Tubulin Deacetylation Research Articles

Abstract 4583: Development of selective HDAC6 inhibitors to improve cancer immunotherapy

Abstract Histone Deacetylases (HDACs) are enzymes that modify histones and non-histone proteins. Our group has previously demonstrated that HDAC6 inhibitors (HDAC6is) modulate multiple immune pathways, including those that suppress the antitumoral M2 phenotype, leading to improved antitumor immunity. Despite the potential of HDAC6is, current inhibitors have poor selectivity and off-target effects and only work at micromolar concentrations. In this work, we designed a screening pipeline of HDAC6i, considering selectivity, potency, capacity to prevent the protumoral phenotype of macrophages, and low cytotoxicity on macrophages and other immune cells. This approach has not been tested before, as the standard screening method for antitumoral HDACis has focused on its cytotoxic effect on cancer cells. We initially designed in silico 980 compound derivatives from previously reported HDAC6i, such as SS208, Nexturastat A, and Suprastat. Twenty-four compounds with a high capacity to bind HDAC6 and chemical stability were screened by their ability to inhibit HDAC6 in cell-free conditions. Sixteen compounds demonstrated high potency and were further evaluated in murine macrophages by their cytotoxic effect using CellTox. The capacity to impede tubulin deacetylation was assessed by western blot and deacetylase activity by HDAC-Glo. The top six candidates were then evaluated by their selectivity compared to other HDAC members and by their effect on repressing the expression of protumoral M2 macrophages Arginase 1 and Fizz1, and activation of antitumoral markers TNFa and iNOS by western blot and qRT-PCR. The best three candidates were then evaluated at nanomolar concentrations to explore their immunomodulatory potential of M1 against M2 by performing antigen presentation (SIINFEKL presentation) and phagocytic capacity using microscopy. Two of these compounds, SM-06-09 and SM-05-947, will be further tested in syngeneic murine melanoma models for their ability in tumor regression and antitumor immunity. This study has yielded important insights into developing new and improved HDAC6is with minimal cytotoxicity, and we believe our work will help advance cancer research and ultimately lead to better patient outcomes. Citation Format: Nithya Gajendran, Manasa Suresh, David Quiceno, Xintang Li, Marie Durr, Karen Tan, Satish Kumar Reddy Noonepalle, Alejandro Villagra. Development of selective HDAC6 inhibitors to improve cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4583.

Genome-wide study reveals novel roles for formin-2 in axon regeneration as a microtubule dynamics regulator and therapeutic target for nerve repair

Peripheral nerves regenerate successfully; however, clinical outcome after injury is poor. We demonstrated that low-dose ionizing radiation (LDIR) promoted axon regeneration and function recovery after peripheral nerve injury (PNI). Genome-wide CpG methylation profiling identified LDIR-induced hypermethylation of the Fmn2 promoter, exhibiting injury-induced Fmn2 downregulation in dorsal root ganglia (DRGs). Constitutive knockout or neuronal Fmn2 knockdown accelerated nerve repair and function recovery. Mechanistically, increased microtubule dynamics at growth cones was observed in time-lapse imaging of Fmn2-deficient DRG neurons. Increased HDAC5 phosphorylation and rapid tubulin deacetylation were found in regenerating axons of neuronal Fmn2-knockdown mice after injury. Growth-promoting effect of neuronal Fmn2 knockdown was eliminated by pharmaceutical blockade of HDAC5 or neuronal Hdac5 knockdown, suggesting that Fmn2deletion promotes axon regeneration via microtubule post-translational modification. In silico screening of FDA-approved drugs identified metaxalone, administered either immediately or 24-h post-injury, accelerating function recovery. This work uncovers a novel axon regeneration function of Fmn2 and a small-molecule strategy for PNI.

Development of First-in-Class Dual Sirt2/HDAC6 Inhibitors as Molecular Tools for Dual Inhibition of Tubulin Deacetylation.

