Heterocyclic Moieties as HDAC Inhibitors: Role in Cancer Therapeutic

‘Epigenetic’ regulation of genes via post-translational modulation of proteins is a well explored approach for the disease therapies, particularly cancer chemotherapeutics. Histone deacetylases (HDACs) are one of the important epigenetic targets and are mainly responsible for balancing the acetylation/deacetylation of lysine amino acids on histone/nonhistone proteins along with histone acetyltransferase (HAT). HDAC inhibitors (HDACIs) have become an important biologically active compounds for the treatment of cancers due to cell cycle arrest, differentiation and apoptosis in tumor cells and thus leads to anticancer activity. Out of the four classes of HDAC i.e. Class I, II, III and IV, HDACIs act on Class-IV (Zinc dependent HDAC) and various FDA-approved drugs belong to this category. The required canonical pharmacophore model (zinc binding group, surface recognition cap and appropriate linker) supported by HDACIs, various heterocyclic moieties containing compounds exhibiting HDAC inhibitory activity and structure activity relationship of different synthetic derivatives reported during last twelve years have been summarized in this review.

Posttranslational modification and beyond: interplay between histone deacetylase 6 and heat-shock protein 90

Posttranslational modification (PTM) and regulation of protein stability are crucial to various biological processes. Histone deacetylase 6 (HDAC6), a unique histone deacetylase with two functional catalytic domains (DD1 and DD2) and a ZnF-UBP domain (ubiquitin binding domain, BUZ), regulates a number of biological processes, including gene expression, cell motility, immune response, and the degradation of misfolded proteins. In addition to the deacetylation of histones, other nonhistone proteins have been identified as substrates for HDAC6. Hsp90, a molecular chaperone that is a critical modulator of cell signaling, is one of the lysine deacetylase substrates of HDAC6. Intriguingly, as one of the best-characterized regulators of Hsp90 acetylation, HDAC6 is the client protein of Hsp90. In addition to regulating Hsp90 at the post-translational modification level, HDAC6 also regulates Hsp90 at the gene transcription level. HDAC6 mainly regulates the Hsp90-HSF1 complex through the ZnF-UBP domain, thereby promoting the HSF1 entry into the nucleus and activating gene transcription. The mutual interaction between HDAC6 and Hsp90 plays an important role in the regulation of protein stability, cell migration, apoptosis and other functions. Plenty of of studies have indicated that blocking HDAC6/Hsp90 has a vital regulatory role in multifarious diseases, mainly in cancers. Therefore, developing inhibitors or drugs against HDAC6/Hsp90 becomes a promising development direction. Herein, we review the current knowledge on molecular regulatory mechanisms based on the interaction of HDAC6 and Hsp90 and inhibition of HDAC6 and/or Hsp90 in oncogenesis and progression, antiviral and immune-related diseases and other vital biological processes.

Dynamics and Functional Interplay of Non-histone Lysine Crotonylome and Ubiquitylome in Vascular Smooth Muscle Cell Phenotypic Remodeling

BackgroundsCrotonylation of histones is a recently discovered type of post-translational modification that can regulate gene expression. However, the function of crotonylation on nonhistone proteins in vascular smooth muscle cells (VSMC) is unclear. Here, we aim to use modification and proteomic analysis to find the cellular characteristic of crotonylated nonhistone proteins and the crosstalk with ubiquitinated proteins in vascular smooth muscle cell (VSMC) phenotypic remodeling. ResultsWe performed modification and proteomic analysis of VSMCs before and after platelet-derived growth factor-BB (PDGF-BB) stimulation. The crotonylated and ubiquitinated pan-antibody was used to enrich the protein and then subjected to high-throughput mass spectrometry analysis. Then we compared the enrichment analysis of differentially modified proteins in regards to GO terms, KEGG pathway and protein domain. As a result, there were 2138 crotonylation sites in 534 proteins and 1359 ubiquitination sites corresponding to 657 proteins. The crotonylated proteins participate in a variety of important cellular pathways and perform different functions in VSMCs. Among them, some proteins were found to be closely involved in the physiological process of VSMC phenotypic remodeling including glycolysis/gluconeogenesis, vascular smooth muscle contraction, and PI3K-Akt signaling pathway. Furthermore, the KEGG pathway enrichment analysis showed that the ubiquitinated proteins were found to be closely involved in the physiological process of VSMC phenotypic remodeling including glycolysis/gluconeogenesis, vascular smooth muscle contraction, RAS signaling pathway or PI3K-Akt signaling pathway. Crosstalk analysis showed that there were 199 sites within 177 proteins modified by crotonylation and ubiquitination simultaneously. PPI network analysis indicated that crotonylated and ubiquitinated proteins played an important role in cellular bioprocess commonly and possible synergistic effect. ConclusionsIn summary, our bioinformatics show that nonhistone crotonylation and ubiquitination play an important role in the VSMC phenotypic transformation induced by PDGF-BB stimulation. The crosstalk of crotonylation and ubiquitination in glycolysis is possibly a novel mechanism underlying the VSMC phenotypic remodeling.

