Isoform-selective Histone Deacetylase Research Articles

Dual-targeting class I HDAC inhibitor and ATM activator, SP-1-303, preferentially inhibits estrogen receptor positive breast cancer cell growth.

Dual-targeting chromatin regulation and DNA damage repair signaling presents a promising avenue for cancer therapy. Applying rational drug design, we synthesized a potent dual-targeting small molecule, SP-1-303. Here, we report SP-1-303 as a class I isoform selective histone deacetylase (HDAC) inhibitor and an activator of the ataxia-telangiectasia mutated protein (ATM). In vitro enzymatic assays demonstrated selective inhibition of HDAC1 and HDAC3. Cellular growth inhibition studies show that SP-1-303 differentially inhibits growth of estrogen receptor positive breast cancer (ER+ BC) cells with effective growth inhibition concentrations (EC50) for MCF-7 and T47D cells ranging from 0.32 to 0.34 μM, compared to 1.2-2.5 μM for triple negative breast cancer cells, and ~12 μM for normal breast epithelial cells. Western analysis reveals that SP-1-303 decreases estrogen receptor alpha (ER-α) expression and increases p53 protein expression, while inducing the phosphorylation of ATM and its substrates, BRCA1 and p53, in a time-dependent manner in ER+ BC cells. Pharmacokinetic evaluation demonstrates an area under the curve (AUC) of 5227.55 ng/ml × h with an elimination half-life of 1.26 h following intravenous administration in a rat model. Collectively, SP-1-303 emerges as a novel second generation class I (HDAC1 and HDAC3) selective HDAC inhibitor, and ATM activator, capable of modulating ER expression, and inhibiting growth of ER+ BC cells. Combined targeting of class I HDACs and ATM by SP-1-303 offers a promising therapeutic approach for treating ER+ breast cancers and supports further preclinical evaluation.

Butyrate functions as a histone deacetylase inhibitor to protect pancreatic beta cells from IL-1β-induced dysfunction.

Butyrate, a gut microbial metabolite, has beneficial effects on glucose homeostasis and has become an attractive drug candidate for type 2 diabetes (T2D). Recently, we showed that butyrate protects pancreatic beta cells against cytokine-induced dysfunction. In this study, we explored the underlying mechanisms of butyrate action. Pancreatic mouse islets were exposed to a non-cytotoxic concentration of interleukin-1β (IL-1β) for 10 days to mimic low-grade inflammation in T2D. Similar to the effect of butyrate, an isoform-selective histone deacetylase 3 (HDAC3) inhibitor normalized IL-1β-reduced glucose-stimulated insulin secretion and insulin content. In contrast, free fatty acid receptor 2 and 3 (FFAR2/3) agonists failed to normalize IL-1β-induced beta cell dysfunction. Furthermore, butyrate inhibited HDAC activity and increased the acetylation of histone H3 and H4 by 3- and 10-fold, respectively. Genome-wide analysis of histone H3 lysine 27 acetylation (H3K27ac) revealed that butyrate mainly increased H3K27ac at promoter regions (74%), while H3K27ac peaks regulated by IL-1β were more equally distributed at promoters (38%), introns (23%) and intergenic regions (23%). Gene ontology analysis showed that butyrate increased IL-1β-reduced H3K27ac levels near several genes related to hormone secretion and reduced IL-1β-increased H3K27ac levels near genes associated with inflammatory responses. Butyrate alone increased H3K27ac near many genes related to MAPK signaling, hormone secretion, and differentiation, and decreased H3K27ac at genes involved in cell replication. Together, these results suggest that butyrate prevents IL-1β-induced pancreatic islet dysfunction by inhibition of HDACs resulting in changes in H3K27ac levels at genes relevant for beta cell function and inflammatory responses.

