Small Molecule HDAC Inhibitors Research Articles

Discovery of Novel HDAC3 Inhibitors with PD-L1 Downregulating/Degrading and Antitumor Immune Effects.

Targeting the programmed cell death-1/ligand 1 (PD-1/PD-L1) pathway is one of the most promising cancer treatment strategies. Studies have shown that HDAC inhibitors can enhance the antitumor immune response by modulating the expression of PD-L1. Herein, we designed and synthesized a series of novel hydrazide-based small molecule HDAC inhibitors; among them, compound HQ-30 showed selective HDAC3 inhibition (IC50 = 89 nM) and remarkable PD-L1-degrading activity (DC50 = 5.7 μM, Dmax = 80% at 10 μM). Further studies revealed that HQ-30 induced the degradation of PD-L1 by regulating cathepsin B (CTSB) in the lysosomes. Further, HQ-30 could enhance the infiltration of CD3+ CD4+ helper T and CD3+ CD8+ cytotoxic T cells in tumors, thus activating the tumor immune microenvironment. Moreover, HQ-30 possessed a benign toxicity profile (LD50 > 1000 mg/kg) and favorable pharmacokinetic properties (F = 57%). Taken together, HQ-30 is worthy of further investigation as a small molecule-based epigenetic modulator of tumor immunotherapy.

A flow cytometry-based screen identifies MBNL1 modulators that rescue splicing defects in myotonic dystrophy type I.

Myotonic dystrophy Type 1 (DM1) is a rare genetic disease caused by the expansion of CTG trinucleotide repeats ((CTG)exp) in the 3' untranslated region of the DMPK gene. The repeat transcripts sequester the RNA binding protein Muscleblind-like protein 1 (MBNL1) and hamper its normal function in pre-mRNA splicing. Overexpressing exogenous MBNL1 in the DM1 mouse model has been shown to rescue the splicing defects and reverse myotonia. Although a viable therapeutic strategy, pharmacological modulators of MBNL1 expression have not been identified. Here, we engineered a ZsGreen tag into the endogenous MBNL1 locus in HeLa cells and established a flow cytometry-based screening system to identify compounds that increase MBNL1 level. The initial screen of small molecule compound libraries identified more than thirty hits that increased MBNL1 expression greater than double the baseline levels. Further characterization of two hits revealed that the small molecule HDAC inhibitors, ISOX and vorinostat, increased MBNL1 expression in DM1 patient-derived fibroblasts and partially rescued the splicing defect caused by (CUG)exp repeats in these cells. These findings demonstrate the feasibility of this flow-based cytometry screen to identify both small molecule compounds and druggable targets for MBNL1 upregulation.

HDAC1 and HDAC2 integrate the expression of p53 mutants in pancreatic cancer.

Mutation of p53 is a frequent genetic lesion in pancreatic cancer being an unmet clinical challenge. Mutants of p53 have lost the tumour-suppressive functions of wild type p53. In addition, p53 mutants exert tumour-promoting functions, qualifying them as important therapeutic targets. Here, we show that the class I histone deacetylases HDAC1 and HDAC2 contribute to maintain the expression of p53 mutants in human and genetically defined murine pancreatic cancer cells. Our data reveal that the inhibition of these HDACs with small molecule HDAC inhibitors (HDACi), as well as the specific genetic elimination of HDAC1 and HDAC2, reduce the expression of mutant p53 mRNA and protein levels. We further show that HDAC1, HDAC2 and MYC directly bind to the TP53 gene and that MYC recruitment drops upon HDAC inhibitor treatment. Therefore, our results illustrate a previously unrecognized class I HDAC-dependent control of the TP53 gene and provide evidence for a contribution of MYC. A combined approach targeting HDAC1/HDAC2 and MYC may present a novel and molecularly defined strategy to target mutant p53 in pancreatic cancer.

