Selective Histone Deacetylase Inhibitors Research Articles

HDAC3 inhibition increases the expression levels of the Amyloid Precursor Protein

AbstractBackgroundEpigenetic‐based interventions have recently garnered attention as potential therapies for Alzheimer’s disease (AD). Histone deacetylases (HDACs) represent an interesting target for neurodegenerative diseases therapies, since HDAC inhibitors have been shown to have the ability to reinstate memory even after neuronal loss. Particularly, HDAC3 inhibition has been shown to enhance long‐term memory and reduce amyloid‐β in AD models, although the molecular mechanisms behind these effects are unclear. The aim of this study was to characterise the effects of HDAC3 inhibition on the mechanisms of generation and degradation of Aβ using in vitro and in ex vivo models of ADMethodN2a mouse neuroblastoma cells overexpressing the Swedish mutation (N2asw) as well as organotypic brain cultures (OBCs) of 5XFAD mice were incubated with various concentrations of the HDAC3 selective inhibitor RGFP966 (0.1‐10 μM) for 24h.ResultTreatment with HDAC3 inhibitor RGFP966, led to a 4% increase on Aβ42 levels in N2a cells, associated with elevated levels carboxy‐terminus fragments (CTFs) and APP protein expressions as well as APP mRNA levels. In vitro chromatin immunoprecipitation (ChIP) analysis in N2a cells, revealed enriched HDAC3 binding at APP promoter regions. The increase in APP expression was also detected in OBCs from 5XFAD mice incubated with 1 μM RGFP966, without significant changes in Aβ secretion. In addition, mRNA sequencing in OBCs revealed that HDAC3 inhibition affects neuronal regenerative pathways, in particular linked to neurogenesis, neuronal differentiation, axonogenesis, resulting in an increase in dendritic spine density.ConclusionThese results suggest that the HDAC3 therapeutic role in AD involves the regulation of APP expression and regenerative pathways, leading to increased synaptic connectivity.

BTK Inhibitors and Chidamide-Combined Therapy in Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Background: Relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) shows poor clinical outcomes, particularly in patients ineligible for stem cell transplantation or who failed induction therapy or salvage therapy. Monotherapy Bruton tyrosine kinase inhibitors (BTKi) have established modest antitumor effect in R/R DLBCL by targeting B-cell receptor signaling, with median progression-free survival (PFS) and overall survival (OS) of 1.64-2.8 months and 6.4-8.4 months, respectively. Chidamide is a selective oral histone deacetylase inhibitors (HDACi) that shows promising treatment for DLBCL and may overcome BTKi resistance. So, we evaluated the efficacy and safety of BTKis and chidamide-combined therapy in R/R DLBCL. Methods: This retrospective study consists of 11 R/R DLBCL who received combination therapy of chidamide and BTK inhibitors between September 2021 and July 2023. Chidamide was orally administered at a dose of 20 mg twice a week, while the BTK inhibitor was administered daily. Patient clinical characteristics were collected upon patient enrollment. Efficacy outcomes included overall response rate (ORR), PFS and OS, whereas safety outcomes included incidence of adverse events. Results: 11 R/R DLBCL patients were enrolled. Median age was 61 years (range, 56-73), and 81.82% of the patients had non-germinal center B-cell-like (non-GCB) DLBCL, one had GCB DLBCL, one had transformed DLBCL. The median number of previous treatment lines was 3 (range, 3-8). 10 patients (90.91%) received treatment with BTK inhibitors before. Four patients（36.36%）failed to chimeric antigen receptor T-cell/NK-cell(CAR-T/CAR-NK) therapy. The best ORR was 81.82%, with 9 patients acquired objective disease remission after 1-2 cycles. Among them, 3 patients (27.27%) achieved complete response, and6 patients (54.55%) achieved partial remission. With a median follow-up of 5 months, the median PFS was 6.0 months (95%CI 3.14-8.86) and median OS was not yet reached. In terms of adverse events, the most common hematologic toxicities observed were grade ≥3 neutropenia (36.36%, 4/11) and grade ≥3 thrombocytopenia (36.36%, 4/11). Two patients (18.18%) had pneumonia, and one patient (9.09%) had herpes zosters. Due to the positive real-world outcomes observed in the retrospective study, prospective clinical studies of chidamide in combination with BTK inhibitors for the treatment of R/R DLBCL are underway (ChiCTR2300073233). Conclusion: This real-world study revealed promising results. Despite the majority of patients had been treated with BTK inhibitors and experienced relapse and resistance, the addition of chidamide brought patients back into remission, suggesting that chidamide can enhance the efficacy of BTK inhibitors and overcome acquired resistance. Notably, chidamide combined with BTK inhibitors showed high efficacy and manageable tolerability, making it a promising treatment option for patients with R/R DLBCL. Keywords: BTK inhibitors, Chidamide, R/R DLBCL, efficacy and safety