Discovery of benzamide-based PI3K/HDAC dual inhibitors with marked pro-apoptosis activity in lymphoma cells

Inhibition of PI3K and histone deacetylase (HDAC) activity simultaneously using a single molecule appears to be a promising approach for cancer treatment. Current PI3K/HDAC dual inhibitors commonly use hydroxamate moiety as zinc binding group, which lack HDAC isoform selectivity and have potential genotoxicity. In this study, a novel series of benzamide-based PI3K/HDAC dual inhibitors were rationally designed and synthesized. Representative compound PH14 showed potent inhibitory activity toward PI3Kα and HDAC3, with IC50 values of 20.3 nM and 24.5 nM, respectively. This was further supported by the blockage of AKT phosphorylation and an increase in acetylated histone H3 levels in Western blot study. The advantage of simultaneously targeting PI3Kα and HDAC is not only reflected in the significant antiproliferative activity, but also in its ability to promote the apoptosis in Jeko-1 cells. Moreover, PH14 had weak inhibitory effects on CYP450 enzymes and hERG. In the pharmacokinetic study, the administration of 1 mg/kg of PH14 the administration of 1 mg/kg of PH14 resulted in a t1/2 of 10 h and an AUC (0-∞) of 2772 h ng/mL. Our study may provide ideas for the further development of novel HDAC/PI3K dual inhibitors.

Discovery of 1-Benzhydryl-Piperazine-Based HDAC Inhibitors with Anti-Breast Cancer Activity: Synthesis, Molecular Modeling, In Vitro and In Vivo Biological Evaluation.

Isoform-selective histone deacetylase (HDAC) inhibition is promoted as a rational strategy to develop safer anti-cancer drugs compared to non-selective HDAC inhibitors. Despite this presumed benefit, considerably more non-selective HDAC inhibitors have undergone clinical trials. In this report, we detail the design and discovery of potent HDAC inhibitors, with 1-benzhydryl piperazine as a surface recognition group, that differ in hydrocarbon linker. In vitro HDAC screening identified two selective HDAC6 inhibitors with nanomolar IC50 values, as well as two non-selective nanomolar HDAC inhibitors. Structure-based molecular modeling was employed to study the influence of linker chemistry of synthesized inhibitors on HDAC6 potency. The breast cancer cell lines (MDA-MB-231 and MCF-7) were used to evaluate compound-mediated in vitro anti-cancer, anti-migratory, and anti-invasive activities. Experiments on the zebrafish MDA-MB-231 xenograft model revealed that a novel non-selective HDAC inhibitor with a seven-carbon-atom linker exhibits potent anti-tumor, anti-metastatic, and anti-angiogenic effects when tested at low micromolar concentrations.

Discovery of Potent and Isoform-selective Histone Deacetylase Inhibitors Using Structure-based Virtual Screening and Biological Evaluation.

Histone deacetylases (HDACs) are key enzymes in epigenetics and promising targets for anticancer therapy. Although several drugs targeting HDAC have been approved for the treatment of tumors, their clinical use has been limited by their deleterious side effects and poor efficacy. Herein, we discover four potent HDAC inhibitors through pharmacophore model screening and molecular docking. These compounds are able to bind HDACs 1, 3, and 6 with nanomolar affinity. Among them, compound 3 shows greater inhibitory effect on HDACs 1, 3, and 6 than that of vorinostat (SAHA). Evaluation of anticancer activity indicates that compound 3 significantly inhibits the growth of solid cancer cells including HGC-27, AGS, MDA-MB-231, A549, MCF-7, and H460 cells. In vivo anticancer study suggests that compound 3 can also markedly inhibit the growth of HGC-27 cells-derived xenograft, with no observable toxicity. These findings suggest that compound 3 may be as a potential HDAC-targeting inhibitor for solid tumor therapy.

Design, synthesis and biological evaluation of dual HDAC and VEGFR inhibitors as multitargeted anticancer agents.

Herein, a novel series of dual histone deacetylase (HDAC) and vascular endothelial growth factor receptor (VEGFR) inhibitors were designed, synthesized and biologically evaluated based on previously reported pazopanib-based HDAC and VEGFR dual inhibitors. Most target compounds showed significant HDAC1, HDAC6 and VEGFR2 inhibition, which contributed to their potent antiproliferative activities against multiple cancer cell lines and significant antiangiogenic potencies in both human umbilical vein endothelial cell (HUVEC) tube formation assays and rat thoracic aorta ring assays. Further HDAC selectivity evaluations indicated that hydroxamic acids 5 and 9epossessed HDAC isoform selectivity profiles similar to that of the approved HDAC inhibitor suberoylanilide hydroxamic acid(SAHA), while hydrazide12 presented an HDAC isoform selectivity profilesimilar to that of the clinical HDAC inhibitor MS-275. The VEGFR inhibition profiles of 5, 9e and 12were similar to that of the approved VEGFR inhibitor pazopanib. The intracellular target engagements of Compounds 5 and 12were confirmed by western blot analysis. The metabolic stabilities of 5, 9e and 12 in mouse liver microsomes were inferior to that of pazopanib. These dual HDAC and VEGFR inhibitors provide lead compounds for further structural optimization to obtainpolypharmacological anticancer agents.

