Class IIb Histone Deacetylase Research Articles

ClassIIb histone deacetylase participates in perioperative neurocognitive disorders in elderly mice via HSP90/GR signaling pathway.

Multiple factors contribute to the development of perioperative neurocognitive disorders (PND). This study was designed to investigate whether Histone Deacetylase 6 (HDAC6) was involved in the formation of postoperative cognitive dysfunction in elderly mice by regulating the degree of acetylation of heat shock protein (HSP90) and related protein functions and quantities. C57BL/6J male mice were randomly divided into six groups: control naive (group Control), anesthesia (group Anesthesia), splenectomy surgery (group Surgery), splenectomy surgery plus dissolvent (group Vehicles), splenectomy surgery plus the inhibitor ACY-1215 (group Ricolinostat), and splenectomy surgery plus the inhibitor RU-486(group Mifepristone). After the mice were trained for Morris Water Maze (MWM) test for five days, anesthesia and operational surgery were carried out the following day. Cognitive function was assessed on the 1st, 3rd and 7th days post-surgery. The hippocampi were harvested on days 1, 3, and 7 post-surgeries for Western blots and ELISA assays. Mice with the splenectomy surgery displayed the activation of the hypothalamic-pituitary-adrenal axis (HPA-axis), marked an increase in adrenocorticotropic hormone (ACTH), glucocorticoid, mineralocorticoid at the molecular level and impaired spatial memory in the MWM test. The hippocampus of surgical groups showed a decrease in acetylated HSP90, a rise in glucocorticoid receptor (GR)-HSP90 association, and an increase in GR phosphorylation and translocation. HDAC6 was increased after the surgical treated. Using two specific inhibitors, HDAC6 inhibitor Ricolinostat (ACY-1215) and GR inhibitor Mifepristone (RU-486), can partially mitigate the effects caused by surgical operation. Abdominal surgery may impair hippocampal spatial memory, possibly through the HDAC6-triggered increase in the function of HSP90, consequently strengthening the negative role of steroids in cognitive function. Targeting HDAC6- HSP90/GR signaling may provide a potential avenue for the treatment of the impairment of cognitive function after surgery.

Synthesis and biological evaluation of ortho-phenyl phenylhydroxamic acids containing phenothiazine with improved selectivity for class IIa histone deacetylases

Class IIa histone deacetylases (HDACs) have been linked to tumorigenesis in various cancers. Previously, we designed phenylhydroxamic acid LH4f as a potent class IIa HDAC inhibitor. However, it also unselectively inhibited class I and class IIb HDACs. To enhance the compound’s selectivity towards class IIa HDACs, the ortho-phenyl group from the selective HDAC7 inhibitor 1 is incorporated into ortho position of the phenylhydroxamic acid in LH4f. Compared to LH4f, most resulting compounds displayed substantially improved selectivity towards the class IIa HDACs. Notably, compound 7 g exhibited the strongest HDAC9 inhibition with an IC50 value of 40 nM. Molecular modelling further identified the key interactions of compound 7 g bound to HDAC9. Compound 7 g significantly inhibited several human cancer cells, induced apoptosis, modulated caspase-related proteins as well as p38, and caused DNA damage. These findings suggest the potential of class IIa HDAC inhibitors as lead compounds for the development of cancer therapeutics.

HDAC6 Deletion Decreases Pristane-induced Inflammation.

Systemic lupus erythematosus is an autoimmune disease characterized by excessive inflammation and production of pathogenic Abs. Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase. It has been reported that selective HDAC6 inhibition decreases inflammation in lupus mouse models. In this study, sex- and age-matched wild-type (WT) and HDAC6-/- mice on the C57BL/6 background were administered 0.5 ml of pristane or PBS i.p. at 8-12 wk of age and were euthanized 10 d later. At sacrifice, body weight and spleen weight were measured, sera were collected, and splenocytes and peritoneal cells were harvested for flow cytometry. We found pristane administration increased the spleen weight with no difference between WT and HDAC6-/- mice. Pristane administration promoted the population of CD11b+Ly6C++ inflammatory monocytes and CD11b+Ly6G+ neutrophils. Peritoneal recruitment of these inflammatory monocytes and neutrophils was significantly decreased in HDAC6-/- mice compared with the WT mice. Flow cytometry results showed that the number of CD69+ T and B cells was increased in HDAC6-/- mice. Pristane administration also induced the IFN signature genes as determined by RT-qPCR. Furthermore, IFN signature genes were not affected in HDAC6-/- mice compared with the WT mice. In vitro studies in J774A.1 cells revealed that the selective HDAC6 inhibitor (ACY-738) increased acetylation of NF-κB while increasing Stat1 phosphorylation, which resulted in inducible NO synthase production in LPS/IFN-γ-stimulated cells. Taken together, these results demonstrate that although HDAC6 inhibition may inhibit some inflammatory pathways, others remain unaffected.