Efficacy and Safety of Chidamide Combined with BEAM Conditioning Regimen in Autologous Transplantation for T-Cell Lymphoma

Background: T cell lymphoma (TCL) is a group of highly heterogeneous aggressive non-Hodgkin lymphomas with different pathogenesis and clinical prognosis. Despite the survival benefits of anthracycline-based chemotherapy bridging autologous stem cell transplantation (ASCT), 40% to 50% of TCL patients fail to respond to treatment and relapse or die within a short period of time. Chidamide is an oral selective histone deacetylase inhibitor (HDACi), interferes with epigenetic regulation by deacetylating nuclear transcription factors, affects proliferation and differentiation of tumor cells, and regulates cytokines in the tumor microenvironment. Chidamide combined with chemotherapy has promising clinical applications in ASCT. Aims: This prospective Phase II trial aims to evaluate the efficacy and safety of chidamide in combination with carmustine, etoposide, cytarabine, and melphalan (Chi-BEAM) conditioning regimen in ASCT of TCL patients received CR/PR to first-line chemotherapy. Method: In this study, eligible patients with TCL (except IPI 0-1 ALK+ anaplastic large cell lymphoma) aged 18-65 years were enrolled. All patients received Chi-BEAM conditioning regimen before ASCT. 30mg of chidamide was administered twice a week for at least 2 years after ASCT.The primary end point was 2-year progression-free survival (PFS) rate, and the secondary end points were 2-year overall survival (OS) rate and safety evaluation. Results: At the time of data cut off, a total of 18 patients were enrolled, with a median age of 48 (15-65) years. The 1-year PFS rate and OS rate after ASCT were 90.0% and 100.0%, respectively. The median implantation time of neutrophils was 10 days (9-13 days), and the median implantation time of platelets was 17 days (11-25 days). Most of the adverse reactions (AEs) were tolerable, including controlled grain deficiency with fever, electrolyte disturbance, and abnormal liver function. Conclusion: Chidamide combined with BEAM as a conditioning regimen for ASCT can significantly improve PFS and OS in TCL patients, and the AEs are controllable. The study is ongoing and further results will be continuously released.

Efficacy of HDAC Inhibitors in Driving Peroxisomal β-Oxidation and Immune Responses in Human Macrophages: Implications for Neuroinflammatory Disorders.

Elevated levels of saturated very long-chain fatty acids (VLCFAs) in cell membranes and secreted lipoparticles have been associated with neurotoxicity and, therefore, require tight regulation. Excessive VLCFAs are imported into peroxisomes for degradation by β-oxidation. Impaired VLCFA catabolism due to primary or secondary peroxisomal alterations is featured in neurodegenerative and neuroinflammatory disorders such as X-linked adrenoleukodystrophy and multiple sclerosis (MS). Here, we identified that healthy human macrophages upregulate the peroxisomal genes involved in β-oxidation during myelin phagocytosis and pro-inflammatory activation, and that this response is impaired in peripheral macrophages and phagocytes in brain white matter lesions in MS patients. The pharmacological targeting of VLCFA metabolism and peroxisomes in innate immune cells could be favorable in the context of neuroinflammation and neurodegeneration. We previously identified the epigenetic histone deacetylase (HDAC) inhibitors entinostat and vorinostat to enhance VLCFA degradation and pro-regenerative macrophage polarization. However, adverse side effects currently limit their use in chronic neuroinflammation. Here, we focused on tefinostat, a monocyte/macrophage-selective HDAC inhibitor that has shown reduced toxicity in clinical trials. By using a gene expression analysis, peroxisomal β-oxidation assay, and live imaging of primary human macrophages, we assessed the efficacy of tefinostat in modulating VLCFA metabolism, phagocytosis, chemotaxis, and immune function. Our results revealed the significant stimulation of VLCFA degradation with the upregulation of genes involved in peroxisomal β-oxidation and interference with immune cell recruitment; however, tefinostat was less potent than the class I HDAC-selective inhibitor entinostat in promoting a regenerative macrophage phenotype. Further research is needed to fully explore the potential of class I HDAC inhibition and downstream targets in the context of neuroinflammation.