Discovery of novel isoform-selective histone deacetylases 5 and 9 inhibitors through combined ligand-based pharmacophore modeling, molecular mocking, and molecular dynamics simulations for cancer treatment

Class IIa histone deacetylases (HDACs) 5 and 9 play crucial roles in several human disorders such as cancer, making them important targets for drug design. Continuous research is pursed to overcome the cytotoxicity side effect that comes with the currently available broad-spectrum HDACs inhibitors. Herein, common features of active HDACs inhibitors in clinical trials and use have been calculated to generate the best pharmacophore hypothesis. Guner-Henry scoring system was used to validate the generated hypotheses. Hypo1 of HDAC5 and Hypo2 of HDAC9 exhibited the most statistically significance hypotheses. Compounds with fit value of 3 and more were examined by QuickVina 2 docking tool to calculate their binding affinity toward all class IIa HDACs. A total of 6 potential selective compounds were subjected to 100 molecular dynamics (MD) simulation to examine their binding modes. The free binding energy calculations were computed according to the MM-PBSA method. Proposed selective compounds displayed good stability with their targets and thus they may offer potent leads for the designing of HDAC5 and HDAC9 isoform selective inhibitors.

Discovery of novel tetrahydrobenzo[b]thiophene-3-carbonitriles as histone deacetylase inhibitors

The discovery and development of isoform-selective histone deacetylase (HDAC) inhibitor is a challenging task because of the sequence homology among HDAC enzymes. In the present work, novel tetrahydro benzo[b]thiophene-3-carbonitrile based benzamides were designed, synthesized, and evaluated as HDAC inhibitors. Pharmacophore modeling was our main design strategy, and two novel series of tetrahydro benzo[b]thiophene-3-carbonitrile derivatives with piperidine linker (series 1) and piperazine linker (series 2) were identified as HDAC inhibitors. Among all the synthesised compounds, 9h with 4-(aminomethyl) piperidine linker and 14n with piperazine linker demonstrated good activity against human HDAC1 and HDAC6, respectively. Both the compounds also exhibited good antiproliferative activity against several human cancer cell lines. Both these compounds (9h and 14n) also induced cell cycle arrest and apoptosis in U937 and MDA-MB-231 cancer cells. Overall, for the first time, this research discovered potent isoform-selective HDAC inhibitors using cyclic linker instead of the aliphatic chain and aromatic ring system, which were reported in known HDAC inhibitors.

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

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

Bifunctional HDAC Therapeutics: One Drug to Rule Them All?

Histone deacetylase (HDAC) enzymes play crucial roles in epigenetic gene expression and are an attractive therapeutic target. Five HDAC inhibitors have been approved for cancer treatment to date, however, clinical applications have been limited due to poor single-agent drug efficacy and side effects associated with a lack of HDAC isoform or complex selectivity. An emerging strategy aiming to address these limitations is the development of bifunctional HDAC therapeutics—single molecules comprising a HDAC inhibitor conjugated to another specificity targeting moiety. This review summarises the recent advancements in novel types of dual-targeting HDAC modulators, including proteolysis-targeting chimeras (PROTACs), with a focus on HDAC isoform and complex selectivity, and the future potential of such bifunctional molecules in achieving enhanced drug efficacy and therapeutic benefits in treating disease.

Anticancer Ruthenium Complexes with HDAC Isoform Selectivity.

The histone deacetylase (HDAC) enzymes have emerged as an important class of molecular targets in cancer therapy, with five inhibitors in clinical use. Recently, it has been shown that a lack of selectivity between the 11 Zn-dependent HDAC isoforms may lead to unwanted side-effects. In this paper, we show that piano stool Ru complexes can act as HDAC inhibitors, and variation in the capping arene leads to differences in HDAC isoform selectivity.