Dysregulation of both tubulin deacetylases sirtuin 2 (Sirt2) and the histone deacetylase 6 (HDAC6) has been associated with the pathogenesis of cancer and neurodegeneration, thus making these two enzymes promising targets for pharmaceutical intervention. Herein, we report the design, synthesis, and biological characterization of the first-in-class dual Sirt2/HDAC6 inhibitors as molecular tools for dual inhibition of tubulin deacetylation. Using biochemical in vitro assays and cell-based methods for target engagement, we identified Mz325 (33) as a potent and selective inhibitor of both target enzymes. Inhibition of both targets was further confirmed by X-ray crystal structures of Sirt2 and HDAC6 in complex with building blocks of 33. In ovarian cancer cells, 33 evoked enhanced effects on cell viability compared to single or combination treatment with the unconjugated Sirt2 and HDAC6 inhibitors. Thus, our dual Sirt2/HDAC6 inhibitors are important new tools to study the consequences and the therapeutic potential of dual inhibition of tubulin deacetylation.

SETDB1 regulates microtubule dynamics.

SETDB1 is a methyltransferase responsible for the methylation of histone H3-lysine-9, which is mainly related to heterochromatin formation. SETDB1 is overexpressed in various cancer types and is associated with an aggressive phenotype. In agreement with its activity, it mainly exhibits a nuclear localization; however, in several cell types a cytoplasmic localization was reported. Here we looked for cytoplasmic functions of SETDB1. SETDB1 association with microtubules was detected by immunofluorescence and co-sedimentation. Microtubule dynamics were analysed during recovery from nocodazole treatment and by tracking microtubule plus-ends in live cells. Live cell imaging was used to study mitotic kinetics and protein-protein interaction was identified by co-immunoprecipitation. SETDB1 co-sedimented with microtubules and partially colocalized with microtubules. SETDB1 partial silencing led to faster polymerization and reduced rate of catastrophe events of microtubules in parallel to reduced proliferation rate and slower mitotic kinetics. Interestingly, over-expression of either wild-type or catalytic dead SETDB1 altered microtubule polymerization rate to the same extent, suggesting that SETDB1 may affect microtubule dynamics by a methylation-independent mechanism. Moreover, SETDB1 co-immunoprecipitated with HDAC6 and tubulin acetylation levels were increased upon silencing of SETDB1. Taken together, our study suggests a model in which SETDB1 affects microtubule dynamics by interacting with both microtubules and HDAC6 to enhance tubulin deacetylation. Overall, our results suggest a novel cytoplasmic role for SETDB1 in the regulation of microtubule dynamics.

Role of histone deacetylase inhibitors in androgenic callus induction of Oryza sativa sub indica, in sight into evolution and mode of action of histone deacetylase genes.

The potential of paddy breeding has reached its pinnacle, and hybrids have been the principal research outcome. Hence, our hypothesis was based on improvising the callus induction efficiency of recalcitrant Oryza sativa sub. indica hybrids by intervening into their cellular functions like cell division and histone regulation for the production of doubled haploids, a better output compared to hybrids. Insight into the mechanism of cell division is the foremost concern in altering the same and hence studies on evolution, expression and action of histone deacetylase and its 12 genes (9 HDA and 3 HD-tunin genes) were chosen in the hypothesis. Expression of HDA genes at three stages (anther dehiscence, 1st callusing and second callusing stages) with inhibitor (trichostatin-A) interventions indicated 1st callusing stage as the most important in influencing callus induction and also the genes HDA19, 6, 15 and 5 were the most important. TSA alone had a significant impact on the regulation of the genes HDT 702, HDA19, HDA9, and HDA6. Higher expression of HDA19 and HDA6 was involved in maximizing callus induction; HDA15 had an antagonistic expression compared to HDA19/6 and might be involved in chlorophyll regulation during regeneration. Results of evolutionary analysis on histone deacetylases indicated a long and single lineage of origin denoting its importance in the basic cellular functions. The tubulin deacetylation gene HDA5, which was exclusively found in dicotyledons, had a recent evolutionary history only from terrestrial plants, and also had significant conservation in its motifs and NLS region. By combating the recalcitrant nature of Indica cultivars, molecular editing on a combination of HDA genes will enhance the callus induction and regeneration efficiency of the next generation of doubled haploids, therby improving the total yield.