A Review on Molecular Docking on HDAC Isoforms: Novel Tool for Designing Selective Inhibitors.

Research into histone deacetylases (HDACs) has experienced a remarkable surge in recent years. These enzymes are key regulators of several fundamental biological processes, often associated with severe and potentially fatal diseases. Inhibition of their activity represents a promising therapeutic approach and a prospective strategy for the development of new therapeutic agents. A critical aspect of their inhibition is to achieve selectivity in terms of enzyme isoforms, which is essential to improve treatment efficacy while reducing undesirable pleiotropic effects. The development of computational chemistry tools, particularly molecular docking, is greatly enhancing the precision of designing molecules with inherent potential for specific activity. Therefore, it was considered necessary to review the molecular docking studies conducted on the major isozymes of the enzyme in order to identify the specific interactions associated with each selective HDAC inhibitor. In particular, the most critical isozymes of HDAC (1, 2, 3, 6, and 8) have been thoroughly investigated within the scope of this review.

EXTH-50. ACUTE AND LONG-TERM RESPONSES TO QUISINOSTAT TREATMENT IN PDX MODELS OF GLIOBLASTOMA

Abstract BACKGROUND Histone deacetylase inhibitors (HDACi) promote reactive oxygen species production, result in DNA damage, and trigger cell fate changes. Quisinostat is a Class I-II selective HDAC inhibitor with high specificity for HDAC1. Here, we evaluated acute and long-term quisinostat treated glioblastoma (GBM) PDX tissues and glioma stem cells for increase in reactive oxygen species and cell fate change. METHODS GBM PDX models (n = 6) and multiple PDX-derived glioma stem cells were treated with quisinostat, radiation, or a combination of quisinostat and radiation. Tissue and cells were processed via LC-MS/MS, RNA-seq, qRT-PCR, immunoblotting, and immunocytochemistry assays to examine increase in cellular reactive oxygen species, oxidative stress biomarkers, and markers of stemness and differentiation. RESULTS RNA sequencing of tumor tissue revealed that chronic co-treatment with quisinostat and radiation led to increased cellular expression of oxidative stress- and neuronal-related genes compared to control group. Immunoblotting confirmed upregulation of neuronal signaling proteins in the quisinostat treated tumor tissue in vivo. qRT-PCR and LC-MS/MS analysis demonstrated an increase in oxidative stress biomarkers following acute quisinostat treatment in vitro. CONCLUSIONS Treatment with quisinostat increased the expression of genes involved in promoting oxidative stress and neuronal signaling in vivo. LC-MS/MS, qRT-PCR, immunoblotting and immunocytochemistry assays confirmed increase in oxidative stress and neuronal markers upon acute treatment in vitro. Cancer cells often express higher levels of reactive oxygen species compared to normal tissue, and oxidative stress is linked to greater survival and proliferation of cancer cells. However, there is much interest in increasing oxidative stress in glioma cells to induce selective cell death in cancer cells. Ongoing studies are aimed at evaluating the significance of these cellular and molecular changes resulting from quisinostat treatment and whether the quisinostat-induced oxidative stress and cell fate changes can be exploited for future combination therapy for GBM.

