Exploration of Novel Inhibitors for Class I Histone Deacetylase Isoforms by QSAR Modeling and Molecular Dynamics Simulation Assays

Histone deacetylases (HDAC) are metal-dependent enzymes and considered as important targets for cell functioning. Particularly, higher expression of class I HDACs is common in the onset of multiple malignancies which results in deregulation of many target genes involved in cell growth, differentiation and survival. Although substantial attempts have been made to control the irregular functioning of HDACs by employing various inhibitors with high sensitivity towards transformed cells, limited success has been achieved in epigenetic cancer therapy. Here in this study, we used ligand-based pharmacophore and 2-dimensional quantitative structure activity relationship (QSAR) modeling approaches for targeting class I HDAC isoforms. Pharmacophore models were generated by taking into account the known IC₅₀ values and experimental energy scores with extensive validations. The QSAR model having an external R² value of 0.93 was employed for virtual screening of compound libraries. 10 potential lead compounds (C1-C10) were short-listed having strong binding affinities for HDACs, out of which 2 compounds (C8 and C9) were able to interact with all members of class I HDACs. The potential binding modes of HDAC2 and HDAC8 to C8 were explored through molecular dynamics simulations. Overall, bioactivity and ligand efficiency (binding energy/non-hydrogen atoms) profiles suggested that proposed hits may be more effective inhibitors for cancer therapy.     

Exploration of Virtual Candidates for Human HMG-CoA Reductase Inhibitors Using Pharmacophore Modeling and Molecular Dynamics Simulations

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a rate-controlling enzyme in the mevalonate pathway which involved in biosynthesis of cholesterol and other isoprenoids. This enzyme catalyzes the conversion of HMG-CoA to mevalonate and is regarded as a drug target to treat hypercholesterolemia. In this study, ten qualitative pharmacophore models were generated based on chemical features in active inhibitors of HMGR. The generated models were validated using a test set. In a validation process, the best hypothesis was selected based on the statistical parameters and used for virtual screening of chemical databases to find novel lead candidates. The screened compounds were sorted by applying drug-like properties. The compounds that satisfied all drug-like properties were used for molecular docking study to identify their binding conformations at active site of HMGR. The final hit compounds were selected based on docking score and binding orientation. The HMGR structures in complex with the hit compounds were subjected to 10 ns molecular dynamics simulations to refine the binding orientation as well as to check the stability of the hits. After simulation, binding modes including hydrogen bonding patterns and molecular interactions with the active site residues were analyzed. In conclusion, four hit compounds with new structural scaffold were suggested as novel and potent HMGR inhibitors.     

Novel Histone Deacetylase Class IIa Selective Substrate Radiotracers for PET Imaging of Epigenetic Regulation in the Brain

Histone deacetylases (HDAC’s) became increasingly important targets for therapy of various diseases, resulting in a pressing need to develop HDAC class- and isoform-selective inhibitors. Class IIa deacetylases possess only minimal deacetylase activity against acetylated histones, but have several other client proteins as substrates through which they participate in epigenetic regulation. Herein, we report the radiosyntheses of the second generation of HDAC class IIa–specific radiotracers: 6-(di-fluoroacetamido)-1-hexanoicanilide (DFAHA) and 6-(tri-fluoroacetamido)-1-hexanoicanilide ([¹⁸F]-TFAHA). The selectivity of these radiotracer substrates to HDAC class IIa enzymes was assessed in vitro, in a panel of recombinant HDACs, and in vivo using PET/CT imaging in rats. [¹⁸F]TFAHA showed significantly higher selectivity for HDAC class IIa enzymes, as compared to [¹⁸F]DFAHA and previously reported [¹⁸F]FAHA. PET imaging with [¹⁸F]TFAHA can be used to visualize and quantify spatial distribution and magnitude of HDAC class IIa expression-activity in different organs and tissues in vivo. Furthermore, PET imaging with [¹⁸F]TFAHA may advance the understanding of HDACs class IIa mediated epigenetic regulation of normal and pathophysiological processes, and facilitate the development of novel HDAC class IIa-specific inhibitors for therapy of different diseases.     

Synthesis and Molecular Modeling of Novel HSV1 Uracil-DNA Glycosylase Inhibitors

Abstract

In a recent paper the first selective inhibitors of HSV1 uracil-DNA glycosylase (UDG) acting in the micromolar range have been reported ¹. A 28.5 kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved². Starting with the optimized model of binding between 6-(4′-n-octylanilino)uracil (octAU) and UDG some new derivatives have been predicted to be active. In vitro studies with the novel synthetized compounds confirm the plausibility of the model and define the structure features for UDG inhibitors.     

