Inhibition Of Histone Deacetylase Activity Research Articles

Design, synthesis and biological evaluation of novel HDAC inhibitors with improved pharmacokinetic profile in breast cancer

The dysfunction of histone deacetylase (HDACs) is closely related to tumorigenesis and development, which has been emerged as an attractive drug design target for cancer therapy. In the present study, we designed and synthesized a series of novel HDAC inhibitors using a substituted quinazoline as the capping group and attaching 3, 5-dimethylbenyl as a potential metabolic site protector. 23g and 23h were demonstrated potent HDAC inhibitory activities and anti-proliferative effects against MDA-MB-231cells. In addition, 23g and 23h both could significantly increase the acetylation level of intracellular proteins, especially in α-Tubulin and HSP90. 23g and 23h displayed a slight different anti-tumor mechanism, 23g mainly induced apoptosis while 23h induced obviously ER-Stress. Furthermore, 23g and 23h both induced autophagy and migration inhibition. In pharmacokinetics assay, 23g showed a significant improvement of pharmacokinetic profile for oral administration. Additionally, 23g presented more potent anti-proliferation and anti-migration activity than SAHA in zebrafish MDA-MB-231cell line-derived xenograft model. Together, these results demonstrate that 23g is a novel oral HDAC inhibitor with a potential capacity of treating breast cancer.

Assessment of Synergistic Contribution of Histone Deacetylases in Prognosis and Therapeutic Management of Sarcoma.

Sarcomas are a rare group of neoplasms with a mesenchymal origin that are mainly characterized by the abnormal growth of connective tissue cells. The standard treatment for local control of sarcomas includes surgery and radiation, while for adjuvant and palliative therapy, chemotherapy has been strongly recommended. Despite the availability of multimodal therapies, the survival rate for patients with sarcoma is still not satisfactory. In recent decades, there has been a considerable effort to overcome chemotherapy resistance in sarcoma cells. This has led to the investigation of more cellular compounds implicated in gene expression and transcription processes. Furthermore, it has been discovered that histone acetylation/deacetylation equilibrium is affected in carcinogenesis, leading to a modified chromatin structure and therefore changes in gene expression. In addition, histone deacetylase inhibition is found to play a key role in limiting the tumor burden in sarcomas, as histone deacetylase inhibitors act on well-described oncogenic signaling pathways. Histone deacetylase inhibitors disrupt the increased cell motility and invasiveness of sarcoma cells, undermining their metastatic potential. Moreover, their activity on evoking cell arrest has been extensively described, with histone deacetylase inhibitors regulating the reactivation of tumor suppressor genes and induction of apoptosis. Promoting autophagy and increasing cellular reactive oxygen species are also included in the antitumor activity of histone deacetylase inhibitors. It should be noted that many studies revealed the synergy between histone deacetylase inhibitors and other drugs, leading to the enhancement of an antitumor effect in sarcomas. Therefore, there is an urgent need for therapeutic interventions modulated according to the distinct clinical and molecular characteristics of each sarcoma subtype. It is concluded that a better understanding of histone deacetylase and histone deacetylase inhibitors could provide patients with sarcoma with more targeted and efficient therapies, which may contribute to significant improvement of their survival potential.

Discovery of a Novel Hybrid of Vorinostat and Riluzole as a Potent Antitumor Agent.

Vorinostat (suberoylanilide hydroxamic acid) was the first approved histone deacetylase (HDAC) inhibitor in a group of validated cancer therapeutic agents targeting epigenetics. Riluzole is a drug used to treat amyotrophic lateral sclerosis, the antitumor potency of which has been recently revealed. Herein, a novel hybrid of vorinostat and riluzole (compound 1) was rationally designed, synthesized, and evaluated. Compared with vorinostat, compound 1 exhibited superior total HDAC inhibitory activity and similar HDAC isoform selective profiles. The intracellular HDAC inhibition of compound 1 was confirmed by Western blot analysis. Moreover, compound 1 possessed more potent in vitro antiproliferative activity against all tested solid and hematological tumor cell lines than vorinostat. In vitro metabolic stability evaluation of compound 1 revealed better human plasma stability and comparable human liver microsomal stability than vorinostat. Additionally, compound 1 demonstrated more significant in vivo antitumor activity in a MDA-MB-231 xenograft model than vorinostat, which could be attributed to its superior in vitro antiproliferative activity and metabolic stability. Taken together, the results presented here support further research and development of compound 1 as a promising antitumor agent.

