ZnF UBP or its role in enhancing autoproteolysis of Tau.

The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries.

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic hydrolase acting mostly on nonhistone protein substrates. Investigations of the substrate specificity of HDAC6 are confounded by the presence of 2 catalytically active deacetylase domains (DD1 and DD2). In this study, acetylome peptide microarrays and peptide libraries were used to map the substrate specificity of DD1 and DD2 of human HDAC6. The results show that DD1 is solely responsible for the deacetylation of substrates harboring the acetyllysine at their C terminus, whereas DD2 exclusively deacetylates peptides with an internal acetyllysine residue. Also, statistical analysis of the deacetylation data revealed amino acid preferences at individual positions flanking the acetyllysine, where glycine and arginine residues are favored at positions N-terminal to the central acetyllysine; negatively charged glutamate is strongly disfavored throughout the sequence. Finally, the deacylation activity of HDAC6 was profiled by using a panel of acyl derivatives of the optimized peptide substrate and showed that HDAC6 acts as a proficient deformylase. Our data thus offer a detailed insight into the substrate preferences of the individual HDAC6 domains at the peptide level, and these findings can in turn help in elucidating the biologic roles of the enzyme and facilitate the development of new domain-specific inhibitors as research tools or therapeutic agents.-Kutil, Z., Skultetyova, L., Rauh, D., Meleshin, M., Snajdr, I., Novakova, Z., Mikesova, J., Pavlicek, J., Hadzima, M., Baranova, P., Havlinova, B., Majer, P., Schutkowski, M., Barinka, C. The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries.

Exploring the inhibitory activity of valproic acid against the HDAC family using an MMGBSA approach.

Valproic acid (VPA) is a compound currently used in clinical practice for the treatment of epilepsy as well as bipolar and mood disorders. VPA targets histone deacetylases (HDACs), which participate in the removal of acetyl groups from lysine in several proteins, regulating a wide variety of functions within the organism. An imbalance or malfunction of these enzymes is associated with the development and progression of several diseases, such as cancer and neurodegenerative diseases. HDACs are divided into four classes, but VPA only targets Class I (HDAC1-3 and 8) and Class IIa (HDAC4-5, 7 and 9) HDACs; however, structural and energetic information regarding the manner by which VPA inhibits these HDACs is lacking. Here, the structural and energetic features that determine this recognition were studied using molecular docking and molecular dynamics (MD) simulation. It was found that VPA reaches the catalytic site in HDAC1-3 and 7, whereas in HDAC6, VPA only reaches the catalytic tunnel. In HDAC4, VPA was bound adjacent to L1 and L2, a zone that participates in corepressor binding, and in HDAC8, VPA was bound to the hydrophobic active site channel (HASC), in line with previous reports.

Discovery of specific HDAC6 inhibitor with anti-metastatic effects in pancreatic cancer cells through virtual screening and biological evaluation.

Histone deacetylase 6 (HDAC6) has been demonstrated to play a major role in cell motility and aggresome formation, and HDAC6 inhibition is therefore considered as a promising epigenetic strategy for cancer treatment. At present, only a minority of compounds have been reported as HDAC6 inhibitors, so specific HDAC6 inhibitors with safety profile need to be discovered urgently. In this paper, HDAC6 inhibitors with diverse structures were used to generate the pharmacophore model by ligand-based method, which contained two hydrogen bond acceptors and two hydrophobic groups. A combined virtual screening based on pharmacophore model and molecular docking was adopted to screen potential HDAC6 inhibitors. Subsequently, the HDAC6 inhibitory activity of the hit compounds were evaluated using an in vitro enzyme binding inhibition assay. The experimental results illustrated that cefoperazone sodium had the strongest inhibitory effect on HDAC6 among the six screened compounds, and its IC50 value was 8.59 ± 1.06 µM. Cefoperazone sodium significantly catalyzed the hyperacetylation of α-tubulin but not histone H3, proving that cefoperazone sodium was a selective inhibitor of HDAC6. Since the expression of HDAC6 plays an important role in cancer metastasis, the effects of cefoperazone sodium on migration and invasion of human pancreatic cancer PANC-1 cells were further investigated by wound healing and transwell chamber assays. It was found that cefoperazone sodium could evidently inhibit the migration and invasion of PANC-1 cells. Furthermore, the binding pattern of inhibitor at the active site of the crystal structure was revealed by molecular docking, providing a reference value for the structural design and optimization of HDAC6 inhibitors. This study provides a systematic virtual screening approach for discovering HDAC6 active inhibitors, and by which the specific effect of cefoperazone sodium against HDAC6 was found, suggesting its potential application on cancer therapy.