Characterization of Histone Deacetylase Mechanisms in Cancer Development

Over decades of studies, accumulating evidence has suggested that epigenetic dysregulation is a hallmark of tumours. Post-translational modifications of histones are involved in tumour pathogenesis and development mainly by influencing a broad range of physiological processes. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are pivotal epigenetic modulators that regulate dynamic processes in the acetylation of histones at lysine residues, thereby influencing transcription of oncogenes and tumour suppressor genes. Moreover, HDACs mediate the deacetylation process of many nonhistone proteins and thus orchestrate a host of pathological processes, such as tumour pathogenesis. In this review, we elucidate the functions of HDACs in cancer.

Discovery of the SMYD3 Inhibitor BAY-6035 Using Thermal Shift Assay (TSA)-Based High-Throughput Screening

SMYD3 (SET and MYND domain-containing protein 3) is a protein lysine methyltransferase that was initially described as an H3K4 methyltransferase involved in transcriptional regulation. SMYD3 has been reported to methylate and regulate several nonhistone proteins relevant to cancer, including mitogen-activated protein kinase kinase kinase 2 (MAP3K2), vascular endothelial growth factor receptor 1 (VEGFR1), and the human epidermal growth factor receptor 2 (HER2). In addition, overexpression of SMYD3 has been linked to poor prognosis in certain cancers, suggesting SMYD3 as a potential oncogene and attractive cancer drug target. Here we report the discovery of a novel SMYD3 inhibitor. We performed a thermal shift assay (TSA)-based high-throughput screening (HTS) with 410,000 compounds and identified a novel benzodiazepine-based SMYD3 inhibitor series. Crystal structures revealed that this series binds to the substrate binding site and occupies the hydrophobic lysine binding pocket via an unprecedented hydrogen bonding pattern. Biochemical assays showed substrate competitive behavior. Following optimization and extensive biophysical validation with surface plasmon resonance (SPR) analysis and isothermal titration calorimetry (ITC), we identified BAY-6035, which shows nanomolar potency and selectivity against kinases and other PKMTs. Furthermore, BAY-6035 specifically inhibits methylation of MAP3K2 by SMYD3 in a cellular mechanistic assay with an IC50 <100 nM. Moreover, we describe a congeneric negative control to BAY-6035. In summary, BAY-6035 is a novel selective and potent SMYD3 inhibitor probe that will foster the exploration of the biological role of SMYD3 in diseased and nondiseased tissues.

Protein arginine methyltransferases: promising targets for cancer therapy

Protein methylation, a post-translational modification (PTM), is observed in a wide variety of cell types from prokaryotes to eukaryotes. With recent and rapid advancements in epigenetic research, the importance of protein methylation has been highlighted. The methylation of histone proteins that contributes to the epigenetic histone code is not only dynamic but is also finely controlled by histone methyltransferases and demethylases, which are essential for the transcriptional regulation of genes. In addition, many nonhistone proteins are methylated, and these modifications govern a variety of cellular functions, including RNA processing, translation, signal transduction, DNA damage response, and the cell cycle. Recently, the importance of protein arginine methylation, especially in cell cycle regulation and DNA repair processes, has been noted. Since the dysregulation of protein arginine methylation is closely associated with cancer development, protein arginine methyltransferases (PRMTs) have garnered significant interest as novel targets for anticancer drug development. Indeed, several PRMT inhibitors are in phase 1/2 clinical trials. In this review, we discuss the biological functions of PRMTs in cancer and the current development status of PRMT inhibitors in cancer therapy.