Tcf4 Regulates Synaptic Plasticity, DNA Methylation, and Memory Function

Human haploinsufficiency of the transcription factor Tcf4 leads to a rare autism spectrum disorder called Pitt-Hopkins syndrome (PTHS), which is associated with severe language impairment and development delay. Here, we demonstrate that Tcf4 haploinsufficient mice have deficits in social interaction, ultrasonic vocalization, prepulse inhibition, and spatial and associative learning and memory. Despite learning deficits, Tcf4(+/-) mice have enhanced long-term potentiation in the CA1 area of the hippocampus. In translationally oriented studies, we found that small-molecule HDAC inhibitors normalized hippocampal LTP and memory recall. A comprehensive set of next-generation sequencing experiments of hippocampal mRNA and methylated DNA isolated from Tcf4-deficient and WT mice before or shortly after experiential learning, with or without administration of vorinostat, identified "memory-associated" genes modulated by HDAC inhibition and dysregulated by Tcf4 haploinsufficiency. Finally, weobserved that Hdac2 isoform-selective knockdown was sufficient to rescue memory deficits in Tcf4(+/-) mice.

Thailandepsins are new small molecule class I HDAC inhibitors with potent cytotoxic activity in ovarian cancer cells: a preclinical study of epigenetic ovarian cancer therapy

BackgroundNew treatment strategies are emerging to target DNA damage response pathways in ovarian cancer. Our group has previously shown that the class I biased HDAC inhibitor romidepsin (FK228) induces DNA damage response and has potent cytotoxic effects in ovarian cancer cells. Here, we investigated newly discovered HDAC inhibitors, thailandepsin A (TDP-A) and thailandepsin B (TDP-B), to determine the effects on cell viability, apoptosis and DNA damage response in ovarian cancer cells.MethodsFK228, TDP-A and TDP-B were tested in five ovarian cancer cell lines. Cellular viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Immunofluorescence assays were used to assess activated caspase 3. Western blots were performed to detect protein expression of PARP cleavage, pH2AX, P-glycoprotein and tubulin acetylation.ResultsTreatment with TDPs decreased cell viability at nanonomolar concentrations in four of the five ovarian cancer cell lines studied. Similar to FK228, both TDP compounds exerted minimal effects on NCI/ADR-RES ovarian cancer cells. Across the four cell lines sensitive to the TDPs, TDP-B consistently had a greater inhibitory effect than TDP-A on cell viability. TDP-B also had relatively greater effects on promoting cell apoptosis and induction of pH2AX (a mark of DNA damage response), than TDP-A. These antitumor effects of TDP-B were of similar magnitude to those induced by an equal concentration of FK228. Similar to FK228, the nanomolar concentrations of the TDPs had little effect on tubulin acetylation (a mark of class II HDAC6 inhibition).ConclusionsThe new small molecule HDAC inhibitors TDP-A and TDP-B are FK228 analogues that suppress cell viability and induce apoptosis at nanomolar drug concentrations. TDP-B showed the most similarity to the biological activity of FK228 with greater cytotoxic effects than TDP-A in vitro. Our results indicate that FK228-like small molecule class I HDAC-biased HDAC inhibitors have therapeutic potential for ovarian cancer.

The DNA damage mark pH2AX differentiates the cytotoxic effects of small molecule HDAC inhibitors in ovarian cancer cells