Unusual zinc-binding mode of HDAC6-selective hydroxamate inhibitors.

Histone deacetylases (HDACs) regulate myriad cellular processes by catalyzing the hydrolysis of acetyl-l-lysine residues in histone and nonhistone proteins. The Zn2+-dependent class IIb enzyme HDAC6 regulates microtubule function by deacetylating α-tubulin, which suppresses microtubule dynamics and leads to cell cycle arrest and apoptosis. Accordingly, HDAC6 is a target for the development of selective inhibitors that might be useful in new therapeutic approaches for the treatment of cancer, neurodegenerative diseases, and other disorders. Here, we present high-resolution structures of catalytic domain 2 from Danio rerio HDAC6 (henceforth simply "HDAC6") complexed with compounds that selectively inhibit HDAC6 while maintaining nanomolar inhibitory potency: N-hydroxy-4-[(N(2-hydroxyethyl)-2-phenylacetamido)methyl)-benzamide)] (HPB), ACY-1215 (Ricolinostat), and ACY-1083. These structures reveal that an unusual monodentate Zn2+ coordination mode is exploited by sterically bulky HDAC6-selective phenylhydroxamate inhibitors. We additionally report the ultrahigh-resolution structure of the HDAC6-trichostatin A complex, which reveals two Zn2+-binding conformers for the inhibitor: a major conformer (70%) with canonical bidentate hydroxamate-Zn2+ coordination geometry and a minor conformer (30%) with monodentate hydroxamate-Zn2+ coordination geometry, reflecting a free energy difference of only 0.5 kcal/mol. The minor conformer is not visible in lower resolution structure determinations. Structural comparisons of HDAC6-inhibitor complexes with class I HDACs suggest active site features that contribute to the isozyme selectivity observed in biochemical assays.

Structural Basis of Catalysis and Inhibition of HDAC6 CD1, the Enigmatic Catalytic Domain of Histone Deacetylase 6.

Histone deacetylase 6 (HDAC6) is emerging as a target for inhibition in therapeutic strategies aimed at treating cancer, neurodegenerative disease, and other disorders. Among the metal-dependent HDAC isozymes, HDAC6 is unique in that it contains two catalytic domains, CD1 and CD2. CD2 is a tubulin deacetylase and a tau deacetylase, and the development of HDAC6-selective inhibitors has focused exclusively on this domain. In contrast, there is a dearth of structural and functional information regarding CD1, which exhibits much narrower substrate specificity in comparison with CD2. As the first step in addressing the CD1 information gap, we now present X-ray crystal structures of seven inhibitor complexes with wild-type, Y363F, and K330L HDAC6 CD1. These structures broaden our understanding of molecular features that are important for catalysis and inhibitor binding. The active site of HDAC6 CD1 is wider than that of CD2, which is unexpected in view of the narrow substrate specificity of CD1. Amino acid substitutions between HDAC6 CD1 and CD2, as well as conformational differences in conserved residues, define striking differences in active site contours. Catalytic activity measurements with HDAC6 CD1 confirm the preference for peptide substrates containing C-terminal acetyllysine residues. However, these measurements also show that CD1 exhibits weak activity for peptide substrates bearing certain small amino acids on the carboxyl side of the scissile acetyllysine residue. Taken together, these results establish a foundation for understanding the structural basis of HDAC6 CD1 catalysis and inhibition, pointing to possible avenues for the development of HDAC6 CD1-selective inhibitors.

Histone deacetylase 6 (HDAC6) deacetylates extracellular signal-regulated kinase 1 (ERK1) and thereby stimulates ERK1 activity.