Acute Valproate Exposure Induces Sex-Specific Changes in Steroid Hormone Metabolism in the Cerebral Cortex of Juvenile Mice.

Valproic acid (VPA), an antiepileptic and mood stabilizer, modulates neurotransmission and gene expression by inhibiting histone deacetylase activity. It is reported that VPA may affects the steroid hormone level. In this study, VPA-induced acute metabolic alterations were investigated using liquid chromatography-tandem mass spectrometry in prepubertal mice brain. In VPA-treated (400 mg/kg in saline solution, intraperitoneal) mice, cortisol levels were increased (female: P < 0.004, male: P < 0.003) and 17β-estradiol levels were decreased (Both P < 0.03). Furthermore, in the VPA-treated male mice, dihydrotestosterone levels were increased (P < 0.02) and testosterone were decreased (P < 0.002). The 4-hydroxylase activity was upregulated in the female VPA-treated mice (P < 0.01) and the 5α-reductase activity was increased in the male VPA-treated mice (P < 0.003). These results indicate sex specific differences in VPA-induced steroid metabolism in the brain cortex.

Novel Histone Deacetylase Inhibitors and HIV-1 Latency-Reversing Agents Identified by Large-Scale Virtual Screening.

Current antiretroviral therapies used for HIV management do not target latent viral reservoirs in humans. The experimental “shock-and-kill” therapeutic approach involves use of latency-reversal agents (LRAs) that reactivate HIV expression in reservoir-containing cells, followed by infected cell elimination through viral or host immune cytopathic effects. Several LRAs that function as histone deacetylase (HDAC) inhibitors are reported to reverse HIV latency in cells and in clinical trials; however, none to date have consistently reduced viral reservoirs in humans, prompting a need to identify new LRAs. Toward this goal, we describe here a virtual screening (VS) approach which uses 14 reported HDAC inhibitors to probe PubChem and identifies 60 LRA candidates. We then show that four screening “hits” including (S)-N-Hydroxy-4-(3-methyl-2-phenylbutanamido)benzamide (compound 15), N-(4-Aminophenyl)heptanamide (16), N-[4-(Heptanoylamino)phenyl]heptanamide (17), and 4-(1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N-(2-hydroxyethyl)butanamide (18) inhibit HDAC activity and/or reverse HIV latency in vitro. This study demonstrates and supports that VS-based approaches can readily identify novel HDAC inhibitors and LRAs, which in turn may help toward inhibitor design and chemical optimization efforts for improved HIV shock-and-kill-based efforts.

Design, synthesis and biological evaluation of coumarin-based N-hydroxycinnamamide derivatives as novel histone deacetylase inhibitors with anticancer activities

A series of novel coumarin-based N-hydroxycinnamamide derivatives were designed and synthesized as histone deacetylase (HDAC) inhibitors. Most of the synthesized compounds showed potent HDAC inhibitory activity and significant antiproliferative activity against human cancer cell lines MCF-7, HepG2, HeLa and HCT-116. Among them, compound 14f displayed the most potent HDAC inhibition, especially against HDAC1 with IC50 value of 0.19 μM, which was better than that of SAHA (IC50 = 0.23 μM). It also showed the strongest antiproliferative activity towards HeLa cells and more than 26-fold selectivity for HDAC1 compared with HDAC6. Molecular docking studies revealed the possible binding modes of compound 14f into the two isoforms and provided a reasonable explanation for the selectivity. In addition, compound 14f could inhibit colony formation, upregulate the acetylation level of histone H3, and induce apoptosis and cell cycle arrest at G2/M phase in HeLa cells. Taken together, these results highlighted that compound 14f might be a promising HDAC inhibitor for cancer therapy.