Acetyl-CoA Carboxylase Inhibitor CP640.186 Increases Tubulin Acetylation and Impairs Thrombin-Induced Platelet Aggregation.

Acetyl-CoA carboxylase (ACC) is the first enzyme regulating de novo lipid synthesis via the carboxylation of acetyl-CoA into malonyl-CoA. The inhibition of its activity decreases lipogenesis and, in parallel, increases the acetyl-CoA content, which serves as a substrate for protein acetylation. Several findings support a role for acetylation signaling in coordinating signaling systems that drive platelet cytoskeletal changes and aggregation. Therefore, we investigated the impact of ACC inhibition on tubulin acetylation and platelet functions. Human platelets were incubated 2 h with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. We have herein demonstrated that CP640.186 treatment does not affect overall platelet lipid content, yet it is associated with increased tubulin acetylation levels, both at the basal state and after thrombin stimulation. This resulted in impaired platelet aggregation. Similar results were obtained using human platelets that were pretreated with tubacin, an inhibitor of tubulin deacetylase HDAC6. In addition, both ACC and HDAC6 inhibitions block key platelet cytoskeleton signaling events, including Rac1 GTPase activation and the phosphorylation of its downstream effector, p21-activated kinase 2 (PAK2). However, neither CP640.186 nor tubacin affects thrombin-induced actin cytoskeleton remodeling, while ACC inhibition results in decreased thrombin-induced reactive oxygen species (ROS) production and extracellular signal-regulated kinase (ERK) phosphorylation. We conclude that when using washed human platelets, ACC inhibition limits tubulin deacetylation upon thrombin stimulation, which in turn impairs platelet aggregation. The mechanism involves a downregulation of the Rac1/PAK2 pathway, being independent of actin cytoskeleton.

The Aneugenicity of Ketone Bodies in Colon Epithelial Cells Is Mediated by Microtubule Hyperacetylation and Is Blocked by Resveratrol.

Diabetes mellitus (DM) is considered to be associated with an increased risk of colorectal cancer. Recent studies have also revealed that tubulin hyperacetylation is caused by a diabetic status and we have reported previously that, under microtubule hyperacetylation, a microtubule severing protein, katanin-like (KL) 1, is upregulated and contributes to tumorigenesis. To further explore this phenomenon, we tested the effects of the ketone bodies, acetoacetate and β-hydroxybutyrate, in colon and fibroblast cells. Both induced microtubule hyperacetylation that responded differently to a histone deacetylase 3 knockdown. These two ketone bodies also generated intracellular reactive oxygen species (ROS) and hyperacetylation was commonly inhibited by ROS inhibitors. In a human fibroblast-based microtubule sensitivity test, only the KL1 human katanin family member showed activation by both ketone bodies. In primary cultured colon epithelial cells, these ketone bodies reduced the tau protein level and induced KL1- and α-tubulin acetyltransferase 1 (ATAT1)-dependent micronucleation. Resveratrol, known for its tumor preventive and tubulin deacetylation effects, inhibited this micronucleation. Our current data thus suggest that the microtubule hyperacetylation induced by ketone bodies may be a causal factor linking DM to colorectal carcinogenesis and may also represent an adverse effect of them that needs to be controlled if they are used as therapeutics.