Histone deacetylase 6 (HDAC6), a class IIb HDAC, plays an important role in many biological and pathological processes. Previously, we found that ERK1, a downstream kinase in the mitogen-activated protein kinase signaling pathway, phosphorylates HDAC6, thereby increasing HDAC6-mediated deacetylation of α-tubulin. However, whether HDAC6 reciprocally modulates ERK1 activity is unknown. Here, we report that both ERK1 and -2 are acetylated and that HDAC6 promotes ERK1 activity via deacetylation. Briefly, we found that both ERK1 and -2 physically interact with HDAC6. Endogenous ERK1/2 acetylation levels increased upon treatment with a pan-HDAC inhibitor, an HDAC6-specific inhibitor, or depletion of HDAC6, suggesting that HDAC6 deacetylates ERK1/2. We also noted that the acetyltransferases CREB-binding protein and p300 both can acetylate ERK1/2. Acetylated ERK1 exhibits reduced enzymatic activity toward the transcription factor ELK1, a well-known ERK1 substrate. Furthermore, mass spectrometry analysis indicated Lys-72 as an acetylation site in the ERK1 N terminus, adjacent to Lys-71, which binds to ATP, suggesting that acetylation status of Lys-72 may affect ERK1 ATP binding. Interestingly, an acetylation-mimicking ERK1 mutant (K72Q) exhibited less phosphorylation than the WT enzyme and a deacetylation-mimicking mutant (K72R). Of note, the K72Q mutant displayed decreased enzymatic activity in an in vitro kinase assay and in a cellular luciferase assay compared with the WT and K72R mutant. Taken together, our findings suggest that HDAC6 stimulates ERK1 activity. Along with our previous report that ERK1 promotes HDAC6 activity, we propose that HDAC6 and ERK1 may form a positive feed-forward loop, which might play a role in cancer.

Development of Thiazolidinedione-Based HDAC6 Inhibitors to Overcome Methamphetamine Addiction.

Thiazolidinedione is a five-membered heterocycle that is widely used in drug discovery endeavors. In this study, we report the design, synthesis, and biological evaluation of a series of thiazolidinedione-based HDAC6 inhibitors. In particular, compound 6b exerts an excellent inhibitory activity against HDAC6 with an IC50 value of 21 nM, displaying a good HDAC6 selectivity over HDAC1. Compound 6b dose-dependently induces the acetylation level of α-tubulin via inhibition of HDAC6 in human neuroblastoma SH-SY5Y cell line. Moreover, compound 6b efficiently reverses methamphetamine-induced morphology changes of SH-SY5Y cells via regulating acetylation landscape of α-tubulin. Collectively, compound 6b represents a novel HDAC6-isoform selective inhibitor and demonstrates promising therapeutic potential for the treatment of methamphetamine addiction.

Entropy as a Driver of Selectivity for Inhibitor Binding to Histone Deacetylase 6.

Among the metal-dependent histone deacetylases, the class IIb isozyme HDAC6 is remarkable because of its role in the regulation of microtubule dynamics in the cytosol. Selective inhibition of HDAC6 results in microtubule hyperacetylation, leading to cell cycle arrest and apoptosis, which is a validated strategy for cancer chemotherapy and the treatment of other disorders. HDAC6 inhibitors generally consist of a Zn2+-binding group such as a hydroxamate, a linker, and a capping group; the capping group is a critical determinant of isozyme selectivity. Surprisingly, however, even "capless" inhibitors exhibit appreciable HDAC6 selectivity. To probe the chemical basis for this selectivity, we now report high-resolution crystal structures of HDAC6 complexed with capless cycloalkyl hydroxamate inhibitors 1-4. Each inhibitor hydroxamate group coordinates to the catalytic Zn2+ ion with canonical bidentate geometry. Additionally, the olefin moieties of compounds 2 and 4 bind in an aromatic crevice between the side chains of F583 and F643. Reasoning that similar binding could be achieved in the representative class I isozyme HDAC8, we employed isothermal titration calorimetry to study the thermodynamics of inhibitor binding. These measurements indicate that the entropy of inhibitor binding is generally positive for binding to HDAC6 and negative for binding to HDAC8, resulting in ≤313-fold selectivity for binding to HDAC6 relative to HDAC8. Thus, favorable binding entropy contributes to HDAC6 selectivity. Notably, cyclohexenyl hydroxamate 2 represents a promising lead for derivatization with capping groups that may further enhance its impressive 313-fold thermodynamic selectivity for HDAC6 inhibition.