5-(Trifluoromethyl)-1,2,4-oxadiazole (TFMO)-based highly selective class IIa HDAC inhibitors exhibit synergistic anticancer activity in combination with bortezomib

Clinically used pan and class I HDACi cause severe side effects, whereas class IIa HDACi are less cytotoxic. Here, we present the synthesis and anticancer effects of a series of 5-(trifluoromethyl)-1,2,4-oxadiazole (TFMO)-based amides and alkoxyamides derived from the previously reported class IIa HDACi YAK540. The most active class IIa inhibitor 1a showed nanomolar inhibition of the class IIa enzymes 4, 5, 7 (IC50 HDAC4: 12 nM) and high selectivity (selectivity index >318 for HDAC4) over non-class IIa HDACs. Instead of a hydroxamic acid group, 1a has a trifluoromethyloxadiazolyl (TFMO) moiety as a non-chelating Zinc-binding group (ZBG). Applying the Chou-Talalay-method we found an increased synergistic cytotoxic effect of 1a in combination with bortezomib in THP1 cells. 1a in combination with bortezomib enhanced expression of p21 leading to increased caspase-induced apoptosis. Eventually, growth inhibition by 1a of the head-neck cancer cell line Cal27 was increased upon HDAC4 overexpression in Cal27 in cell culture and using the in vivo chorioallantoic membrane model. The class IIa HDACi 1a outperforms previously described HDAC class IIa inhibitor YAK540 regarding anticancer effects and may constitute a novel option compared to pan and class I HDACi in anticancer combination treatments.

Design and synthesis of bioreductive prodrugs of class I histone deacetylase inhibitors and their biological evaluation in virally transfected acute myeloid leukemia cells.

Although histone deacetylase (HDAC) inhibitors show promise in treating various types of hematologic malignancies, they have some limitations, including poor pharmacokinetics and off-target side effects. Prodrug design has shown promise as an approach to improve pharmacokinetic properties and to improve target tissue specificity. In this work, several bioreductive prodrugs for class I HDACs were designed based on known selective HDAC inhibitors. The zinc-binding group of the HDAC inhibitors was masked with various nitroarylmethyl residues to make them substrates of nitroreductase (NTR). The developed prodrugs showed weak HDAC inhibitory activity compared to their parent inhibitors. The prodrugs were tested against wild-type and NTR-transfected THP1 cells. Cellular assays showed that both 2-nitroimidazole-based prodrugs 5 and 6 were best activated by the NTR and exhibited potent activity against NTR-THP1 cells. Compound 6 showed the highest cellular activity (GI50 = 77 nM) and exhibited moderate selectivity. Moreover, activation of prodrug 6 by NTR was confirmed by liquid chromatography-mass spectrometry analysis, which showed the release of the parent inhibitor after incubation with Escherichia coli NTR. Thus, compound 6 can be considered a novel prodrug selective for class I HDACs, which could be used as a good starting point for increasing selectivity and for further optimization.

Targeting Histone Deacetylases 6 in Dual-Target Therapy of Cancer.

Histone deacetylases (HDACs) are the major regulators of the balance of acetylation of histone and non-histone proteins. In contrast to other HDAC isoforms, HDAC6 is mainly involved in maintaining the acetylation balance of many non-histone proteins. Therefore, the overexpression of HDAC6 is associated with tumorigenesis, invasion, migration, survival, apoptosis and growth of various malignancies. As a result, HDAC6 is considered a promising target for cancer treatment. However, none of selective HDAC6 inhibitors are in clinical use, mainly because of the low efficacy and high concentrations used to show anticancer properties, which may lead to off-target effects. Therefore, HDAC6 inhibitors with dual-target capabilities represent a new trend in cancer treatment, aiming to overcome the above problems. In this review, we summarize the advances in tumor treatment with dual-target HDAC6 inhibitors.