Cellular Gene Modulation of HIV-Infected CD4 T Cells in Response to Serial Treatment with the Histone Deacetylase Inhibitor Vorinostat.

Histone deacetylase inhibitors (HDACi) are the most widely studied HIV latency-reversing agents (LRAs). The HDACi suberoylanilide hydroxamic acid (vorinostat [VOR]) has been employed in several clinical HIV latency reversal studies, as well as in vitro models of HIV latency, and has been shown to effectively induce HIV RNA and protein expression. Despite these findings, response to HDACi can vary, particularly with intermittent dosing, and information is lacking on the relationship between the host transcriptional response and HIV latency reversal. Here, we report on global gene expression responses to VOR and examine the longevity of the transcriptional response in various cellular models. We found that many genes are modulated at 6 h post-VOR treatment in HCT116, Jurkat, and primary resting CD4 T cells, yet return to baseline levels after an 18-h VOR-free period. With repeat exposure to VOR in resting CD4 T cells, we found similar and consistent transcriptional changes at 6 h following each serial treatment. In addition, serial exposure in HIV-infected suppressed donor CD4 T cells showed consistent transcriptional changes after each exposure to VOR. We identified five host genes that were strongly and consistently modulated following histone deacetylase (HDAC) inhibition; three (H1F0, IRGM, and WIPI49) were upregulated, and two (PHF15 and PRDM10) were downregulated. These genes demonstrated consistent modulation in peripheral blood mononuclear cell (PBMC) samples from HIV-positive (HIV+) participants who received either single or multiple doses of 400 mg of VOR. Interestingly, the host transcriptional response did not predict induction of cell-associated HIV RNA, suggesting that other cellular factors play key roles in HIV latency reversal in vivo despite robust HDACi pharmacological activity.IMPORTANCE Histone deacetylase inhibitors are widely studied HIV latency-reversing agents (LRAs). VOR, an HDACi, induces histone acetylation and chromatin remodeling and modulates host and HIV gene expression. However, the relationship between these events is poorly defined, and clinical studies suggest diminished HIV reactivation in resting CD4 T cells with daily exposure to VOR. Our study provides evidence that VOR induces a consistent level of host cell gene transcription following intermittent exposure. In addition, in response to VOR exposure a gene signature that was conserved across single and serial exposures both in vitro and in vivo was identified, indicating that VOR can consistently and reproducibly modulate transcriptional host responses. However, as the HIV response to HDACi declines over time, other factors modulate viral reactivation in vivo despite robust HDAC activity. The identified host gene VOR biomarkers can be used for monitoring the pharmacodynamic activity of HDAC inhibitors.

Synthesis and biological evaluation of a novel photo-activated histone deacetylase inhibitor

Hydroxamic acid-based histone deacetylase inhibitors (HDACi) are a class of epigenetic agents with potentially broad therapeutic application to several disease states including post angioplasty mediated neointimal hyperplasia (NIH). Precise spatiotemporal control over the release of HDACi at the target blood vessel site is required for the safe and successful therapeutic use of HDACi in the setting of drug eluting balloon catheter (DEBc) angioplasty treatment of NIH. We aimed to develop and characterise a novel photoactive HDACi, as a potential coating agent for DEBc.Metacept-3 1 was caged with a photo-labile protecting group (PPG) to synthesise a novel UV365nm active HDACi, caged metacept-3 15. Conversion of caged metacept-3 15 to active/native metacept-3 1 by UV365nm was achievable in significant quantities and at UV365nm power levels in the milliwatt (mW) range.In vitro evaluation of the biological activity of pre and post UV365nm activation of caged metacept-3 15 identified significant HDACi activity in samples exposed to short duration, mW range UV365nm. Toxicity studies performed in human umbilical vein endothelial cells (HUVEC’s) identified significantly reduced toxicity of caged metacept-3 15 pre UV365nm exposure compared with native metacept-3 1 and paclitaxel (PTX).Taken together these findings identify a novel photo-activated HDACi, caged metacept-3 15, with pharmacokinetic activation characteristics and biological properties which may make it suitable for evaluation as a novel coating for targeted DEBc angioplasty interventions.