Exploring structural requirements of isoform selective histone deacetylase inhibitors: a comparative in silico study

Histone deacetylases (HDACs) are a widely popular class of epigenetic regulators, second only in importance to DNA methyltransferases. They are responsible for deacetylating the lysine residues of a wide range of proteins, both nuclear and cytoplasmic. Therefore, deregulated HDAC activity is implicated in disruption of important biological functions leading to cancerous, neuropathological, infectious and inflammatory diseased states. The current therapeutic strategies aimed at combating HDAC related pathologies consist of pan HDAC inhibitors that target multiple HDAC isoforms. Many side-effects of such therapeutics have been reported due to off-target effects. Hence, efforts need to be focused towards developing therapeutics targeting single isoforms. This work aims at recognizing structural features, both of receptors and inhibitors, that would help achieve selective inhibition of HDAC isoforms. Protein alignment studies have been carried out to define the receptor structure differences that can be exploited for this purpose. Binding modes of highly isoform selective inhibitors have been established through molecular docking studies to characterize the receptor-ligand interactions responsible for selective inhibition. This information is represented with the help of pharmacophore models.

Design, Synthesis and In Vitro Biological Evaluation Of N-(2-Aminophenyl)-3-Quinolin-4-yl -Prop-2-enamide Derivatives As Novel Colon Anticancer Agents

Acetylation and deacetylation of histone proteins is regulated through two enzymes; acetylation is mediated through histone acetyl transferase (HAT) whereas deacetylation is mediated through histone deacetylase (HDAC). Histone deacetylase removes acetyl group of lysine residue on histone proteinthis makes negatively charged histone able to bind with DNA containing positive centers. In this way DNA remain in compact form with histone proteins. Therefore the transcription or replication enzymes and cofactors not able to bind such compact DNA structure. Histone deacetylase inhibition results into inhibition of various transcription activities which may get accelerated during cancer development. The aim of proposed study is to develop effective isoform selective Histone deacetylase inhibitor. Synthesis of N-(2-Aminophenyl)-3-Quinolin-4-yl-Prop-2-Enamide derivatives from isatin and its derivatives was achieved. Synthesis was carried out in two steps with good yield. 21 Compounds were synthesized and found to be effective with IC50s in between 3.694 - 38.4µmol in human HCT-116, COLO 205 and COLO 320 DM colon cancer cells in vitro. For the cytotoxicity activity study MTT assay method was adopted. Docking studies were performed initially with HDAC8 enzyme using V-life MDS software for synthesized compounds and had selected best fit molecules for synthesis. The synthesized molecules are effective as colon anticancer agents.

Development of classification models for identification of important structural features of isoform-selective histone deacetylase inhibitors (class I).

As one of the hot topics in the epigenetic studies, histone deacetylases inhibitors (HDACIs) have been introduced to treat a variety of diseases such as cancer, immune disorder and neuronal diseases. Given the high numbers of available pathways in which HDACs are involved, the HDACIs that act particularly on Class I or Class II enzymes are considered as possible candidates for anticancer drugs. Due to their effective roles in the onset of cancer and its progression, HDAC Class I isoforms (HDAC 1, 2, 3 and 8) were considered in this study. Herein, our objective is to determine the important isoform-selective and isoform-active structural features of HDACIs using the valid classification models. For this purpose, a diverse dataset comprising 8224 HDAC modulators was collected from the binding database. To identify the significant discriminative features, five classification models were generated by supervised Kohonen network and support vector machine methods. Variable importance in projection method was used as a variable selection approach. The results obtained from descriptor analysis show that physicochemical properties, such as hydrogen bonding, number of branches, size, flexibility, polarity and sphericity in the structure of molecules, were closely related to the bioactivity of HDACIs. The reliability and predictive ability of the conducted models were evaluated using the tenfold cross-validation techniques, test sets and applicability domain analysis. All of the obtained classification models represented high statistical quality and predictive ability with accuracy greater than 85% for the test sets. The proposed strategy and the selective patterns represented in this paper can be applied by researchers in the pharmaceutical sciences who aim to use the same idea for the design of drugs with improved anticancer properties.

Histone Deacetylase Inhibition Sensitizes PD1 Blockade-Resistant B-cell Lymphomas.