Maintenance Therapy with Chidamide Plus Azacitidine after Allogeneic Hematopoietic Stem Cell Transplantation for High-Risk Acute Myeloid Leukemia Patients

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is theoretically the only curative option for high-risk acute myeloid leukemia (AML) patients. However, many patients relapse after allo-HSCT, and treatment options are very limited. Azacitidine, a DNA methyltransferase inhibitor exhibits anti-leukemia activity in myeloid diseases. Chidamide, a selective histone deacetylase inhibitor, effectively induces apoptosis of AML cells. They have synergistic inhibitory effect on AML cells when combined together. Therefore, we conducted a multicentre, prospective study (ChiCTR2300067593) to examine the tolerability and efficacy of a combination of both chidamide and azacitidine as a maintenance therapy for high-risk AML patients after allo-HSCT. Patients and methods： Patients ≤ 60 years of age with high-risk AML post- allo-HSCT were enrolled. The high-risk characteristics of AML were defined as one or more of the following criteria: a) Patients with poor prognosis according to the genetic risk stratification assessment of European Leukmia Net (ELN) in 2022; b) Primary refractory or recurrent AML; c) Secondary AML (secondary to MDS or treatment-related AML). All patients received Chidamide 5 mg/d combined with azacitidine 75 mg/m2/d for 5 days. The cycle interval was 6-8 weeks. A total of 6 cycles was recommended. Treatment started as early as 3 months after transplantation. The primary endpoint of this study was cumulative incidence of relapse (CIR). The secondary endpoints included rates of overall survival (OS) and event-free survival (EFS). Survival outcomes were estimated using Kaplan-Meier analysis. Results： Thirty-six patients have been recruited. The median age was 42 years (range, 16-59). Baseline characteristics are shown in Table 1. With a median follow-up time of 412（185-762）days, the 1-year CIR was 12.3 (6.5-18.1) %, the 1-year OS and EFS rates were 100.0% and 87.7 (81.9-93.5) %, respectively. The median OS and EFS time were not reached. Four patients relapsed, two died, and two are still alive and ready for the second transplantation. The most common adverse events (AEs) were thrombocytopenia reaction and gastrointestinal tract reaction. Grade 2 or 3 AEs were observed in 16.7% (6/36) and 25.0% (9/36) of the patients, respectively. No grade >3 AEs were noted. 9 patients (6/36,16.7%) developed grade 3-4 thrombocytopenia, without active hemorrhage. All of them received blood transfusion and thrombopoietin receptor agonist. The median persistence of thrombocytopenia was 3 (2-5) d. Chronic graft-versus-host-disease (cGVHD) occurred in 25.0% (9/36) of patients. Four cases (4/36, 11.1%) were complicated with infection. They were one case of intestinal infection, one case of pulmonary and EBV infections, one case of hepatitis B virus activation and one case of herpes zoster. And all infected patients recovered safely after anti-infection and symptom-oriented treatment. CMV activation was not observed. Non relapse mortality (NRM) was zero. Conclusions： We conclude that chidamide plus azacitidine can be administered safely after allo- HSCT with no evidence of an increased incidence of GVHD, and this combination may decrease the relapse rate in high-risk AML patients. This novel maintenance therapy may be a promising way to prevent relapse in high-risk AML patients.

New insights into possible HDAC inhibitor resistance in DLBCL - Comment on 'defining cellular responses to HDAC-selective inhibitors reveals that efficient targeting of HDAC3 is required to elicit cytotoxicity and overcome naïve resistance to pan-HDACi in diffuse large B cell lymphoma' by Havas et al.

New insights into possible HDAC inhibitor resistance in DLBCL - Comment on 'defining cellular responses to HDAC-selective inhibitors reveals that efficient targeting of HDAC3 is required to elicit cytotoxicity and overcome naïve resistance to pan-HDACi in diffuse large B cell lymphoma' by Havas et al.

Virtual screening for early identification of potent and selective histone deacetylase 6 inhibitor series

Virtual screening was leveraged to identify novel series of histone deacetylase 6 (HDAC6) inhibitors prior to conducting a high-throughput screen. The virtual screen was designed to augment and expand on chemical matter that would otherwise be unavailable for high-throughput screening. Through this effort we succeeded in identifying four novel, potent and highly selective HDAC6 inhibitor series. These series displayed favorable ligand binding efficiencies and good potential for further optimization. The virtual design specifications and results are discussed herein.