Histone deacetylase 6's function in viral infection, innate immunity, and disease: latest advances.

In the family of histone-deacetylases, histone deacetylase 6 (HDAC6) stands out. The cytoplasmic class IIb histone deacetylase (HDAC) family is essential for many cellular functions. It plays a crucial and debatable regulatory role in innate antiviral immunity. This review summarises the current state of our understanding of HDAC6's structure and function in light of the three mechanisms by which it controls DNA and RNA virus infection: cytoskeleton regulation, host innate immune response, and autophagy degradation of host or viral proteins. In addition, we summed up how HDAC6 inhibitors are used to treat a wide range of diseases, and how its upstream signaling plays a role in the antiviral mechanism. Together, the findings of this review highlight HDAC6's importance as a new therapeutic target in antiviral immunity, innate immune response, and some diseases, all of which offer promising new avenues for the development of drugs targeting the immune response.

Anticancer Study of a Novel Pan-HDAC Inhibitor MPT0G236 in Colorectal Cancer Cells.

Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies and a leading cause of cancer worldwide. Histone deacetylases (HDACs), which regulate cell proliferation and survival, are associated with the development and progression of cancer. Moreover, HDAC inhibitors are promising therapeutic targets, with five HDAC inhibitors approved for cancer treatment to date. However, their safety profile necessitates the exploration of well-tolerated HDAC inhibitors that can be used in cancer therapeutic strategies. In this study, the pan-HDAC inhibitor MPT0G236 reduced the viability and inhibited the proliferation of human colorectal cancer cells, and normal human umbilical vein endothelial cells (HUVECs) showed reduced sensitivity. These findings indicated that MPT0G236 specifically targeted malignant tumor cells. Notably, MPT0G236 significantly inhibited the activities of HDAC1, HDAC2, and HDAC3, Class I HDACs, as well as HDAC6, a Class IIb HDAC, at low nanomolar concentrations. Additionally, it promoted the accumulation of acetyl-α-tubulin and acetyl-histone H3 in HCT-116 and HT-29 cells in a concentration-dependent manner. Furthermore, MPT0G236 treatment induced G2/M cell cycle arrest in CRC cells by initially regulating the levels of cell-cycle-related proteins, such as p-MPM2; specifically reducing p-cdc2 (Y15), cyclin B1, and cdc25C levels; and subsequently inducing apoptosis through the caspase-dependent pathways and PARP activation. Our findings demonstrate that MPT0G236 exhibits significant anticancer activity in human colorectal cancer cells.

Exploring structural requirements of HDAC10 inhibitors through comparative machine learning approaches

Histone deacetylase (HDAC) inhibitors are in the limelight of anticancer drug development and research. HDAC10 is one of the class-IIb HDACs, responsible for cancer progression. The search for potent and effective HDAC10 selective inhibitors is going on. However, the absence of human HDAC10 crystal/NMR structure hampers the structure-based drug design of HDAC10 inhibitors. Different ligand-based modeling techniques are the only hope to speed up the inhibitor design. In this study, we applied different ligand-based modeling techniques on a diverse set of HDAC10 inhibitors (n = 484). Machine learning (ML) models were developed that could be used to screen unknown compounds as HDAC10 inhibitors from a large chemical database. Moreover, Bayesian classification and Recursive partitioning models were used to identify the structural fingerprints regulating the HDAC10 inhibitory activity. Additionally, a molecular docking study was performed to understand the binding pattern of the identified structural fingerprints towards the active site of HDAC10. Overall, the modeling insight might offer helpful information for medicinal chemists to design and develop efficient HDAC10 inhibitors.