PD1 blockade is effective in a subset of patients with B-cell lymphoma (e.g., classical-Hodgkin lymphomas); however, most patients do not respond to anti-PD1 therapy. To study PD1 resistance, we used an isoform-selective histone deacetylase inhibitor (HDACi; OKI-179), and a mouse mature B-cell lymphoma, G1XP lymphoma, immunosuppressive features of which resemble those of human B-cell lymphomas, including downregulation of MHC class I and II, exhaustion of CD8+ and CD4+ tumor-infiltrating lymphocytes (TIL), and PD1-blockade resistance. Using two lymphoma models, we show that treatment of B-cell lymphomas refractory to PD1 blockade with both OKI-179 and anti-PD1 inhibited growth; furthermore, sensitivity to single or combined treatment required tumor-derived MHC class I, and positively correlated with MHC class II expression level. We conclude that OKI-179 sensitizes lymphomas to PD1-blockade by enhancing tumor immunogenicity. In addition, we found that different HDACis exhibited distinct effects on tumors and T cells, yet the same HDACi could differentially affect HLA expression on different human B-cell lymphomas. Our study highlights the immunologic effects of HDACis on antitumor responses and suggests that optimal treatment efficacy requires personalized design and rational combination based on prognostic biomarkers (e.g., MHCs) and the individual profiles of HDACi.

Design, synthesis and biological evaluation of novel thioquinazolinone-based 2-aminobenzamide derivatives as potent histone deacetylase (HDAC) inhibitors

A series of novel 2-aminobenzamide derivatives decorated with thioquinazolinone were designed and synthesized as histone deacetylase (HDAC) inhibitors. These derivatives were evaluated for their antiproliferative activities against several human cancer cell lines including A375, Hela, A549, HCT116 and SMMC7721. It's significantly indicated that some inhibitors exhibited potent antiproliferative activities towards all the studied cancer cell lines. Compounds 7a, 4i, 4o, and 4p exhibited higher antiproliferative activities towards three cancer cell lines: A375, A549 and SMMC7721 compared to CS055, MS275, and CI994. Compound 4p showed more than 4000-fold the isoform selectivity for HDAC1 and more than 250-fold selectivity for HDAC2 compared with HDAC6. The molecular docking analysis reasonably explained the HDAC inhibitory activity and isoform selectivity. In addition, compounds 7a, 4i, 4o, and 4p showed potent inhibitory activities in migration assay and colony formation analysis, and also promoted cell apoptosis. Moreover, compounds 7a, 4i, and 4o inhibited the growth of SMMC7721 cells at S phase of the cell cycle. The immunofluorometric analysis indicated that compounds 7a, 4i, 4o, and 4p could increase the acetylation status of H3K9. Furthermore, in vivo anticancer efficacy of compound 4p was assessed in the A549 xenograft models, and 4p demonstrated potent antitumor activity (TGI = 62.5%). This study provided an effective strategy for further development of tumor-targeting therapy.

Identification of isoform-selective hydroxamic acid derivatives that potently reactivate HIV from latency

ObjectivesCurrent antiretroviral therapy can suppress HIV replication, increase CD4 count and result in increased lifespan. However, it cannot eradicate the virus due to the presence of latent provirus in cellular reservoirs, such as resting CD4+ T cells. Using combination latency-reversing agents to shock the virus out of latency for elimination through immune clearance or viral cytopathic effect is one of the most promising strategies for HIV eradication. Specifically, recent evidence shows that isoform-selective histone deacetylase inhibitors may be more effective than their non-selective counterparts. Therefore, identification and characterisation of new isoform-selective compounds are of prime importance. Here, we sought to determine the ability of two new isoform-targeted hydroxamic acid derivatives to reactivate HIV from latency. MethodsWe used cell lines and infected primary resting CD4+ T cells. These were treated with these compounds with HIV reactivation measured using fluorescence-activated cell sorting, Western blots and luciferase luminescence. Isoform selectivity and acetylation of the HIV promoter were measured by Western blotting and chromatic immunoprecipitation. Results and conclusionsThe two new hydroxamic acid derivatives, MC2625 and MC1742, potently reactivate HIV from latency. These compounds are isoform-selective histone deacetylate inhibitors that increase the levels of histone acetylation at the HIV promoter. In addition, they synergise effectively with the protein kinase C modulators bryostatin-1 and INDY, an inhibitor of the dual-specificity tyrosine phosphorylation regulated kinase 1A. We conclude that the combinations of new hydroxamic acid derivatives and bryostatin-1 or INDY could be a new tool for HIV reactivation in the cure efforts.