A novel orally active HDAC6 inhibitor T-518 shows a therapeutic potential for Alzheimer\u2019s disease and tauopathy in mice

Accumulation of tau protein is a key pathology of age-related neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Those diseases are collectively termed tauopathies. Tau pathology is associated with axonal degeneration because tau binds to microtubules (MTs), a component of axon and regulates their stability. The acetylation state of MTs contributes to stability and histone deacetylase 6 (HDAC6) is a major regulator of MT acetylation status, suggesting that pharmacological HDAC6 inhibition could improve axonal function and may slow the progression of tauopathy. Here we characterize N-[(1R,2R)-2-{3-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]-5-oxo-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl}cyclohexyl]-2,2,3,3,3-pentafluoropropanamide (T-518), a novel, potent, highly selective HDAC6 inhibitor with clinically favorable pharmacodynamics. T-518 shows potent inhibitory activity against HDAC6 and superior selectivity over other HDACs compared with the known HDAC6 inhibitors in the enzyme and cellular assays. T-518 showed brain penetration in an oral dose and blocked HDAC6-dependent tubulin deacetylation at Lys40 in mouse hippocampus. A 2-week treatment restored impaired axonal transport and novel object recognition in the P301S tau Tg mouse, tauopathy model, while a 3-month treatment also decreased RIPA-insoluble tau accumulation. Pharmaceutical inhibition of HDAC6 is a potential therapeutic strategy for tauopathy, and T-518 is a particularly promising drug candidate.

Direct Amidation to Access 3-Amido-1,8-Naphthalimides Including Fluorescent Scriptaid Analogues as HDAC Inhibitors.

Methodology to access fluorescent 3-amido-1,8-naphthalimides using direct Buchwald–Hartwig amidation is described. The protocol was successfully used to couple a number of substrates (including an alkylamide, an arylamide, a lactam and a carbamate) to 3-bromo-1,8-naphthalimide in good yield. To further exemplify the approach, a set of scriptaid analogues with amide substituents at the 3-position were prepared. The new compounds were more potent than scriptaid at a number of histone deacetylase (HDAC) isoforms including HDAC6. Activity was further confirmed in a whole cell tubulin deacetylation assay where the inhibitors were more active than the established HDAC6 selective inhibitor Tubastatin. The optical properties of these new, highly active, compounds make them amenable to cellular imaging studies and theranostic applications.

Tektin4 loss promotes triple-negative breast cancer metastasis through HDAC6-mediated tubulin deacetylation and increases sensitivity to HDAC6 inhibitor.

Progression of triple-negative breast cancer (TNBC) constitutes a major unresolved clinical challenge, and effective targeted therapies are lacking. Because microtubule dynamics play pivotal roles in breast cancer metastasis, we performed RNA sequencing on 245 samples from TNBC patients to characterize the landscape of microtubule-associated proteins (MAPs). Here, our transcriptome analyses revealed that low expression of one MAP, tektin4, indicated poor patient outcomes. Tektin4 loss led to a marked increase in TNBC migration, invasion, and metastasis and a decrease in microtubule stability. Mechanistically, we identified a novel microtubule-associated complex containing tektin4 and histone deacetylase 6 (HDAC6). Tektin4 loss increased the interaction between HDAC6 and α-tubulin, thus decreasing microtubule stability through HDAC6-mediated tubulin deacetylation. Significantly, we found that tektin4 loss sensitized TNBC cells, xenograft models, and patient-derived organoid models to the HDAC6-selective inhibitor ACY1215. Furthermore, tektin4 expression levels were positively correlated with microtubule stability levels in clinical samples. Together, our findings uncover a metastasis suppressor function of tektin4 and support clinical development of HDAC6 inhibition as a new therapeutic strategy for tektin4-deficient TNBC patients.

Sub-toxic levels of cobalt ions impair chondrocyte mechanostranduction via HDAC6-dependent primary cilia shortening