Targeting HDAC6 in the Dorsal Root Ganglia Attenuates Peripheral Nerve Injury‐induced Hypersensitivity

Histone deacetylase 6 (HDAC6) is a Class IIb histone deacetylase, which is primarily localized to the cytoplasm and plays an important role in cell structure and dynamism, transcriptional repression, exocytosis and endocytocis. Preclinical evidence has suggested that HDAC6 inhibitors alleviate signs of chemotherapy‐induced peripheral neuropathy (CIPN), such as mechanical allodynia. However, no group to our knowledge has investigated the mechanism of action of HDAC6 inhibitors in a severe nerve injury model, which has a different pathophysiology than milder models such as CIPN. In this study we use genetically modified mice and the tibial spared nerve injury (SNI) model to determine the impact of dorsal root ganglion (DRG) specific HDAC6 knockout in the induction and maintenance of sensory hypersensitivity. Downregulation of HDAC6 in the DRG was achieved by injection of the left sciatic nerve of adult male HDAC6fl\fl mice with AAV8‐CMV‐Cre‐EGFP or AAV8‐CMV‐EGFP vectors. DRG‐knockdown of HDAC6 prevented the development of mechanical allodynia after SNI. Using in situ hybridization (RNAScope), we also demonstrate that HDAC6 is upregulated at three weeks after the induction of nerve injury in L3‐6 DRG neurons. Furthermore, treatment with the peripherally acting HDAC6 inhibitor ACY1215 (Regenacy Pharmaceuticals, Waltham, MA) after SNI leads to full recovery from mechanical allodynia. We are continuing this work by performing bulk RNA sequencing on L3‐6 DRGs from mice who received 21 days of treatment with ACY1215 or vehicle starting at three weeks after SNI or Sham operation, in order to understand the transcriptomic events and upstream pathways associated with recovery from prolonged neuropathic states.Our findings highlight a promising therapeutic role of HDAC6 inhibitors for the prevention or recovery from sensory hypersensitivity behaviors associated with prolonged peripheral nerve injury. Our future work aims to define the mechanisms underlying HDAC6 action in the DRG and identify new pathways associated with recovery from sensory hypersensitivity to influence novel treatment strategies for pain.

HDAC6 knockout alleviates pristane-induced lupus

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by excessive inflammation and production of pathogenic antibodies. Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase. It has been reported that selective HDAC6 inhibition decreases inflammation in lupus mice models. C57BL/6 mice develop SLE-like symptoms following pristane injection. In this study, sex and age-matched wild type and HDAC6−/− mice on the C57BL/6 background were administered 0.5 ml pristane or PBS intraperitoneally (i.p.) at 8–12 weeks-of-age and were euthanized 10 days later. At sacrifice, body weight and spleen weight were measured, sera were collected, splenocytes and peritoneal cells were harvested for flow cytometry. We found pristane administration increased the spleen weight with no difference between WT mice and HDAC6−/− mice. Flow cytometry results showed that there was no difference in T cell or B cell populations in the spleen. Pristane administration promoted the population of CD11b+ Ly6Chigh inflammatory monocytes and CD11b+Ly6G+ neutrophils. Peritoneal recruitment of these inflammatory monocytes and neutrophils in HDAC6−/− mice was significantly decreased compared to the WT mice. Pristane administration also induced the interferon (IFN) signature genes, like Mx1, Oas1a, Irf7, Irf9, Cxcl10, Isg15. qRT-PCR revealed that these IFN signature genes were decreased in HDAC6−/− mice compared to the WT mice. In summary, our results show that HDAC6 knockout inhibits the recruitment of inflammatory monocytes and neutrophils in the peritoneum in early inflammation response to pristane. HDAC6 deletion also inhibited the expression of IFN signature genes with pristine stimulation.

HDAC6: A Key Link Between Mitochondria and Development of Peripheral Neuropathy.

Peripheral neuropathy, which is the result of nerve damage from lesions or disease, continues to be a major health concern due to the common manifestation of neuropathic pain. Most investigations into the development of peripheral neuropathy focus on key players such as voltage-gated ion channels or glutamate receptors. However, emerging evidence points to mitochondrial dysfunction as a major player in the development of peripheral neuropathy and resulting neuropathic pain. Mitochondrial dysfunction in neuropathy includes altered mitochondrial transport, mitochondrial metabolism, as well as mitochondrial dynamics. The mechanisms that lead to mitochondrial dysfunction in peripheral neuropathy are poorly understood, however, the Class IIb histone deacetylase (HDAC6), may play an important role in the process. HDAC6 is a key regulator in multiple mechanisms of mitochondrial dynamics and may contribute to mitochondrial dysregulation in peripheral neuropathy. Accumulating evidence shows that HDAC6 inhibition is strongly associated with alleviating peripheral neuropathy and neuropathic pain, as well as mitochondrial dysfunction, in in vivo and in vitro models of peripheral neuropathy. Thus, HDAC6 inhibitors are being investigated as potential therapies for multiple peripheral neuropathic disorders. Here, we review emerging studies and integrate recent advances in understanding the unique connection between peripheral neuropathy and mitochondrial dysfunction through HDAC6-mediated interactions.