Safety and Tolerability of Histone Deacetylase (HDAC) Inhibitors in Oncology

Histone deacetylases (HDACs) are expressed at increased levels in cells of various malignancies, and the use of HDAC inhibitors has improved outcomes in patients with haematological malignancies (T-cell lymphomas and multiple myeloma). However, they are not as effective in solid tumours. Five agents are currently approved under various jurisdictions, namely belinostat, chidamide, panobinostat, romidepsin and vorinostat. These agents are associated with a range of class-related and agent-specific serious and/or severe adverse effects, notably myelosuppression, diarrhoea and various cardiac effects. Among the cardiac effects are ST-T segment abnormalities and QTc interval prolongation of the electrocardiogram, isolated cases of atrial fibrillation and, in rare instances, ventricular tachyarrhythmias. In order to improve the safety profile of this class of drugs as well as their efficacy in indications already approved and to further widen their indications, a large number of newer HDAC inhibitors with varying degrees of HDAC isoform selectivity have been synthesised and are currently under clinical development. Preliminary evidence from early studies suggests that they may be effective in non-haematological cancers as well when used in combination with other therapeutic modalities, but that they too appear to be associated with the above class-related adverse effects. As the database accumulates, the safety, efficacy and risk/benefit of the newer agents and their indications will become clearer.

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).

Abstract 5801: Unexpected selectivity of histone deacetylase inhibitors in live MDA-MD-231 triple-negative breast cancer cells

Abstract While inhibition of histone deacetylases (HDAC) is a promising strategy for the treatment of cancer, HDACs do play essential roles in normal cellular function, limiting their use as therapeutic targets. Increasing evidence shows that each of the HDAC isoforms carry out unique biological functions; therefore, by inhibiting a single isoform, one could regulate a focused subset of genes and achieve the desired therapeutic effect and lower toxicity. To investigate this aspect, many isoform-selective inhibitors have been synthesized, characterized for selectivity against recombinant HDACs in biochemical assays, and tested for anti-cancer activity. Despite significant progress, the actual engagement of HDAC isoforms is poorly characterized since methods to determine selectivity of HDAC inhibitors in live cells with unperturbed epigenetic machinery and in vivo are missing. Recently, using a novel photoreactive HDAC probe (PRP), we demonstrated that its isoform selectivity in live breast cancer cells is cell-type dependent, differs from that determined in the biochemical assays, and, in the case of HDAC3, is regulated by phosphorylation by c-Jun N-terminal kinase (JNK). Similar cell-type dependent variability in HDAC isoform selectivity was also observed for other HDAC isoforms. In this report, we extended our photolabeling approach to characterize HDAC isoform selectivity of the commonly used in pre-clinical and clinical research non-selective and selective inhibitors: SAHA, panobinostat, pimelic diphenylamide 106, PCI-34051, and TMP-269. Seven novel PRPs based on the chemical scaffolds of these HDAC inhibitors were synthesized and tested in the biochemical assay and the cell-based photolabeling assay in live triple negative breast cancer cells MDA-MB-231. We found that the cell-based profiles of these PRPs were skewed toward inhibition of HDAC3 irrespective of their biochemical selectivity, chemotypes, or isoform abundance, suggesting that the actual HDAC isoform selectivity and target engagement in live cells is different from that perceived based on the results of the biochemical assay. These studies suggest that a substantial correction in our understanding of the role HDAC isoforms and their inhibitors play in live cells in culture and in vivo may be necessary. Further expansion of these studies to other types of cells and animal tissues ex vivo and in vivo as well as identification of the mechanisms responsible for modulation of HDAC isoform selectivity and target engagement are in progress. Citation Format: Shaimaa M. Aboukhatwa, Thomas W. Hanigan, Jayaprakash Neerasa, Taha Y. Taha, Nathan D. Brown, Eman E. El-Bastawissy, Mohamed A. Elkersh, Tarek F. El-Moselhy, Jonna Frasor, Pavel A. Petukhov. Unexpected selectivity of histone deacetylase inhibitors in live MDA-MD-231 triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5801.