SAT-311 Role of SREBP in Regulation of Corticotroph Tumor ACTH Secretion and Cell Proliferation

Cushing Disease (CD) is a life-threatening condition with suboptimal medical treatment. To identify drugs that not only inhibit ACTH secretion to attain eucortisolemia but also inhibit tumor growth, we conducted a high throughput screen employing a novel “gain of signal” ACTH AlphaLISA assay. From a kinase inhibitor library containing 430 compounds, we identified the dual PI3K/HDAC inhibitor, CUDC-907, as a potent inhibitor of both in vitro and in vivo corticotroph tumor ACTH secretion and growth. By stepwise comparison of CUDC-907 with mono-functional PI3K and HDAC inhibitors, we demonstrated that CUDC-907 exerts its inhibitory effect on ACTH secretion primarily through its inhibition of HDAC activity at the POMC transcriptional level; while PI3K-mediated inhibition of corticotroph cell viability further contributes to reduced ACTH secretion. We also used RNA-seq to characterize the global transcriptiome changes associated with CUDC-907 treatment. Hierarchical clustering showed that 1432 differentially expressed genes (DEGs, p≤0.05 and fold-change≥1.5) were altered by CUDC-907 treatment in comparison to the vehicle-treated control cells. Gene ontology (GO) analysis of 456 downregulated and 976 upregulated DEGs revealed that the most enriched biological processes were cholesterol biosynthesis (GO:0006695, p=1.977e-17) and the type I interferon signaling pathway (GO: 0060337, p=4.928e-7) respectively. Further analysis demonstrated downregulation of the membrane-bound transcription factor sterol regulatory element binding proteins (SREBPs). Downregulation of SREBP-2 by CUDC-907 as well as the several other target enzymes in the cholesterol biosynthesis and uptake pathway including IDI2, NSDHL, MVD, and HMGCR, was confirmed by real-time PCR. To further characterize a role for SREBP-2 in regulation of corticotroph tumor ACTH secretion and proliferation, we employed siRNA targeting endogenous SREBP-2 (SREBP-2 mRNA, Control vs. siRNA 1±0.03 vs. 0.6±0.08, p<0.05), and demonstrated that knockdown of SREBP-2 not only inhibited POMC mRNA expression (POMC mRNA, 1±0.03 vs. 0.7±0.01, p<0.01), and ACTH secretion (ACTH (ng/mL) 29±0.4 vs. 23±0.3, p<0.005), but also suppressed cell proliferation (Relative Proliferation Rate, 1±0.01 vs. 0.7±0.01, p<0.005). This was further confirmed by overexpression of cleaved mature SREBP-2, which led to increased POMC expression and cell proliferation. We demonstrate for the first time the role of the SREBP-mediated cholesterol biosynthesis pathway in regulation of corticotroph tumor POMC regulation and growth. Our studies identify SREBP and cholesterol biosynthesis as a therapeutic target in CD.

Short chain fatty acids regulate T cell metabolism to promote IL-22 production and gut immunity

Abstract Interleukin-22 (IL-22) is critical in gut immunity. Both innate lymphocyte cells (ILCs) and CD4+ T cells produce IL-22. ILCs provide a rapid source of IL-22 that is essential for early protection of epithelial barrier function upon enteric infection and inflammatory insult, while T cells become the dominant source of IL-22 in the intestinal lamina propria during chronic intestinal inflammation. Microbiota is central in IL-22 production in the intestines, however, the factors that regulate IL-22 production in T cells and ILCs in the intestines, and the mechanisms involved remain unclear. We found that microbiota-derived short-chain fatty acids (SCFAs) promoted IL-22 production in T cells and ILCs through their inhibition of HDAC activity but not GPR43. Additionally, SCFAs upregulated IL-22 production by promoting aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor (HIF)1α expression, which were differentially regulated by mTOR and Stat3 pathways. Furthermore, HIF1α directly bind to the il22 promoter, and SCFAs increased HIF1α binding to the il22 promoter through histone modification. SCFAs enhanced oxygen consumption and glycolysis, which mediated SCFA-induced IL-22 production. Hypixic condition enhanced the SCFAs induction of IL-22, and glucose facilitated SCFAs induction of IL-22 production in a dose-dependent manner. SCFA supplementation protected intestines from Citrobacter Rodentium infection and dextran sodium sulfate (DSS) insult, which was mediated by enhanced IL-22. SCFAs promoted T cell IL-22 production from patients with Crohn’s disease through inducing AHR and HIF1α. These findings establish the roles of SCFAs in inducing IL-22 production in T cells and ILCs to maintain the intestinal homeostasis.