Inhibition of Class IIa HDACs improves endothelial barrier function in endotoxin-induced acute lung injury.

Acute lung injury (ALI) is an acute inflammatory process arises from a wide range of lung insults. A major cause of ALI is dysfunction of the pulmonary vascular endothelial barrier but the mechanisms involved are incompletely understood. The therapeutic potential of histone deacetylase (HDAC) inhibitors for the treatment of cardiovascular and inflammatory diseases is increasingly apparent, but the mechanisms by which HDACs regulate pulmonary vascular barrier function remain to be resolved. We found that specific Class IIa HDACs inhibitor, TMP269, significantly attenuated the lipopolysaccharide (LPS)-induced human lung microvascular endothelial cells (HLMVEC) barrier compromise in vitro and improved vascular barrier integrity and lung function in murine model of ALI in vivo. TMP269 decreased LPS-induced myosin light chain phosphorylation suggesting the role for Class IIa HDACs in LPS-induced cytoskeleton reorganization. TMP269 did not affect microtubule structure and tubulin acetylation in contrast to the HDAC6-specific inhibitor, Tubastatin A suggesting that Class IIa HDACs and HDAC6 (Class IIb) regulate endothelial cytoskeleton and permeability via different mechanisms. Furthermore, LPS increased the expression of ArgBP2 which has recently been attributed to HDAC-mediated activation of Rho. Depletion of ArgBP2 abolished the ability of LPS to disrupt barrier function in HLMVEC and both TMP269 and Tubastatin A decreased the level of ArgBP2 expression after LPS stimulation suggesting that both Class IIa and IIb HDACs regulate endothelial permeability via ArgBP2-dependent mechanism. Collectively, our data strongly suggest that Class IIa HDACs are involved in LPS-induced ALI in vitro and in vivo via specific mechanism which involved contractile responses, but not microtubule reorganization.

Structural Basis for the Selective Inhibition of HDAC10, the Cytosolic Polyamine Deacetylase.

The cytosolic class IIb histone deacetylase HDAC10 is an emerging target for drug design. As an inducer of autophagy, its selective inhibition suppresses the autophagic response that otherwise attenuates the efficacy of cytotoxic cancer chemotherapy drugs. HDAC10 is a zinc-dependent polyamine deacetylase exhibiting maximal catalytic activity against N8-acetylspermidine. As revealed in the structure of Danio rerio (zebrafish) HDAC10, two conserved structural motifs direct this narrow substrate specificity: a 310 helix containing the P(E,A)CE motif that sterically constricts the active site and an electrostatic "gatekeeper," E274, that confers selectivity for cationic polyamine substrates. To accelerate drug design efforts targeting human HDAC10, we now report the preparation of "humanized" zebrafish HDAC10 in which two amino acid substitutions, A24E and D94A, yield an active site contour more similar to that of human HDAC10. X-ray crystal structures of this HDAC10 variant complexed with Tubastatin A and indole analogues bearing pendant tertiary amines reveal that inhibitors capable of hydrogen bonding with gatekeeper E274 exhibit high affinity and selectivity for HDAC10 over HDAC6 (the other class IIb isozyme). Moreover, these structures reveal that the P(E,A)CE motif helix can shift by up to 2 Å to accommodate the binding of bulky inhibitors. Thus, slender polyamine-like inhibitor structures are not exclusively required for selective, high affinity binding to HDAC10. Indeed, the flexibility of the P(E,A)CE motif helix could conceivably enable the binding of certain protein substrates.

Inhibition of cell migration and induction of apoptosis by a novel class II histone deacetylase inhibitor, MCC2344