High grade epithelial ovarian cancers are relatively sensitive to DNA damaging platinum-based chemotherapy, suggesting that the dependencies of ovarian tumors on DNA damage response pathways can be harnessed for therapeutic purposes. Our goal was to determine if the DNA damage mark gamma-H2AX phosphorylation (pH2AX) could be used to identify suitable cytotoxic histone deacetylase inhibitors (HDACi) for ovarian cancer treatment. Nineteen chemically diverse HDACi compounds were tested in 7 ovarian cancer cell lines. Fluorescent, biochemical and cell-based assays were performed to assess DNA damage by induction of pH2AX and to measure cell viability and apoptosis. The relationships between pH2AX and the cellular effects of cell viability and apoptosis were calculated. Selected HDACi were tested in combination with cisplatin and other DNA damaging agents to determine if the HDACi improved upon the effects of the DNA damaging agents. The HDACi compounds induced differing levels of pH2AX expression. High levels of pH2AX in HDACi-treated ovarian cancer cells were tightly associated with decreased cell viability and increased apoptosis. Consequently, a ketone-based HDACi was chosen and found to enhance the effects of cisplatin, even in ovarian cancer cells with extreme resistance to DNA damaging drugs. In conclusion, a fluorescent-based assay for pH2AX can be used to determine cellular responses to HDACi in vitro and may be a useful tool to identify potentially more effective HDACi for the treatment of ovarian cancer. In addition, these results lend support to the inclusion of ketone-derived HDACi compounds for future development.

Hdac-Mediated Control of Endochondral and Intramembranous Ossification

Histone deacetylases (Hdacs) remove acetyl groups (CH3CO-) from ε-amino groups in lysine residues within histones and other proteins. This posttranslational (de) modification alters protein stability, protein-protein interactions, and chromatin structure. Hdac activity plays important roles in the development of all organs and tissues, including the mineralized skeleton. Bone is a dynamic tissue that forms and regenerates by two processes: endochondral and intramembranous ossification. Chondrocytes and osteoblasts are responsible for producing the extracellular matrices of skeletal tissues. Several Hdacs contribute to the molecular pathways and chromatin changes that regulate tissue-specific gene expression during chondrocyte and osteoblast specification, maturation, and terminal differentiation. In this review, we summarize the roles of class I and class II Hdacs in chondrocytes and osteoblasts. The effects of small molecule Hdac inhibitors on the skeleton are also discussed.

Identification of Core Gene Signature Associated with Synergism Between ABT-869, a FLT3 Inhibitor and SAHA, a HDAC Inhibitor in AML with FLT3-ITD Mutation.

Internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) mutation occurs in about 25% of acute myeloid leukemia (AML) patients and is associated with poor prognosis. Various FLT3 inhibitors of different chemical structure are under clinical investigation for the treatment of AML patients with FLT3 mutations. In contrast to their impressive potency in cell culture systems, FLT3 inhibitors as single agents in clinical trials only induce transient reduction of peripheral blasts, but not bone marrow blasts. Combination with other small molecule drugs represents a promising strategy to improve therapeutic efficacy of FLT3 inhibitors in the clinic. Small molecule HDAC inhibitors (HDACi) have proven to be a promising new class of anticancer drugs against hematological malignancies, as well as solid tumors. The HDACi, suberoylanilide hydroxamic acid (SAHA, Vorinostat) has been examined in a combinatory fashion with other classes of anticancer agents in acute leukemias, including cyclin-dependent kinase (CDK) inhibitors (flavopiridol), HSP 90 antagonists (17-allylamino- 17-demethoxygeldanamycin, 17-AAG), and estradiol analogs (2-methoxyestradiol, 2-ME). ABT-869, a multi-targeted receptor tyrosine kinase inhibitor, inhibits FLT3 phosphorylation and signaling and now is in active clinical development. In the current study, cell proliferation and apoptosis assays revealed that combining ABT-869 with SAHA led to synergistic killing of AML cells with FLT3 mutations in conventional cell culture and human stromal cell coculture models. To elucidate the molecular mechanism of the synergistic lethality between ABT-869 and SAHA, we compared the gene expression profiles of cells from FLT3 mutated leukemia cell lines, MV4-11 and MOLM-14, treated with DMSO control, ABT-869, SAHA and combination therapy using the Affymetrix microarray platform. Here, we focused on delineating a core gene signature unique to combination therapy and common to both MV4-11 and MOLM-14 cell lines, which could be crucial for the positive cooperation we observed. The core gene signature differentially induced more than two-fold by combination therapy in both cell lines included upregulation of FRY, LMO4, IFI16, ACADSB, and S100A8, and downregulation of PTP4A3 (PRL-3), ORC1L, MND1, ZNF85, DENND3, FAM119A, ACAT2, HMGCS1, DCS2, GSPG4, CBS, TUBE1. Overexpression of PRL-3, a metastasis-associated gene, has been found in different types of solid tumors and multiple myeloma. Modulation of PRL-3 expression level using genetic approaches demonstrated that PRL-3 played an essential role in the synergism in the therapeutic effect of ABT-869 and SAHA.