Cobalt ions are the main wear particles associated with orthopaedic implants, causing adverse complications due to cytotoxicity and inflammatory mediators. Recent studies have shown that sub-toxic levels of cobalt ions regulate matrix synthesis and inflammation, but the influence of cobalt ions on mechanotransduction remains unclear. Previously, we reported that sub-toxic levels of cobalt ions modulated primary cilia, which are crucial for mechanotransduction. This study therefore aimed to investigate the effect of cobalt ions on chondrocyte mechanosensation in response to cyclic tensile strain and the association with primary cilia. Sub-toxic levels of cobalt ions impaired chondrocyte mechanosensation and affected the gene expression of aggrecan, collagen II and MMP-13. Moreover, cobalt ions induced HDAC6-dependent primary cilia disassembly, which was associated with either cytoplasmic or ciliary α-tubulin deacetylation. Pharmaceutical HDAC6 inhibition with tubacin restored primary cilia length and mechanotransduction, whereas chemical depletion of primary cilia by chloral hydrate prevented mechanosignalling. Thus, sub-toxic levels of cobalt ions impaired chondrocyte mechanotransduction via HDAC6 activation, which was associated with tubulin deacetylation and primary cilia shortening.

A phase II study of selective HDAC6 inhibition with KA2507 for second-line treatment of advanced biliary tract cancer (ABC-11).

TPS4652 Background: The ABC-02 trial provided Level A evidence supporting the use of cisplatin plus gemcitabine as first-line chemotherapy for advanced biliary tract cancer (ABC) [Valle, 2010]. In second line therapy oxaliplatin and 5FU and ivosedinib for IDH1 mutated cancers are options [Lamarca, 2019; Abou-Alfa, 2019] however there remains significant unmet need for patients without actionable alterations. Histone deacetylase 6 (HDAC6) is over-expressed in cholangiocarcinoma, reducing primary cilia. This is mediated through increased resorption in normal human cholangiocytes via tubulin deacetylation in the ciliary axoneme. Inhibition of HDAC6 elicits both cell intrinsic and extrinsic anti-cancer activity. HDAC6 inhibition reversed oncogenic loss of ciliation and demonstrated preclinical efficacy in a syngeneic rat orthotopic biliary cancer model [Gradilone, 2013]. KA2507 is a potent and selective small molecule inhibitor of HDAC6. Phase I dose escalation study identified an oral dose of 800mg bid for further development, being well tolerated and showing evidence of selective target engagement. Methods: ABC-11 is a Phase II multi-centre, open-label study of KA2507 in 40 evaluable patients with advanced biliary tract cancer previously treated with standard of care chemotherapy. The study follows a single-arm single-stage design using A’Hern’s methodology. Eligible patients receive continuous 28-day cycles of fixed daily oral dose of KA2507 until death, disease progression or other pre-defined reason for study drug discontinuation. Tumour assessment is made at baseline and at 8-weekly intervals using RECIST 1.1 criteria until disease progression; primary endpoint is PFS at 4 months. Independent Data Monitoring Committee will review 4 month PFS and other data after first six patients, after a total of 17 patients (futility analysis, corresponding to cut-off of the Simon’s minimax 2-stage design; 33% was set as the target 4-month PFS rate expected with KA2507) and at least annually thereafter. Subject to availability of adequate tissue, mandatory pre-treatment and on-study tumour biopsy samples will undergo multiparameter flow cytometry of immune cell subsets, immunofluorescence analysis of immune cell subsets (activation status and topology) and T cell repertoire studies. The study received regulatory and ethical approval to proceed in January 2020 and enrolment is in progress. Clinical trial information: NCT04186156 .

The disordered N-terminus of HDAC6 is a microtubule-binding domain critical for efficient tubulin deacetylation

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic enzyme having tubulin deacetylase activity that has been unequivocally assigned to the second of the tandem catalytic domains. However, virtually no information exists on the contribution of other HDAC6 domains on tubulin recognition. Here, using recombinant protein expression, site-directed mutagenesis, fluorimetric and biochemical assays, microscale thermophoresis, and total internal reflection fluorescence microscopy, we identified the N-terminal, disordered region of HDAC6 as a microtubule-binding domain and functionally characterized it to the single-molecule level. We show that the microtubule-binding motif spans two positively charged patches comprising residues Lys-32 to Lys-58. We found that HDAC6-microtubule interactions are entirely independent of the catalytic domains and are mediated by ionic interactions with the negatively charged microtubule surface. Importantly, a crosstalk between the microtubule-binding domain and the deacetylase domain was critical for recognition and efficient deacetylation of free tubulin dimers both in vitro and in vivo Overall, our results reveal that recognition of substrates by HDAC6 is more complex than previously appreciated and that domains outside the tandem catalytic core are essential for proficient substrate deacetylation.