Epigenetic modifiers provide a new target for the development of anti-cancer drugs. The eraser histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase that targets various non-histone proteins such as transcription factors, nuclear receptors, cytoskeletal proteins, DNA repair proteins, and molecular chaperones. Therefore, it became an attractive target for cancer treatment. In this study, virtual screening was applied to the MicroCombiChem database with 1162 drug-like compounds to identify new HDAC6 inhibitors. Five compounds were tested in silico and in vitro as HDAC6 inhibitors. Both analyses revealed 1-cyclohexene-1-carboxamide, 2-hydroxy-4,4-dimethyl-N-1-naphthalenyl-6-oxo- (MCC2344) as the best HDAC6 inhibitor among the five ligands. The binding affinity of MCC2344 to HDAC6 was further confirmed by microscale thermophoresis. Additionally, the anti-cancer activity of MCC2344 was tested in several tumor cell lines. Leukemia cells were the most sensitive cells towards MCC2344, particularly the P-glycoprotein-overexpressing multidrug-resistant cell line CEM/ADR5000 exhibited remarkable collateral sensitivity towards MCC2344. Transcriptome analysis using microarray hybridization was performed for investigating downstream mechanisms of action of MCC2344 in leukemia cells. MCC2344 affected microtubule dynamics and suppressed cell migration in the wound healing assay as well as in a spheroid model by hyper-acetylation of tubulin and HSP-90. MCC2344 induced cell death in CEM/ADR5000 cells by activation of PARP, caspase-3, and p21 in addition to the downregulation of p62. MCC2344 significantly inhibited tumor growth in vivo in zebrafish larvae without mortality until 20 pM. We propose MCC2344 as a novel HDAC6 inhibitor for cancer treatment.

Identification of novel class I and class IIb histone deacetylase inhibitor for Alzheimer's disease therapeutics

Histone deacetylase enzymes were prominent chromatin remodeling drug that targets in the pathophysiology of Alzheimer's disease associated with transcriptional dysregulation. In vitro and in vivo models of AD have demonstrated overexpression of HDAC activity. Non-specificity and non-selectivity of HDAC are the major problems of existing HDAC inhibitors. Hence, we aim to set up a methodology describing the rational development of isoform-selective HDAC inhibitor targeting class, I and class IIb. A convenient multistage virtual screening followed by machine learning and IC50 screenings were used to classify the 5064 compounds into inhibitors and non-inhibitors classes retrieved from the ChEMBL database. ADMET analysis identified the pharmacokinetics and pharmacodynamics properties of selected compounds. Molecular docking, along with mutational analysis of eleven compounds, characterized the inhibiting potency. Herein, for the first time, we reported ChEMBL1834473 (2-[[5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl]amino]-N-hydroxypyrimidine-5-carboxamide) as the isoform-selective HDAC inhibitor, which interact central Zn2+ atom. The negative energy and interacting residue of the ChEMBL1834473 with six HDAC isoform has also been tabulated and mapped. Moreover, our findings concluded histidine, glycine, phenylalanine, and aspartic acid as key residues in protein-ligand interaction and classify 2347 compounds as HDAC inhibitors. Later, a protein-protein interaction network of six HDAC with the key proteins involved in the progression of an AD and signaling pathway, which describes the relationship between ChEMBL1834473 and AD, has been demonstrated using PPI network where the chosen inhibitor will work. Altogether, we conclude that the compound ChEMBL1834473 may be capable of inhibiting all isoforms of class I and class IIb HDAC based on computational analysis for AD therapeutics.

HDAC6-selective inhibitors decrease nerve-injury and inflammation-associated mechanical hypersensitivity in mice.

HDAC6 is a class IIB histone deacetylase expressed at many levels of the nociceptive pathway. This study tested the ability of novel and selective HDAC6 inhibitors to alleviate sensory hypersensitivity behaviors in mouse models of peripheral nerve injury and peripheral inflammation. We utilized the murine spared nerve injury (SNI) model for peripheral nerve injury and the Complete Freund's Adjuvant (CFA) model of peripheral inflammation. We applied the Von Frey assay to monitor mechanical allodynia. Using the SNI model, we demonstrate that daily administration of the brain-penetrant HDAC6 inhibitor, ACY-738, abolishes mechanical allodynia in male and in female mice. Importantly, there is no tolerance to the antiallodynic actions of these compounds as they produce a consistent increase in Von Frey thresholds for several weeks. We observed a similar antiallodynic effect when utilizing the HDAC6 inhibitor, ACY-257, which shows limited brain expression when administered systemically. We also demonstrate that ACY-738 and ACY-257 attenuate mechanical allodynia in the CFA model of peripheral inflammation. Overall, our findings suggest that inhibition of HDAC6 provides a promising therapeutic avenue for the alleviation of mechanical allodynia associated with peripheral nerve injury and peripheral inflammation.