KD5170, a novel mercaptoketone-based histone deacetylase inhibitor that exhibits broad spectrum antitumor activity in vitro and in vivo

Histone deacetylase (HDAC) inhibitors have garnered significant attention as cancer drugs. These therapeutic agents have recently been clinically validated with the market approval of vorinostat (SAHA, Zolinza) for treatment of cutaneous T-cell lymphoma. Like vorinostat, most of the small-molecule HDAC inhibitors in clinical development are hydroxamic acids, whose inhibitory activity stems from their ability to coordinate the catalytic Zn2+ in the active site of HDACs. We sought to identify novel, nonhydroxamate-based HDAC inhibitors with potentially distinct pharmaceutical properties via an ultra-high throughput small molecule biochemical screen against the HDAC activity in a HeLa cell nuclear extract. An alpha-mercaptoketone series was identified and chemically optimized. The lead compound, KD5170, exhibits HDAC inhibitory activity with an IC50 of 0.045 micromol/L in the screening biochemical assay and an EC50 of 0.025 micromol/L in HeLa cell-based assays that monitor histone H3 acetylation. KD5170 also exhibits broad spectrum classes I and II HDAC inhibition in assays using purified recombinant human isoforms. KD5170 shows significant antiproliferative activity against a variety of human tumor cell lines, including the NCI-60 panel. Significant tumor growth inhibition was observed after p.o. dosing in human HCT-116 (colorectal cancer), NCI-H460 (non-small cell lung carcinoma), and PC-3 (prostate cancer) s.c. xenografts in nude mice. In addition, a significant increase in antitumor activity and time to end-point occurred when KD5170 was combined with docetaxel in xenografts of the PC-3 prostate cancer cell line. The biological and pharmaceutical profile of KD5170 supports its continued preclinical and clinical development as a broad spectrum anticancer agent.

Histone deacetylase inhibitors: the Abbott experience.

Histone deacetylase inhibitors have generated significant interest as anti-cancer agents due to their ability to cause growth arrest, terminal differentiation and/ or apoptosis in carcinoma cells. Abbott entered this area after the serendipitous discovery of the biaryl hydroxamate A-161906 in a TGF beta mimetic screen and the subsequent identification of this compound as an inhibitor of selected HDACs. The complex biology of these enzymes became evident when cloning and expression of the HDACs demonstrated that they were present as multiprotein and, in some cases, multi-HDAC containing complexes in their active forms. This discovery suggested that any selectivity determinations would have to be considered in the context of these multi-protein/HDAC complexes. However, siRNA gene knockdown studies did demonstrate that reduction of the Class I HDACs resulted in a phenotype similar to that observed with small molecule HDAC inhibitors. Evaluation of the Abbott small molecule HDAC inhibitors utilized a Class I HDAC (HDAC 1/2) preparation and antiproliferation assays using HT1080 fibrosarcoma and MDA435 breast carcinoma cells. Characterization of several series of hydroxamic acids indicated that while many of these analogs possessed potent enzymatic and cellular activity, in general these compounds had unacceptable pharmacokinetic profiles and marginal antitumor effects. Replacement of the potentially labile hydroxamic acid moiety with a trifluoromethyl ketone or a ketooxazole gave measurable HDAC potency but only modest cellular and in vivo activity. However, hydroxamate replacement with an alpha-ketoamide moiety provided potent HDAC inhibitors (IC(50) values as low as 3 nM) with excellent cellular activity (IC50 values < 0.2 microM) and measurable anti-tumor activity in a flank tumor growth model.