Inhibition of HDAC6 activity through interaction with RanBPM and its associated CTLH complex.

BACKGROUND: Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase that promotes many cellular processes that lead to cell transformation and tumour development. We previously documented an interaction between Ran-Binding Protein M (RanBPM) and HDAC6 and found that RanBPM expression inhibits HDAC6 activity. RanBPM is part of a putative E3 ubiquitin ligase complex, termed the C-terminal to LisH (CTLH) complex. Here, we investigated the involvement of the CTLH complex on HDAC6 inhibition and assessed the outcome of this regulation on the cellular motility induced by HDAC6. METHODS: Cell lines (Hela, HEK293 and immortalized mouse embryonic fibroblasts) stably or transiently downregulated for several components of the CTLH complex were employed for the assays used in this study. Interactions of HDAC6, RanBPM and muskelin were assessed by co-immunoprecipitations. Quantifications of western blot analyses were employed to evaluate acetylated α-tubulin levels. Confocal microscopy analyses were used to determine microtubule association of HDAC6 and CTLH complex members. Cell migration was evaluated using wound healing assays. RESULTS: We demonstrate that RanBPM-mediated inhibition of HDAC6 is dependent on its association with HDAC6. We show that, while HDAC6 does not require RanBPM to associate with microtubules, RanBPM association with microtubules requires HDAC6. Additionally, we show that Twa1 (Two-hybrid-associated protein 1 with RanBPM) and MAEA (Macrophage Erythroblast Attacher), two CTLH complex members, also associate with α-tubulin and that muskelin, another component of the CTLH complex, is able to associate with HDAC6. Downregulation of CTLH complex members muskelin and Rmnd5A (Required for meiotic nuclear division homolog A) resulted in decreased acetylation of HDAC6 substrate α-tubulin. Finally, we demonstrate that the increased cell migration resulting from downregulation of RanBPM is due to the relief in inhibition of HDAC6 α-tubulin deacetylase activity. CONCLUSIONS: Our work shows that RanBPM, together with the CTLH complex, associates with HDAC6 and restricts cell migration through inhibition of HDAC6 activity. This study uncovers a novel function for the CTLH complex and suggests that it could have a tumour suppressive role in restricting HDAC6 oncogenic properties.

An assessment of crucial structural contributors of HDAC6 inhibitors through fragment-based non-linear pattern recognition and molecular dynamics simulation approaches.

Amidst the Zn2+-dependant isoforms of the HDAC family, HDAC6 has emerged as a potential target associated with an array of diseases, especially cancer and neuronal disorders like Rett's Syndrome, Alzheimer's disease, Huntington's disease, etc. Also, despite the availability of a handful of HDAC inhibitors in the market, their non-selective nature has restricted their use in different disease conditions. In this situation, the development of selective and potent HDAC6 inhibitors will provide efficacious therapeutic agents to treat different diseases. In this context, this study has been carried out to evaluate the potential structural contributors of quinazoline-cap-containing HDAC6 inhibitors via machine learning (ML), conventional classification-dependant QSAR, and MD simulation-based binding mode of interaction analysis toward HDAC6 binding. This combined conventional and modern molecular modeling study has revealed the significance of the quinazoline moiety, substitutions present at the quinazoline cap group, as well as the importance of molecular property, number of hydrogen bond donor-acceptor functions, carbon-chlorine distance that significantly affects the HDAC6 binding of these inhibitors, subsequently affecting their potency . Interestingly, the study also revealed that the substitutions such as the chloroethyl group, and bulky quinazolinyl cap group can affect the binding of the cap function with the amino acid residues present in the loops proximal to the catalytic site of HDAC6. Such contributions of cap groups can lead to both stabilization and destabilization of the cap function after occupying the hydrophobic catalytic site by the aryl hydroxamate linker-ZBG functions.