Histone deacetylase 10 knockout activates chaperone-mediated autophagy and accelerates the decomposition of its substrate

Histone deacetylase (HDAC) 10 is a member of class IIb HDACs, but its deacetylation targets and functions are poorly characterized. Recent investigation has proposed that HDAC10 deacetylates heat shock cognate protein 70 kDa (HSC70) after interaction. HSC70 plays an important role in chaperone-mediated autophagy (CMA), binding CMA substrates and transporting them to lysosomes. However, it has not been clarified whether HDAC10 is involved in CMA. In this study, we established the HDAC10 knockout HeLa cell line and evaluated its CMA activity to determine whether HDAC10 participates in regulating CMA. In HDAC10 knockout cells, lysosome-associated protein type 2A (LAMP2A) protein level increased and LAMP2A-positive lysosomes accumulated around the nucleus. Moreover, GAPDH, which is a well-known CMA substrate, was delivered to LAMP2A-positive lysosomes and degraded in HDAC10 knockout cells more efficiently than in wild type HeLa cells. These results suggest that CMA is activated in HDAC10 knockout cells. Meanwhile, turnover assay using LC3 and p62, which are macroautophagy markers, indicated that autophagic flux was fully functioning in HDAC10 knockout cells as well as in wild type HeLa cells. In conclusion, HDAC10 participated in regulating CMA, and HDAC10 knockout activated CMA and accelerated degradation of a CMA substrate.

Novel Therapeutic Rationale for Targeting HDAC1 and PIM2 in Multiple Myeloma

Multiple myeloma (MM) cells preferentially grow and expand in the bone marrow (BM) to elicit the alteration of gene expression thereby acquiring drug resistance. The serine/threonine kinase PIM2 is constitutively overexpressed which is further up-regulated as a critical anti-apoptotic mediator in MM cells by interacting with BM stromal cells (BMSCs) and/or osteoclasts (Leukemia 2011, 2015). Histone deacetylases (HDACs) generally repress gene expression through deacetylation of lysine residues in histone tails. Therefore, HDAC inhibitors are able to restore the expression of tumor suppressor genes, and utilized as anti-cancer agents for various types of malignancies, including MM. Importantly, class-I and a class-IIb (HDAC6) HDACs have been shown as important therapeutic targets in MM (Nat Chem Biol 2010). Among class-I HDAC isoforms, HDAC1 and HDAC3 are highly expressed in MM cells (GSE5900 and GSE2113) and we have already reported that the HDAC3-DNMT1 axis is a critical therapeutic target (Leukemia 2017). However, the significance of HDAC1 expression in MM cell growth and survival is still largely unknown. In the present study, we aimed to clarify the epigenetic regulation of PIM2 and the therapeutic implication of HDAC1 in MM cells. We observed that HDAC1- and HDAC3-selective inhibitor MS-275 (Entinostat) inhibited MM cell growth in a dose-dependent fashion. HDAC1 knockdown using a lentiviral shRNA system induced apoptosis in MM cell lines, indicating a crucial role of HDAC1 in MM cell growth and survival. To identify downstream targets of HDAC1 mediating MM cell survival, we next carried out RNA-Seq using RPMI 8226 cells after HDAC1 knockdown. Expression of a number of genes were altered (adjusted P values < 0.05, log fold change > 0.5). Among these genes, we found that PIM2 and IRF4 were significantly downregulated in HDAC1 knocked down cells. The downregulation of IRF4 and PIM2 was further confirmed at mRNA and protein levels in additional MM cell lines. It has been shown that MS-275 impaired the viability of primary MM cells associated with downregulation of IRF4 and PIM2 expression. However, importantly, HDAC1 knocked down-induced growth inhibition was not observed in RPMI8226 cells with IRF4 overexpression, indicating that IRF4 is a key MM cell survival mediator targeted by HDAC1 inhibition. Previous study shows that HDAC1 is abundantly enriched around at H3K27 acetylation or RNA Pol II- binding sites compared to HDAC2 or HDAC3 (GSE86450), However, our data assessed by ChIP-Seq indicated that HDAC1-occupied genes were not completely upregulated but rather downregulated in HDAC1-knockdown cells. Indeed, MS-275 and a histone acetyltransferase inhibitor C646 downregulated IRF4 and PIM2 expression in MM cells despite upregulation and downregulation of histone H3 acetylation, respectively. The ChIP-Seq data showed HDAC1 binding is enriched around the promotor regions of IRF4 and PIM2 in MM cells; however, MS-275 significantly reduced the HDAC1 enrichment as determined in ChIP-Q-PCR assays, suggesting that IRF4 and PIM2 expression is regulated by the balance between acetylation and deacetylation status of histones in MM cells. In addition, we found that IRF4 binds to the promoter of PIM2 and IRF4 knockdown reduced PIM2 expression, suggesting that IRF4 transcriptionally regulates PIM2. Although PIM2 expression is robustly upregulated in MM cells in an ambient microenvironment with BMSCs and/or osteoclasts, MS-275 and the PIM inhibitor SMI-16a cooperatively induce MM cell death. In conclusion, our data provides a basis of rationale combination strategy targeting of class-I HDAC and PIM2 to improve MM patient outcome. Disclosures Anderson: Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board.