Study of the antilymphoma activity of pracinostat reveals different sensitivities of DLBCL cells to HDAC inhibitors

Histone deacetylase inhibitors (HDACis) are antitumor agents with distinct efficacy in hematologic tumors. Pracinostat is a pan-HDACi with promising early clinical activity. However, similar to other HDACis, its activity as a single agent is limited. Diffuse large B-cell lymphoma (DLBCL) includes distinct molecular subsets or metabolically defined subtypes that rely in different ways on the B-cell receptor signaling pathway, oxidative phosphorylation, and glycolysis for their survival. The antitumor activity of pracinostat has not been determined in lymphomas. We performed preclinical in vitro activity screening of 60 lymphoma cell lines that included 25 DLBCLs. DLBCL cells belonging to distinct metabolic subtypes were treated with HDACis for 6 hours or 14 days followed by transcriptional profiling. DLBCL xenograft models enabled assessment of the in vivo antilymphoma activity of pracinostat. Combination treatments with pracinostat plus 10 other antilymphoma agents were performed. Western blot was used to assess acetylation levels of histone and nonhistone proteins after HDACi treatment. Robust antiproliferative activity was observed across all lymphoma histotypes represented. Focusing on DLBCL, we identified a low-sensitivity subset that almost exclusively consists of the oxidative phosphorylation (OxPhos)-DLBCL metabolic subtype. OxPhos-DLBCL cells also showed poorer sensitivity to other HDACis, including vorinostat. Transcriptomic analysis revealed fewer modulated transcripts but an enrichment of antioxidant pathway genes after HDACi treatment of OxPhos-DLBCLs compared with high-sensitivity B-cell receptor (BCR)–DLBCLs. Pharmacologic inhibition of antioxidant production rescued sensitivity of OxPhos-DLBCLs to pracinostat whereas BCR-DLBCLs were unaffected. Our study provides novel insights into the antilymphoma activity of pracinostat and identifies a differential response of DLBCL metabolic subtypes to HDACis.

Global Profiling of Lysine Crotonylation in Vascular Smooth Muscle Cell Phenotypic Remodeling

Objective: Crotonylation of histones is a recently discovered type of post-translational modification that can regulate gene expression. However, the function of crotonylation on nonhistone proteins in vascular smooth muscle cells (VSMC) is unclear. In this study, we aim to use modification and proteomic analysis to find the cellular characteristic of crotonylated nonhistone proteins for deep research.Methods: In this article, we performed modification and proteomic analysis of VSMCs before and after platelet-derived growth factor-BB (PDGF-BB) stimulation. The crotonylated pan-antibody was used to enrich the protein and then subjected to high-throughput mass spectrometry analysis. Results: As a result, there were 2138 crotonylation sites in 534 proteins. These proteins are widely distributed in the cytoplasm, nucleus, and mitochondria, and participate in the cell metabolism processes such as amino acid synthesis, glycolysis, and glycogen synthesis. Conclusions: In summary, our bioinformatics shows that nonhistone crotonylation plays an important role in the VSMC phenotypic transformation induced by PDGF-BB stimulation.

SMYD2 promotes tumorigenesis and metastasis of lung adenocarcinoma through RPS7

The protein methyltransferase SET and MYND domain-containing protein 2 (SMYD2) is a transcriptional regulator that methylates histones and nonhistone proteins. As an oncogene, SMYD2 has been investigated in numerous types of cancer. However, its involvement in lung cancer remains elusive. The prognostic value of SMYD2 expression in lung adenocarcinoma (LUAD) was determined through bioinformatics analysis, reverse-transcription polymerase chain reaction, western blotting, and immunohistochemistry. The effect of SMYD2 on LUAD cell proliferation and metastasis was explored in vivo and in vitro, and the underlying mechanisms were investigated via RNA-seq, and chromatin immunoprecipitation-quantitative PCR. SMYD2 expression was significantly upregulated in LUAD cell lines and tissues. High SMYD2 expression was associated with shorter overall and disease-free survival in LUAD patients. Inhibition of SMYD2 with SMYD2 knockdown or AZ505 dramatically inhibited the proliferation, migration, and invasion ability of GLC-82 and SPC-A1 cells and remarkably reduced tumor growth in mice. Mechanically, SMYD2 may activate the transcription of ribosomal small subunit protein 7 (RPS7) by binding to its promoter. Following overexpression of SMYD2, the proliferation, migration, and invasion of cells increased, which was partially reversed by RPS7. Thus, SMYD2 might modulate tumorigenesis and metastasis mediated by RPS7 LUAD. SMYD2 might be a prognostic biomarker and therapeutic target in LUAD.