Identification of Novel Aldose Reductase Inhibitors from Spices: A Molecular Docking and Simulation Study

Hyperglycemia in diabetic patients results in a diverse range of complications such as diabetic retinopathy, neuropathy, nephropathy and cardiovascular diseases. The role of aldose reductase (AR), the key enzyme in the polyol pathway, in these complications is well established. Due to notable side-effects of several drugs, phytochemicals as an alternative has gained considerable importance for the treatment of several ailments. In order to evaluate the inhibitory effects of dietary spices on AR, a collection of phytochemicals were identified from Zingiber officinale (ginger), Curcuma longa (turmeric) Allium sativum (garlic) and Trigonella foenum graecum (fenugreek). Molecular docking was performed for lead identification and molecular dynamics simulations were performed to study the dynamic behaviour of these protein-ligand interactions. Gingerenones A, B and C, lariciresinol, quercetin and calebin A from these spices exhibited high docking score, binding affinity and sustained protein-ligand interactions. Rescoring of protein ligand interactions at the end of MD simulations produced binding scores that were better than the initially docked conformations. Docking results, ligand interactions and ADMET properties of these molecules were significantly better than commercially available AR inhibitors like epalrestat, sorbinil and ranirestat. Thus, these natural molecules could be potent AR inhibitors.     

Identification of Novel Human Dipeptidyl Peptidase-IV Inhibitors of Natural Origin (Part I): Virtual Screening and Activity Assays

Background

There has been great interest in determining whether natural products show biological activity toward protein targets of pharmacological relevance. One target of particular interest is DPP-IV whose most important substrates are incretins that, among other beneficial effects, stimulates insulin biosynthesis and secretion. Incretins have very short half-lives because of their rapid degradation by DPP-IV and, therefore, inhibiting this enzyme improves glucose homeostasis. As a result, DPP-IV inhibitors are of considerable interest to the pharmaceutical industry. The main goals of this study were (a) to develop a virtual screening process to identify potential DPP-IV inhibitors of natural origin; (b) to evaluate the reliability of our virtual-screening protocol by experimentally testing the in vitro activity of selected natural-product hits; and (c) to use the most active hit for predicting derivatives with higher binding affinities for the DPP-IV binding site.

Methodology/Principal Findings

We predicted that 446 out of the 89,165 molecules present in the natural products subset of the ZINC database would inhibit DPP-IV with good ADMET properties. Notably, when these 446 molecules were merged with 2,342 known DPP-IV inhibitors and the resulting set was classified into 50 clusters according to chemical similarity, there were 12 clusters that contained only natural products for which no DPP-IV inhibitory activity has been previously reported. Nine molecules from 7 of these 12 clusters were then selected for in vitro activity testing and 7 out of the 9 molecules were shown to inhibit DPP-IV (where the remaining two molecules could not be solubilized, preventing the evaluation of their DPP-IV inhibitory activity). Then, the hit with the highest activity was used as a lead compound in the prediction of more potent derivatives.

Conclusions/Significance

We have demonstrated that our virtual-screening protocol was successful in identifying novel lead compounds for developing more potent DPP-IV inhibitors.     

Clinical Potential of Histone Deacetylase Inhibitors as Stand Alone Therapeutics and in Combination with other Chemotherapeutics or Radiotherapy for Cancer

Histone deacetylase inhibitors are emerging as a new class of cancer chemotherapeutics and already are being heralded as the first anti-cancer drugs targeting the epigenome. Through histone hyperacetylation-mediated changes in chromatin conformation and gene expression, histone deacetylase inhibitors induce differentiation, cell cycle arrest, apoptosis, growth inhibition and cell death, which are more pronounced in transformed cell-lines than in normal cells. Additional anti-cancer effects of HDAC inhibitors include inhibition of migration, invasion and angiogenesis in vivo. Indeed, clinical anti-cancer activity has been observed using HDAC inhibitors as single agents or in combination with conventional chemotherapeutics, in phase I and II trials. Furthermore, numerous pre-clinical studies are suggesting a potential clinical role for HDAC inhibitors in radiotherapy either as radiation sensitizers or protectors. In this article the molecular basis for the clinical potential of HDAC inhibitors, either as stand alone cancer therapeutics or in combination with other chemotherapy agents or ionizing radiation will be overviewed.      

Novel Cancer Chemotherapy Hits by Molecular Topology: Dual Akt and Beta-Catenin Inhibitors

Background and Purpose

Colorectal and prostate cancers are two of the most common types and cause of a high rate of deaths worldwide. Therefore, any strategy to stop or at least slacken the development and progression of malignant cells is an important therapeutic choice. The aim of the present work is the identification of novel cancer chemotherapy agents. Nowadays, many different drug discovery approaches are available, but this paper focuses on Molecular Topology, which has already demonstrated its extraordinary efficacy in this field, particularly in the identification of new hit and lead compounds against cancer. This methodology uses the graph theoretical formalism to numerically characterize molecular structures through the so called topological indices. Once obtained a specific framework, it allows the construction of complex mathematical models that can be used to predict physical, chemical or biological properties of compounds. In addition, Molecular Topology is highly efficient in selecting and designing new hit and lead drugs. According to the aforementioned, Molecular Topology has been applied here for the construction of specific Akt/mTOR and β-catenin inhibition mathematical models in order to identify and select novel antitumor agents.