The histone deacetylase inhibitor tubacin mitigates endothelial dysfunction by up-regulating the expression of endothelial nitric oxide synthase

Endothelial nitric oxide (NO) synthase (eNOS) plays a critical role in the maintenance of blood vessel homeostasis. Recent findings suggest that cytoskeletal dynamics play an essential role in regulating eNOS expression and activation. Here, we sought to test whether modulation of cytoskeletal dynamics through pharmacological regulation of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation affects eNOS expression and endothelial function in vitro and in vivo We found that tubulin acetylation inducer (tubacin), a compound that appears to selectively inhibit HDAC6 activity, dramatically increased eNOS expression in several different endothelial cell lines, as determined by both immunoblotting and NO production assays. Mechanistically, we found that these effects were not mediated by tubacin's inhibitory effect on HDAC6 activity, but rather were due to its ability to stabilize eNOS mRNA transcripts. Consistent with these findings, tubacin also inhibited proinflammatory cytokine-induced degradation of eNOS transcripts and impairment of endothelium-dependent relaxation in the mouse aorta. Furthermore, we found that tubacin-induced up-regulation in eNOS expression in vivo is associated with improved endothelial function in diabetic db/db mice and with a marked attenuation of ischemic brain injury in a murine stroke model. Our findings indicate that tubacin exhibits potent eNOS-inducing effects and suggest that this compound might be useful for the prevention or management of endothelial dysfunction-associated cardiovascular diseases.

Histone deacetylase 6 inhibition rescues axonal transport impairments and prevents the neurotoxicity of HIV-1 envelope protein gp120

Despite successful antiretroviral drug therapy, a subset of human immunodeficiency virus-1 (HIV)-positive individuals still display synaptodendritic simplifications and functional cognitive impairments referred to as HIV-associated neurocognitive disorders (HANDs). The neurological damage observed in HAND subjects can be experimentally reproduced by the HIV envelope protein gp120. However, the complete mechanism of gp120-mediated neurotoxicity is not entirely understood. Gp120 binds to neuronal microtubules and decreases the level of tubulin acetylation, suggesting that it may impair axonal transport. In this study, we utilized molecular and pharmacological approaches, in addition to microscopy, to examine the relationship between gp120-mediated tubulin deacetylation, axonal transport, and neuronal loss. Using primary rat cortical neurons, we show that gp120 decreases acetylation of tubulin and increases histone deacetylase 6 (HDAC6), a cytoplasmic enzyme that regulates tubulin deacetylation. We also demonstrate that the selective HDAC6 inhibitors tubacin and ACY-1215, which prevented gp120-mediated deacetylation of tubulin, inhibited the ability of gp120 to promote neurite shortening and cell death. We further observed by co-immunoprecipitation and confirmed with mass spectroscopy that exposure of neurons to gp120 decreases the association between tubulin and motor proteins, a well-established consequence of tubulin deacetylation. To assess the physiological consequences of this effect, we examined the axonal transport of brain-derived neurotrophic factor (BDNF). We report that gp120 decreases the velocity of BDNF transport, which was restored to baseline levels when neurons were exposed to HDAC6 inhibitors. Overall, our data suggest that gp120-mediated tubulin deacetylation causes impairment of axonal transport through alterations to the microtubule cytoskeleton.

HDAC6 Inhibition Protects against OGDR-Induced Golgi Fragmentation and Apoptosis.