Influence of side-chain changes on histone deacetylase inhibitory and cytotoxicity activities of curcuminoid derivatives

Using curcuminoids as lead compounds, fifty-nine curcuminoid derivatives with different side chains at the phenolic moiety were synthesized. All compounds were investigated for their histone deacetylase (HDAC) inhibitory activities. The potent pan-HDAC inhibitors were further tested against three human cancer cell lines including Hela, HCT116 and MCF-7 with MTT-based assay. The bisethylamide 4z and the mono-sec-butyl derivative 5j manifested good antiproliferative activities against HCT116 cancer cells with the IC50 values as 14.60 ± 1.19 μg/mL and 7.33 ± 0.98 μg/mL, respectively. Molecular docking study of both compounds with Class I HDACs revealed that the compounds might bind tightly to the binding pocket of HDAC2. These findings suggested that these compounds can be putative candidates for the development of anticancer drugs via inhibiting HDACs.

Synthesis of Tetramic Acid Derivatives via a Tandem Umpolung Alkylation/Reduction/Cyclization Reaction of γ-Hydrazono β-Ketoester.

An efficient method for the one-pot synthesis of tetramic acid derivatives was developed utilizing tandem umpolung N-alkylation/reduction/cyclization of γ-hydrazono β-ketoester. By using this reaction as a key step, the total synthesis of the 3-spiro 7-hydroxamic acid tetralin which possesses an HDAC inhibitory activity was also achieved.

Design, synthesis and anticancer activity of novel valproic acid conjugates with improved histone deacetylase (HDAC) inhibitory activity

Twenty-five valproic acid conjugates have been designed and synthesized. All target compounds were explored for their in vitro anti-proliferative activities using the MTT-based assay against four human cancer cell lines includingliver (HePG2), colon (HCT116), breast (MCF7) and cervical (HeLa) carcinoma cell lines. Out of six valproic acid-amino acid conjugates 2a-f. Only cysteine containing conjugate 2f showed the significant activity (IC50 9.10µM against HePG2 and 6.81µM against HCT116). However conjugate 2j showed broad-spectrum antitumor activity against all cell lines tested. In addition, conjugates 4j and 4k which contains phenyl hydrazide and hydroxamic acid group, respectively, also showed broad spectrum activity. Furthermore, six compounds were screened for HDAC 1-9 isozymes inhibitory activities. Compounds 2j, 4j and 4k manifested a higher inhibitory activity more than valproic acid but less than SAHA. In addition, the in vivo antitumor screening of 2j, 4j and 4k was done and the results have shown that 2j, 4j and 4k, particularly 4j, showed a significant decrease in tumor size and presented a considerable decrease in viable EAC count. Docking study of selectedcompound 4j revealed that it can bind nicely to the binding pocket of HDAC 1, 2, 3, 4 and HDAC 8. The results suggest that compounds 2j, 4j and 4k, particularly 4j, may be promising lead candidates for the development of novel targeted anti-tumor drug potentially via inhibiting HDACs.

Synthesis, Characterization and Biological Evaluation of Novel Dihydropyranoindoles Improving the Anticancer Effects of HDAC Inhibitors.