Defining cellular responses to HDAC-selective inhibitors reveals that efficient targeting of HDAC3 is required to elicit cytotoxicity and overcome naïve resistance to pan-HDACi in diffuse large B cell lymphoma

Approved histone deacetylase (HDAC) inhibitors have low efficacy against the most commonly-diagnosed non-Hodgkin lymphoma, diffuse large B cell lymphoma (DLBCL), but the mechanisms underlying clinical resistance are poorly understood. Using a DLBCL cell-based model, we previously demonstrated that resistance to pan-HDAC inhibitors (HDACi) is characterized by reversible growth arrest and sensitivity by mitotic arrest and apoptosis. The goal of the current study is to better define mechanisms of sensitivity and resistance to the cytotoxic effects of HDACi by using HDAC-selective inhibitors to determine which HDACs need to be targeted to achieve the sensitive and resistant phenotypes. We find that an inhibitor selective for HDACs 1 and 2 induces G1 arrest across DLBCL cell lines used, which is consistent with the resistant phenotype. In contrast an HDAC3-selective inhibitor induces DNA damage and cytotoxicity in a cell line that is sensitive to pan-HDACi but has no effect on resistant cell lines. RNAi-mediated depletion of HDAC3 indicate the presence of a long-lived population of HDAC3 in DLBCL cell lines. Finally, doses of pan-HDACi 3–5 times higher than the IC50 established for reversible growth inhibition induce the sensitive phenotype in resistant cell lines, suggesting that resistance may be associated with failure to efficiently inhibit HDAC3. Our findings indicate that selective inhibition of HDACs 1 and 2 is associated with G1 arrest and resistance to pan-HDACi while efficient targeting of HDAC3 could be key to achieving a cytotoxic response. Thus, our work reveals a potential novel mechanism of resistance to pan-HDACi.

HDAC3 aberration-incurred GPX4 suppression drives renal ferroptosis and AKI-CKD progression

Acute kidney injury (AKI) progression to chronic kidney disease (CKD) represents a unique renal disease setting characterized by early renal cellular injury and regulated cell death, and later renal fibrosis, of which the critical role and nature of ferroptosis are only partially understood. Here, we report that renal tubular epithelial ferroptosis caused by HDAC3 (histone deacetylase 3) aberration and the resultant GPX4 suppression drives AKI-CKD progression. In mouse models of AKI-CKD transition induced by nephrotoxic aristolochic acid (AA) and folic acid (FA), renal tubular epithelial ferroptosis occurred early that coincided with preferential HDAC3 elevation and marked suppression of a core anti-ferroptosis enzyme GPX4 (glutathione peroxidase 4). Intriguingly, genetic Hdac3 knockout or administration of a HDAC3-selective inhibitor RGFP966 effectively mitigated the GPX4 suppression, ferroptosis and the fibrosis-associated renal functional loss. In cultured tubular epithelial cells, HDAC3 over-expression or inhibition inversely affected GPX4 abundances. Further analysis revealed that Gpx4 promoter contains a typical binding motif of transcription factor KLF5 (Kruppel-like factor 5). HDAC3 and KLF5 inducibly associated and bound to Gpx4 promoter upon AA treatment, leading to local histone hypoacetylation and GPX4 transactivation inhibition, which was blocked by RGFP966 and a KLF5 inhibitor ML264, respectively, suggesting that KLF5 co-regulated the HDAC3-incurred Gpx4 transcription inhibition. More importantly, in AKI-CKD mice receiving a GPX4 inactivator RSL3, the anti-ferroptosis and renoprotective effects of RGFP966 were largely abrogated, indicating that GPX4 is an essential downstream mediator of the HDAC3 aberration and renal ferroptosis during AKI-CKD transition. Together, our study identified a critical epigenetic pathway of ferroptosis during AKI-CKD transition and suggested that the strategies preserving GPX4 by HDAC3 inhibition are potentially effective to reduce renal ferroptosis and slow AKI-CKD progression.