Experimental Approach

Based on the results obtained by the selected mathematical models, six novel potential inhibitors of the Akt/mTOR and β-catenin pathways were identified. These compounds were then tested in vitro to confirm their biological activity.

Conclusion and Implications

Five of the selected compounds, CAS n° 256378-54-8 (Inhibitor n°1), 663203-38-1 (Inhibitor n°2), 247079-73-8 (Inhibitor n°3), 689769-86-6 (Inhibitor n°4) and 431925-096 (Inhibitor n°6) gave positive responses and resulted to be active for Akt/mTOR and/or β-catenin inhibition. This study confirms once again the Molecular Topology’s reliability and efficacy to find out novel drugs in the field of cancer.     

Computer Simulation by Molecular Dynamics as a Tool for Modelling of Molecular Systems

Abstract

A survey is given of methods for simulation of molecular systems on a computer. The various assumptions, approximations and limitations are discussed and the possibility of making comparisons with experimental quantities is assessed. Finally, a number of practical applications of molecular dynamics simulation techniques in chemistry are reviewed.     

Novel Interaction of Class IIb Histone Deacetylase 6 (HDAC6) with Class IIa HDAC9 Controls Gonadotropin Releasing Hormone (GnRH) Neuronal Cell Survival and Movement

The impact of histone deacetylases (HDACs) in the control of gonadotropin releasing hormone (GnRH) neuronal development is unknown. We identified an increase in many HDACs in GT1-7 (differentiated) compared with NLT (undifferentiated) GnRH neuronal cell lines. Increased HDAC9 mRNA and protein and specific deacetylase activity in GT1-7 cells suggested a functional role. Introduction of HDAC9 in NLT cells protected from serum withdrawal induced apoptosis and impaired basal neuronal cell movement. Conversely, silencing of endogenous HDAC9 in GT1-7 cells increased apoptosis and cell movement. Comparison of WT and mutant HDAC9 constructs demonstrated that the HDAC9 pro-survival effects required combined cytoplasmic and nuclear localization, whereas the effects on cell movement required a cytoplasmic site of action. Co-immunoprecipitation demonstrated a novel interaction of HDAC9 selectively with the Class IIb HDAC6. HDAC6 was also up-regulated at the mRNA and protein levels, and HDAC6 catalytic activity was significantly increased in GT1-7 compared with NLT cells. HDAC9 interacted with HDAC6 through its second catalytic domain. Silencing of HDAC6, HDAC9, or both, in GT1-7 cells augmented apoptosis compared with controls. HDAC6 and -9 had additive effects to promote cell survival via modulating the BAX/BCL2 pathway. Silencing of HDAC6 resulted in an activation of movement of GT1-7 cells with induction in acetylation of α-tubulin. Inhibition of HDAC6 and HDAC9 together resulted in an additive effect to increase cell movement but did not alter the acetylation of αtubulin. Together, these studies identify a novel interaction of Class IIa HDAC9 with Class IIb HDAC6 to modulate cell movement and survival in GnRH neurons.     

Novel inhibitors of enkephalin-degrading enzymes. I: Inhibitors of enkephalinase by penicillins

Several penicillins have been found to have pro-antinociceptive properties and also to be enkephalinase (neutral endopeptidase-24.11) inhibitors, carfecillin being the most potent. Carfecillin i.c.v. (but not i.p.) had significant antinociceptive activity in the mouse tail immersion test and completely suppressed abdominal constrictions (acetic acid) in mice (IC50 = 23 micrograms/animal). In combination with (D-Ala2-D-leu5)-enkephalin (DADL) i.c.v. in the abdominal constriction test the complete protection observed was reversed by the opioid receptor antagonist naltrexone. Carfecillin was a competitive inhibitor of enkephalinase from mouse brain striata (IC50 = 207 + 57 nM, cf thiorphan 10.6 +/- 1.9 nM) but did not inhibit other known enkephalin- degrading enzymes. Carfecillin provides a new lead structure for the development of more potent enkephalinase inhibitors.     

RedMDStream: Parameterization and Simulation Toolbox for Coarse-Grained Molecular Dynamics Models

Coarse-grained (CG) models in molecular dynamics (MD) are powerful tools to simulate the dynamics of large biomolecular systems on micro- to millisecond timescales. However, the CG model, potential energy terms, and parameters are typically not transferable between different molecules and problems. So parameterizing CG force fields, which is both tedious and time-consuming, is often necessary. We present RedMDStream, a software for developing, testing, and simulating biomolecules with CG MD models. Development includes an automatic procedure for the optimization of potential energy parameters based on metaheuristic methods. As an example we describe the parameterization of a simple CG MD model of an RNA hairpin.