The dihydropyranoindole scaffold was identified as a promising target for improving the anti-cancer activity of HDAC inhibitors from the preliminary screening of a library of compounds. A suitable methodology has been developed for the preparation of novel dihydropyranoindoles via the Hemetsberger indole synthesis using azido-phenylacrylates, derived from the reaction of corresponding alkynyl-benzaldehydes with methyl azidoacetate, followed by thermal cyclization in high boiling solvents. Anti-cancer activity of all the newly synthesized compounds was evaluated against the SH-SY5Y and Kelly neuroblastoma cells as well as the MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines. Biological studies showed that the tetracyclic systems had significant cytotoxic activity at higher concentration against the neuroblastoma cancer cells. More importantly, these systems, at the lower concentration, considerably enhanced the SAHA toxicity. In addition to that, the toxicity of designated systems on the healthy human cells was found to be significantly less than the cancer cells.

SUN-040 MANIPULATING THE GUT MICROBIOME BY DIETARY FIBRE TO PREVENT FOLIC ACID INDUCED KIDNEY DISEASE

Gut dysbiosis has been associated with inflammation and mortality in chronic kidney disease. Bacterial fermentation of dietary fibre produces short-chain fatty acids (SCFAs) that regulate inflammation and immunity. Here we evaluated the effect of dietary fibre or SCFA supplementation on development of AKI and subsequent fibrosis in a murine model of folic acid nephropathy (FAN). FAN was induced in C57BL/6 (WT) and GRP43-/- mice by intraperitoneal folic acid injection. Mice were fed a high fibre (HF) diet, normal chow (NF), or supplemented with oral SCFAs (150mM sodium acetate (SA), 150mM propionate (SP), or 100mM butyrate (SB) in drinking water). Diets and SCFA treatment commenced 2 weeks prior to injection and continued throughout the duration of all experiments. Samples were collected on day 2 and 28 after FAN induction. Gut microbiota composition was assessed by 16S rRNA sequencing of faecal DNA. Metabolic profiling of serum and faeces was performed using 1H NMR spectroscopy. HF diet protected against FAN, with improvement of renal function and histological parameters versus NF diet, as evidenced by lower serum creatinine (day2: 5.7±3.0 vs 69.3±54.2; day28: 6.6±2.2 vs 12.3±4.4, P<0.0001), reduced tubular injury (P<0.05) and less cellular infiltration at days 2 and 28. FAN caused intestinal dysbiosis, while gut microbiota balance was restored by HF diet with increased relative abundance of phylum Actinobacteria at the expense of Firmicutes, and expansion of SCFA producing Bifidobacterium (p<0.0001) and Prevotella (p<0.0005) genera compared to FAN controls on NF diet. This change in microbial ecology correlated with a significant increase in faecal SCFAs (p<0.0005) and serum acetate (1.00±0.18vs0.53±0.15, p<0.0001) relative to NF fed controls. Supplementation with SCFAs in FAN achieved similar degrees of protection from loss of renal function (P<0.0001) and histological injury at days 2 and 28, and interstitial fibrosis at day 28. mRNA expression of the innate immune receptor (TLR2), proinflammatory cytokines (IL-6, TNFα) and chemokine (MIP2) at day 2 and 28, and fibrotic genes (TGFb, MMP2 & MMP9 ) at day 28 were downregulated in HF diet and SCFA mice compared with NF diet. SA supplementation was equally effective in GPR43+/+ and GPR43-/- FAN mice. Kidney tissue histone deacetylase (HDAC) activity was inhibited in HF FAN mice (P<0.01) at day 2, with significantly reduced mRNA expression of HDAC4 (P<0.005) and HDAC10 (P<0.01) compared to NF FAN mice. HF diet prevented development of folic acid induced acute and chronic kidney injury through enrichment of SCFA producing gut bacteria and increased SCFA production that inhibited HDAC activity. The protective effects of dietary modulation of the gut microbiota identify this as a simple, potentially translatable therapy for AKI and CKD.