The Golgi apparatus (GA) is a pivotal organelle, and its fragmentation is an essential process in the development of apoptosis. GA is a potential target in the treatment of cerebral ischemia-reperfusion injury. Histone deacetylase 6 (HDAC6) catalyzes the removal of functional acetyl groups from proteins and plays an important role in cell homeostasis. In this study, the neuroprotective effects and the underlying mechanisms of HDAC6 inhibition were assessed in an ischemia-reperfusion injury model. Mouse neuroblastoma N2a cells and cultured neurons were subjected to oxygen-glucose deprivation/reperfusion (OGDR) insult. OGDR induces Golgi fragmentation and reduces tubulin acetylation in N2a cells and cultured neurons. Golgi fragmentation is prior to nuclear chromatin condensation after OGDR injury. Overexpression of GBF1 not only protects against OGDR-induced Golgi fragmentation but also protects against OGDR-induced apoptosis, suggesting that Golgi fragmentation is not secondary to apoptosis but plays a causal role for subsequent apoptosis. HDAC6 inhibition suppresses OGDR-induced tubulin deacetylation, p115 cleavage, and caspase 3 activation and protects against OGDR-induced Golgi fragmentation and apoptosis. This work opens a new avenue for potential clinical application of HDAC6 inhibitors for cerebral ischemia-reperfusion-related disorders.

Kif18a regulates Sirt2-mediated tubulin acetylation for spindle organization during mouse oocyte meiosis

BackgroundDuring oocyte meiosis, the cytoskeleton dynamics, especially spindle organization, are critical for chromosome congression and segregation. However, the roles of the kinesin superfamily in this process are still largely unknown.ResultsIn the present study, Kif18a, a member of the kinesin-8 family, regulated spindle organization through its effects on tubulin acetylation in mouse oocyte meiosis. Our results showed that Kif18a is expressed and mainly localized in the spindle region. Knock down of Kif18a caused the failure of first polar body extrusion, dramatically affecting spindle organization and resulting in severe chromosome misalignment. Further analysis showed that the disruption of Kif18a caused an increase in acetylated tubulin level, which might be the reason for the spindle organization defects after Kif18a knock down in oocyte meiosis, and the decreased expression of deacetylase Sirt2 was found after Kif18a knock down. Moreover, microinjections of tubulin K40R mRNA, which could induce tubulin deacetylation, protected the oocytes from the effects of Kif18a downregulation, resulting in normal spindle morphology in Kif18a-knock down oocytes.ConclusionsTaken together, our results showed that Kif18a affected Sirt2-mediated tubulin acetylation level for spindle organization during mouse oocyte meiosis. Our results not only revealed the critical effect of Kif18a on microtubule stability, but also extended our understanding of kinesin activity in meiosis.

Pharmacophore-based virtual screening for identification of potential selective inhibitors of human histone deacetylase 6

Histone deacetylase (HDAC) 6 plays a role in oncogenic transformation and cancer metastasis via tubulin deacetylation, making it a critical target for anticancer drug design. However, lack of selectivity shown by many of the current HDAC6 inhibitors in clinical use and trials prompts the continuous search for selective inhibitors. Here, 10 pharmacophore hypotheses were developed based on the 3D common features of training set of 20 HDAC inhibitors in clinical use and trials. The hypotheses were validated using a test set of another 20 HDAC inhibitors along with 400 inactive (decoys) molecules based on Güner-Henry pharmacophore scoring method. Hypothesis 1 consisting of 1 H-bond donor, 1 H-bond acceptor and 2 hydrophobic features, was used to screen “DruglikeDiverse” database using Biovia Discovery Studio 4.5. The top 10 hit compounds were selected based on the pharmacophore fit values (>3.00). Their binding affinity against HDAC6 compared to class I HDACs (1, 2, 3 & 8) and a class IIa member (HDAC7), was calculated by molecular docking using AutoDock4. The stability of binding modes of 2 potential HDAC6-selective inhibitors (ENA501965 and IBS399024) was examined by 30 ns-molecular dynamics (MD) simulation using nanoscale MD (NAMD) software. Both ligands showed potential stability in HDAC6 active site over time. Therefore, these may provide additional scaffolds for further optimization towards the design of safe, potent and selective HDAC6 inhibitors.

HDAC3 inhibition disrupts the assembly of meiotic apparatus during porcine oocyte maturation.