Integrated molecular modeling and dynamics approaches revealed the mechanism of selective inhibition of HDAC6/8

The high structural homology of histone deacetylases 6 and 8 (HDAC6/8) poses a challenge in achieving isoform selectivity and has resulted in adverse side effects due to pan-inhibition in clinical applications. Additionally, the rational design of dual-target inhibitors, centered on HDAC6/8, demands a profound understanding of their selectivity mechanisms. Addressing the urgent need for enhanced specificity in the development of inhibitors targeting specific isoforms, we elucidate the mechanism underpinning the selective inhibition of HDAC6/8 inhibitors through in-silico strategies. The hydrogen bonding interaction with Asp101 and Tyr306 is a key factor that enables compound 12b to selectively inhibit HDAC8. Its favorable spatial orientation places the Cap group of 12b between Tyr306 and Tyr100, resulting in an overall L-shaped conformation. These two factors significantly contribute to the selective inhibitory activity of 12b against HDAC8. The zinc binding group (ZBG) of compound NN-390 forms a hydrogen bond with His610, a key residue of HDAC6, facilitating stable chelation with zinc ions. In addition, the Cap group of NN-390 interacts with Phe620 and Phe680 via van der Waals forces, leading to an overall Y-shaped conformation. The aforementioned factors are the main reasons for the selective inhibition of HDAC6 by NN-390. Furthermore, whether the Cap group is in the para or meta-position will influence the selective inhibition of either HDAC6 or HDAC8. We believe these clues can offer valuable insights for the rational design of selective inhibitors targeting HDAC6/8 and pave the way for rational design of dual-target HDAC6/8-based inhibitors. Communicated by Ramaswamy H. Sarma

Selective and Bioavailable HDAC6 2-(Difluoromethyl)-1,3,4-oxadiazole Substrate Inhibitors and Modeling of Their Bioactivation Mechanism.

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family mainly targeting cytosolic nonhistone substrates, such as α-tubulin, cortactin, and heat shock protein 90 to regulate cell proliferation, metastasis, invasion, and mitosis in tumors. We describe the identification and characterization of a series of 2-(difluoromethyl)-1,3,4-oxadiazoles (DFMOs) as selective nonhydroxamic acid HDAC6 inhibitors. By comparing structure-activity relationships and performing quantum mechanical calculations of the HDAC6 catalytic mechanism, we show that potent oxadiazoles are electrophilic substrates of HDAC6 and propose a mechanism for the bioactivation. We also observe that the inherent electrophilicity of the oxadiazoles makes them prone to degradation in water solution and the generation of potentially toxic products cannot be ruled out, limiting the developability for chronic diseases. However, the oxadiazoles demonstrate high oral bioavailability and low in vivo clearance and are excellent tools for studying the role of HDAC6 in vitro and in vivo in rats and mice.

Difluoromethyl-1,3,4-oxadiazoles Are Selective, Mechanism-Based, and Essentially Irreversible Inhibitors of Histone Deacetylase 6.

Histone deacetylase 6 (HDAC6) is an important drug target in oncological and non-oncological diseases. Most available HDAC6 inhibitors (HDAC6i) utilize hydroxamic acids as a zinc-binding group, which limits therapeutic opportunities due to its genotoxic potential. Recently, difluoromethyl-1,3,4-oxadiazoles (DFMOs) were reported as potent and selective HDAC6i but their mode of inhibition remained enigmatic. Herein, we report that DFMOs act as mechanism-based and essentially irreversible HDAC6i. Biochemical data confirm that DFMO 6 is a tight-binding HDAC6i capable of inhibiting HDAC6 via a two-step slow-binding mechanism. Crystallographic and mechanistic experiments suggest that the attack of 6 by the zinc-bound water at the sp2 carbon closest to the difluoromethyl moiety followed by a subsequent ring opening of the oxadiazole yields deprotonated difluoroacetylhydrazide 13 as active species. The strong anionic zinc coordination of 13 and the binding of the difluoromethyl moiety in the P571 pocket finally result in an essentially irreversible inhibition of HDAC6.