Design, synthesis and biological evaluation of novel indazole-based derivatives as potent HDAC inhibitors via fragment-based virtual screening

Based on fragment-based virtual screening and bioisoterism strategies, novel indazole and pyrazolo[3,4-b] pyridine derivatives as HDACs inhibitors were designed, synthesized and evaluated. Most of these compounds displayed good to excellent inhibitory activities against HDACs, especially compounds 15k and 15m were identified as potent inhibitors of HDAC1 (IC50 = 2.7 nM and IC50 = 3.1 nM), HDAC2 (IC50 = 4.2 nM and IC50 = 3.6 nM) and HDAC8 (IC50 = 3.6 nM and IC50 = 3.3 nM). Further anti-proliferation assays revealed that compounds 15k and 15m showed better anti-proliferative activities against HCT-116 and HeLa cells than positive control SAHA. The western blot analysis results indicated that compounds 15k and 15m noticeably up-regulated the level of acetylated α-tubulin and histone H3. In addition, the two compounds 15k and 15m could arrest cell cycle in G2/M phase and promote cell apoptosis, which was similar as the reference compound SAHA. Through the molecular docking and dynamic studies, the potent HDAC inhibitory activities mainly caused by van der Waals and electrostatic interactions with the HDACs.

Hydroxygenkwanin Inhibits Class I HDAC Expression and Synergistically Enhances the Antitumor Activity of Sorafenib in Liver Cancer Cells.

Abnormal histone deacetylase (HDAC) expression is closely related to cancer development and progression. Many HDAC inhibitors have been widely used in cancer treatment; however, severe side effects often limit their clinical application. In this study, we attempted to identify natural compounds with HDAC inhibitory activity and low physiological toxicity and explored their feasibility and mechanisms of action in liver cancer treatment. A yeast screening system was used to identify natural compounds with HDAC inhibitory activity. Further, western blotting was used to verify inhibitory effects on HDAC in human liver cancer cell lines. Cell functional analysis was used to explore the effects and mechanisms and the in vitro results were verified in BALB/c nude mice. We found that hydroxygenkwanin (HGK), an extract from Daphne genkwa, inhibited class I HDAC expression, and thereby induced expression of tumor suppressor p21 and promoted acetylation and activation of p53 and p65. This resulted in the inhibition of growth, migration, and invasion of liver cancer cells and promoted cell apoptosis. Animal models revealed that HGK inhibited tumor growth in a synergistic manner with sorafenib. HGK inhibited class I HDAC expression and had low physiological toxicity. It has great potential as an adjuvant for liver cancer treatment and may be used in combination with anticancer drugs like sorafenib to improve therapeutic efficacy.

Discriminations of active from inactive HDAC8 inhibitors Part II: Bayesian classification study to find molecular fingerprints

ABSTRACTIn continuation of our earlier work (Doi: 10.1080/07391102.2019.1661876), a statistically validated and robust Bayesian model was developed on a large diverse set of HDAC8 inhibitors. The training set comprised of 676 small molecules and 293 compounds were considered as test set molecules. The findings of this analysis will help to explore some major directions regarding the HDAC8 inhibitor designing approach. Acrylamide (G1-G3, G9), N-substituted 2-phenylimidazole (G4-G8, G9, G12-G13, G16-G19), benzimidazole (G10-G11), piperidine substituted pyrrole (G13-G14) groups, alkyl/aryl amide (G15) and aryloxy carboxamide (G20) fingerprints were found to play a crucial role in HDAC8 inhibitory activity whereas -CH-N=CH- (B1, B4-B6, B14) motif, benzamide (B2-B3, B9-B13, B16-B17) groups and heptazepine (B7-B8, B15, B18-B20) group were found to influence negatively the HDAC8 inhibitory activity. The importance of such fingerprints was further validated by the HDAC8 enzyme and related inhibitor interactions at the receptor level. These results are in close agreement with those of our previous work that validate each other. Moreover, this comparative learning may enrich future endeavours regarding the designing strategy of HDAC8 inhibitors.

Biochemical and Behavioral Characterization of IN14, a New Inhibitor of HDACs with Antidepressant-Like Properties.