Structure-activity relationship study of thiazolyl-hydroxamate derivatives as selective histone deacetylase 6 inhibitors

Several human diseases are associated with aberrant epigenetic pathways mediated by histone deacetylases (HDACs), especially HDAC6, a class IIb HDACs, which has emerged as an attractive target for neurodegenerative and autoimmune disease therapeutics. In a previous study, we developed the novel HDAC6-selective inhibitor 9a ((E)-N-hydroxy-4-(2-styrylthiazol-4-yl)butanamide) and showed that it has anti-sepsis activity in vivo. In this study, we conducted structure-activity relationship (SAR) studies to optimize the activity and selectivity of HDAC6, synthesizing its derivatives with various aliphatic linker sizes and cap structures. We identified 6u ((E)-N-hydroxy-3-(2-(4-fluorostyryl)thiazol-4-yl)propanamide), which has nanomolar inhibition activity and a 126-fold selectivity for HDAC6 over HDAC1. Through the docking analyses of 6u against HDAC subtypes, we revealed the importance of the optimal aliphatic linker size, as well as the electronic substituent effect and rigidity of the aryl cap group. Thus, we suggest a new rationale for the design of HDAC6-selective inhibitors.

HDAC6 Modulates Signaling Pathways Relevant to Synaptic Biology and Neuronal Differentiation in Human Stem-Cell-Derived Neurons.

Recent studies show that histone deacetylase 6 (HDAC6) has important roles in the human brain, especially in the context of a number of nervous system disorders. Animal models of neurodevelopmental, neurodegenerative, and neuropsychiatric disorders show that HDAC6 modulates important biological processes relevant to disease biology. Pan-selective histone deacetylase (HDAC) inhibitors had been studied in animal behavioral assays and shown to induce synaptogenesis in rodent neuronal cultures. While most studies of HDACs in the nervous system have focused on class I HDACs located in the nucleus (e.g., HDACs 1,2,3), recent findings in rodent models suggest that the cytoplasmic class IIb HDAC, HDAC6, plays an important role in regulating mood-related behaviors. Human studies suggest a significant role for synaptic dysfunction in the prefrontal cortex (PFC) and hippocampus in depression. Studies of HDAC inhibitors (HDACi) in human neuronal cells show that HDAC6 inhibitors (HDAC6i) increase the acetylation of specific lysine residues in proteins involved in synaptogenesis. This has led to the hypothesis that HDAC6i may modulate synaptic biology not through effects on the acetylation of histones, but by regulating acetylation of non-histone proteins.

Class IIb HDAC Inhibition Enhances the Inhibitory Effect of Am80, a Synthetic Retinoid, in Prostate Cancer.

Combination therapy is often an effective strategy to treat cancer. In this study, we examined the growth-inhibitory effects of Am80 (tamibarotene), a specific retinoic acid receptor (RAR) α/β agonist, in combination with a histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), or a DNA methyl transferase (DNMT) inhibitor, 5-aza-2'-deoxycytidine, on androgen receptor (AR)-positive and AR-negative prostate cancer cell lines (LNCaP and PC-3, respectively). We found that the combination therapy of SAHA and Am80 showed an enhanced growth-inhibitory effect on LNCaP cells. Further studies with various HDAC isotype-selective inhibitors showed that SAHA and KD5170 (a selective class I and II HDAC inhibitor) each increased the RARα protein level in LNCaP cells. Our results indicate that the target of the enhancing effect belongs to the Class IIb HDACs, especially HDAC6. Dual targeting of Class IIb HDAC and RARα may be a candidate therapeutic strategy for prostate cancer.