Design and synthesis of 1H-benzo[d]imidazole selective HDAC6 inhibitors with potential therapy for multiple myeloma

Pan-HDAC inhibitors exhibit significant inhibitory activity against multiple myeloma, however, their clinical applications have been hampered by substantial toxic side effects. In contrast, selective HDAC6 inhibitors have demonstrated effectiveness in treating multiple myeloma. Compounds belonging to the class of 1H-benzo[d]imidazole hydroxamic acids have been identified as novel HDAC6 inhibitors, with the benzimidazole group serving as a specific linker for these inhibitors. Notably, compound 30 has exhibited outstanding HDAC6 inhibitory activity (IC50 = 4.63 nM) and superior antiproliferative effects against human multiple myeloma cells, specifically RPMI-8226. Moreover, it has been shown to induce cell cycle arrest in the G2 phase and promote apoptosis through the mitochondrial pathway. In a myeloma RPMI-8226 xenograft model, compound 30 has demonstrated significant in vivo antitumor efficacy (T/C = 34.8%) when administered as a standalone drug, with no observable cytotoxicity. These findings underscore the immense potential of compound 30 as a promising therapeutic agent for the treatment of multiple myeloma.

Mocetinostat activates Krüppel-like factor 4 and protects against tissue destruction and inflammation in osteoarthritis.

Osteoarthritis (OA) is the most common joint disorder, and disease-modifying OA drugs (DMOADs) represent a major need in OA management. Krüppel-like factor 4 (KLF4) is a central transcription factor upregulating regenerative and protective functions in joint tissues. This study was aimed to identify small molecules activating KLF4 expression and to determine functions and mechanisms of the hit compounds. High-throughput screening (HTS) with 11,948 clinical-stage compounds was performed using a reporter cell line detecting endogenous KLF4 activation. Eighteen compounds were identified through the HTS and confirmed in a secondary screen. After testing in SW1353 chondrosarcoma cells and human chondrocytes, mocetinostat - a class I selective histone deacetylase (HDAC) inhibitor - had the best profile of biological activities. Mocetinostat upregulated cartilage signature genes in human chondrocytes, meniscal cells, and BM-derived mesenchymal stem cells, and it downregulated hypertrophic, inflammatory, and catabolic genes in those cells and synoviocytes. I.p. administration of mocetinostat into mice reduced severity of OA-associated changes and improved pain behaviors. Global gene expression and proteomics analyses revealed that regenerative and protective effects of mocetinostat were dependent on peroxisome proliferator-activated receptor γ coactivator 1-α. These findings show therapeutic and protective activities of mocetinostat against OA, qualifying it as a candidate to be used as a DMOAD.

Selective HDAC6 inhibition protects against blood-brain barrier dysfunction after intracerebral hemorrhage.

Blood-brain barrier (BBB) disruption after intracerebral hemorrhage (ICH) significantly induces neurological impairment. Previous studies showed that HDAC6 knockdown or TubA can protect the TNF-induced endothelial dysfunction. However, the role of HDAC6 inhibition on ICH-induced BBB disruption remains unknown. Hemin-induced human brain microvascular endothelial cells (HBMECs) and collagenase-induced rats were employed to investigated the underlying impact of the HDAC6 inhibition in BBB lesion and neuronal dysfunction after ICH. We found a significant decrease in acetylated α-tubulin during early phase of ICH. Both 25 or 40 mg/kg of TubA could relieve neurological deficits, perihematomal cell apoptosis, and ipsilateral brain edema in ICH animal model. TubA or specific siRNA of HDAC6 inhibited apoptosis and reduced the endothelial permeability of HBMECs. HDAC6 inhibition rescued the degradation of TJ proteins and repaired TJs collapses after ICH induction. Finally, the results suggested that the protective effects on BBB after ICH induction were exerted via upregulating the acetylated α-tubulin and reducing stress fiber formation. Inhibition of HDAC6 expression showed beneficial effects against BBB disruption after experimental ICH, which suggested that HDAC6 could be a novel and promising target for ICH treatment.