Evidence suggests that histone deacetylases (HDACs) inhibitors could be used as an effective treatment for some psychiatric and neurological conditions such as depression, anxiety and age-related cognitive decline. However, non-specific HDAC inhibiting compounds have a clear disadvantage regarding their efficacy and safety, thus the need to develop more selective ones. The present study evaluated the toxicity, the capacity to inhibit HDAC activity and antidepressant-like activity of three recently described class I HDAC inhibitors IN01, IN04 and IN14, using A. salina toxicity test, in vitro fluorometric HDAC activity assay and forced-swimming test, respectively. Our data show that IN14 possesses a better profile than the other two. Therefore, the pro-cognitive and antidepressant effects of IN14 were evaluated. In the forced-swimming test model of depression, intraperitoneal administration of IN14 (100 mg/Kg/day) for five days decreased immobility, a putative marker of behavioral despair, significantly more than tricyclic antidepressant desipramine, while also increasing climbing behavior, a putative marker of motivational behavior. On the other hand, IN14 left the retention latency in the elevated T-maze unaltered. These results suggest that novel HDAC class I inhibitor IN14 may represent a promising new antidepressant with low toxicity and encourages further studies on this compound.

Abstract P3-11-08: Targeting LIFR enhances the activity of HDAC inhibitors for the treatment of triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease. TNBC lacks targeted therapies and represents a disproportional share of the breast cancer (BC) mortality rate. Histone deacetylase inhibitors (HDACIs) are emerging as promising multifunctional agents in TNBC to elicit cytotoxic actions. Recent studies have shown that cancer cells elucidate feedback activation of leukemia inhibitory factor receptor (LIFR) which in turn curtails response to HDACIs. We developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIFR downstream signaling. Here, we examined whether the novel LIFR inhibitor, EC359, has the ability to counteract negative effects of LIFR signaling to enhance HDACIs therapeutic efficacy in the treatment of TNBC. Methods: We tested multiple HDACIs currently in clinical trials including vorinostat, panobinostat, romidepsin, and givinostat using multiple TNBC models. The effect of combination therapy of HDACIs and EC359 on TNBC cell viability and invasion was examined using MTT assays and matrigel invasion assays respectively. The efficacy of combination therapy on cell survival and apoptosis was determined using clonogenic assays and Caspase 3/7 assays, respectively. Mechanistic studies were performed using Western blotting, qRT-PCR, and reporter gene assays. The efficacy of combination therapy in vivo was examined using Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDEX) models. Results: We demonstrated that the treatment of TNBC models with HDACIs increased the expression of LIFR. Immunohistochemistry analyses of breast tumors using tissue microarrays revealed significant expression of LIFR in TNBC samples. Knockdown of LIFR or treatment with a small molecule inhibitor of LIFR (EC359) significantly enhanced the efficacy of HDACIs in reducing cell viability, colony formation ability, and invasiveness as well as promoted apoptosis compared to monotherapy of HDACIs or EC359 in TNBC cell lines. Mechanistic studies, reporter gene assays and biochemical studies using multiple TNBC models exhibited activation of the LIFR signaling pathway upon HDACIs treatment but was attenuated by EC359+HDACI combination therapy. Treatment of human breast tumors utilizing PDEX assays showed that EC359 enhanced the ability of HDACIs to decrease the proliferation (Ki-67 positivity) compared to monotherapy. Furthermore, using TNBC xenografts and PDX models, we demonstrated that EC359 treatment enhanced the ability of HDACIs to reduce in vivo tumor growth compared to monotherapy. Conclusions: Our results suggest that the combination therapy of HDACIs and EC359 provides greater therapeutic efficacy than monotherapy. In addition, treatment with EC359 can overcome the feedback activation of LIFR currently observed in the treatment of TNBC with HDACIs. Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Bindu Santhamma, Uday P Pratap, Yiliao Luo, Junhao Liu, Kristin A Altwegg, Xiaonan Li, Hui Yan, Zhenming Xu, Andrew Brenner, Gangadhara R Sareddy, Rajeshwar R Tekmal, Hareesh B Nair, Klaus J Nickisch, Ratna K Vadlamudi. Targeting LIFR enhances the activity of HDAC inhibitors for the treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-08.