Histone deacetylases (HDACs) are involved in a wide array of biological processes. However, the role of HDAC3 in porcine oocytes remains unclear. In the current study, we examine the effects of HDAC3 inhibition on porcine oocyte maturation using RGFP966, a selective HDAC3 inhibitor. We find that suppression of HDAC3 activity prevents not only the expansion of cumulus cells but also the meiotic progression of oocytes. It is interesting to note that HDAC3 displays a spindle-like distribution pattern as the porcine oocytes enter meiosis. In line with this, confocal microscopy reveals the high frequency of spindle defects and chromosomal congression failure in metaphase oocytes exposed to RGFP966. Moreover, HDAC3 inhibition results in the hyperacetylation of α-tubulin during oocyte meiosis. These findings indicate that HDAC3 activity might control the microtubule stability via the deacetylation of tubulin, which is critical for maintaining the proper spindle assembly, accurate chromosome separation, and orderly meiotic progression during porcine oocyte maturation.

Sirtuin 2 Isoform 1 Enhances Hepatitis B Virus RNA Transcription and DNA Synthesis through the AKT/GSK-3β/β-Catenin Signaling Pathway.

Sirtuin 2 (Sirt2), a NAD+-dependent protein deacetylase, is overexpressed in many hepatocellular carcinomas (HCCs) and can deacetylate many proteins, including tubulins and AKT, prior to AKT activation. Here, we found that endogenous Sirt2 was upregulated in wild-type hepatitis B virus (HBV WT)-replicating cells, leading to tubulin deacetylation; however, this was not the case in HBV replication-deficient-mutant-transfected cells and 1.3-mer HBV WT-transfected and reverse transcriptase inhibitor (entecavir or lamivudine)-treated cells, but all HBV proteins were expressed. In HBV WT-replicating cells, upregulation of Sirt2 induced AKT activation, which consequently downregulated glycogen synthase kinase 3β (GSK-3β) and increased β-catenin levels; however, downregulation of Sirt2 in HBV-nonreplicating cells impaired AKT/GSK-3β/β-catenin signaling. Overexpression of Sirt2 isoform 1 stimulated HBV transcription and consequently HBV DNA synthesis, which in turn activated AKT and consequently increased β-catenin levels, possibly through physical interactions with Sirt2 and AKT. Knockdown of Sirt2 by short hairpin RNAs (shRNAs), inhibition by 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide (AGK2), or dominant negative mutant expression inhibited HBV replication, reduced AKT activation, and decreased β-catenin levels. Through HBV infection, we demonstrated that Sirt2 knockdown inhibited HBV replication from transcription. Although HBx itself activates AKT and upregulates β-catenin, Sirt2-mediated signaling and upregulated HBV replication were HBx independent. Since constitutively active AKT inhibits HBV replication, the results suggest that upregulated Sirt2 and activated AKT may balance HBV replication to prolong viral replication, eventually leading to the development of HCC. Also, the results indicate that Sirt2 inhibition may be a new therapeutic option for controlling HBV infection and preventing HCC.IMPORTANCE Even though Sirt2, a NAD+-dependent protein deacetylase, is overexpressed in many HCCs, and overexpressed Sirt2 promotes hepatic fibrosis and associates positively with vascular invasion by primary HCCs through AKT/GSK-3β/β-catenin signaling, the relationship between Sirt2, HBV, HBx, and/or HBV-associated hepatocarcinogenesis is unclear. Here, we show that HBV DNA replication, not HBV expression, correlates positively with Sirt2 upregulation and AKT activation. We demonstrate that overexpression of Sirt2 further increases HBV replication, increases AKT activation, downregulates GSK-3β, and increases β-catenin levels. Conversely, inhibiting Sirt2 decreases HBV replication, reduces AKT activation, and decreases β-catenin levels. Although HBx activates AKT to upregulate β-catenin, Sirt2-mediated effects were not dependent on HBx. The results also indicate that a Sirt2 inhibitor may control HBV infection and prevent the development of hepatic fibrosis and HCC.