SE-7552, a Highly Selective, Non-Hydroxamate Inhibitor of Histone Deacetylase-6 Blocks Multiple Myeloma Growth In Vivo

Despite recent advances, disease progression and treatment resistance in multiple myeloma (MM) remains a significant challenge. Current therapies include proteasome inhibitors, immunomodulatory agents, monoclonal antibodies and more recently histone deacetylase (HDAC) inhibitors.Many HDAC inhibitors have progressed into clinical development, however, there has been limited success with the approval of only three pan-HDAC inhibitors (SAHA, belinostat and panobinostat) and the class I selective inhibitor, romidepsin. A challenge to the development of HDAC inhibitors has been the management of toxicities, many of which are dose limiting. Some of these toxicities can be attributed to the use of the hydroxamate, a potent zinc binding group that has been associated with toxicity and poor pharmacokinetic (PK) properties. The very tight binding potency of the hydroxamate to zinc also leads to limited HDAC isoform selectivity and thus further predisposes to toxicity.HDAC6, a member of the class IIb HDAC family, is a target of high interest and has significant therapeutic potential in oncology, particularly MM. It has a unique protein structure and is predominantly located in the cytoplasm where it has multiple non-histone protein substrates including α-tubulin, HSP90, cortactin, and Foxp3. Here, we describe the identification of a highly selective, non-hydroxamate HDAC6 inhibitor with excellent PK properties capable of inhibiting MM growth in vivo.Extensive medicinal chemistry exploration and optimization led to the discovery of a zinc-binding group that was associated with excellent potency and selectivity for HDAC6. Additional optimization of the chemical scaffold led to the identification of SE-7552, a compound with an IC50 of 33nM against HDAC6 and greater than 850-fold selectivity versus all other known HDAC isozymes. SE-7552 demonstrated superior PK compared to hydroxamate-based HDAC inhibitors, with a maximum exposure of 597 ng/ml and a half-life of 7.2 hours after a single oral dose of 5 mg/kg in the mouse. To characterize inhibition of HDAC6 in vivo, acetylated α-tubulin (a biomarker for HDAC6 inhibition) was measured in the spleen of mice treated with SE-7552 or the hydroxamate-based HDAC6 inhibitor, ricolinostat. After a single oral dose of SE-7552 at 30 mg/kg, levels of acetylated α-tubulin were increased for over 24 hours, whereas ricolinostat at a 50 mg/kg IP dose showed increased levels for less than 8 hours. In the same study, SE-7552 had no effect on the acetylation of H3 (a biomarker for inhibition of Class I HDACs), whereas ricolinostat increased the levels of acetylated H3.Based on the positive in vitro and in vivo profile, SE-7552 was progressed into pre-clinical rodent models of MM. Since previously studied HDAC6 inhibitors were not able to significantly inhibit MM growth as monotherapy, SE-7552 was co-administered with either pomalidomide or bortezomib. In a subcutaneous MM model using human H929 MM cells, SE-7552 dosed orally at 10 mg/kg daily in combination with pomalidomide dosed at 1 mg/kg IP daily significantly delayed tumor growth in comparison to pomalidomide alone (p < 0.01), as well as enhanced the survival of the mice. Utilizing human MM.1S cells that can be quantified by luminescence, a disseminated model of MM was conducted in which SE-7552 dosed orally at 10 mg/kg daily in combination with bortezomib dosed at 1.5 mg/kg IP once a week significantly delayed tumor growth compared to bortezomib alone (p < 0.05).In summary, we have identified a non-hydroxamate HDAC6 inhibitor, SE-7552, with superior selectivity and pharmacokinetics compared to current HDAC6 inhibitors. SE-7552 demonstrated high levels of selectivity in an in vivo biomarker study and blocked MM growth in two pre-clinical models when co-administered with current MM therapies. DisclosuresHolt:Selenity Therapeutics: Employment, Equity Ownership, Patents & Royalties: METALLOENZYME INHIBITOR COMPOUNDS, U.S. Patent Application No.: 15/917,555 (March 9, 2018). Garvey:Selenity Therapeutics: Employment, Equity Ownership. Becherer:Selenity Therapeutics: Employment, Equity Ownership. Hoekstra:Selenity Therapeutics: Employment, Equity Ownership, Patents & Royalties: METALLOENZYME INHIBITOR COMPOUNDS, U.S. Patent Application No.: 15/917,555 (March 9, 2018). Schotzinger:Mycovia Pharma: Membership on an entity's Board of Directors or advisory committees; Innocrin Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Selenity Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: METALLOENZYME INHIBITOR COMPOUNDS, U.S. Patent Application No.: 15/917,555 (March 9, 2018). Yates:Selenity Therapeutics: Employment, Equity Ownership, Patents & Royalties: METALLOENZYME INHIBITOR COMPOUNDS, U.S. Patent Application No.: 15/917,555 (March 9, 2018).