IDENTIFICATION OF POTENTIAL INHIBITORS FOR LOWERING CHOLESTEROL LEVEL BY INHIBITING PCSK9

ABSTRACTObjective: PCSK9 has medical significance in lowering cholesterol levels. Inhibitors target and inactivate PCSK9 in the liver. Knocking out PCSK9 (proprotein convertase subtilisin kexin 9) reduces the amount of harmful LDL cholesterol circulating in the bloodstream. There are two known inhibitors for treating the cardiovascular disease Arilocumab†and Evalocumabâ€. However there are many side-effects. The current study is to identify natural and synthetic inhibitor using the pharmacophoric feature of the known inhibitor and validating the short listed candidates using Molecular dynamics and ADMET properties.Methods: Known inhibitors for the PCSK9 Protein were taken from the BINDING DATABASE. Molecular docking was performed for the known inhibitors with the PCSK9 protein. After docking the best inhibitor was selected and the docking result was then imported to find the pharmacophoric features.Results: The pharmacophore model was generated with 3 features containing  1 hydrogen bond acceptor(A),1 Hydrogen bond donor(B) and 1 Aromatic ring. The constructed e-pharmacophore model was screened with more than 20000 natural compounds. 5 compounds were short listed. Among them ZINC85625485 has  glide  score  of  -13.03  kcal/mol  with  glide  energy  was  -57.62 kcal/mol and ZINC85625406 has glide score of -8.1kcal/mol with glide energy was -39.33kcal/mol were taken as the best Hits.Conclusion: PCSK9 is known to be a therapeutic agent as it controls the plasma LDL cholesterol levels by posttranslational regulation of the LDL receptor. Therefore, up-regulation of PCSk9 can lead to elevated cholesterol level in such case inhibition of PCSK9 will be a effective remedy. In this study already known inhibitors were taken and pharmacophore feature was generated. Zinc database was screened to find out novel compounds with similar pharmacophore features that can act as potentially active compound against PCSK9. ZINC85625485 and ZINC85625406 were short listed as lead compounds with Molecular dynamics simulation and checking the ADMET properties. Keywords: PCSK9, Docking, ADMET, Molecular Dynamics.                                                            Â

Identification of new benzofuran derivatives as STING agonists with broad-spectrum antiviral activity

The Stimulator of Interferon Genes (STING) is involved in cytosolic DNA sensing and type I Interferons (IFN-I) induction. Aiming to identify new STING agonists with antiviral activity and given the known biological activity of benzothiazole and benzimidazole derivatives, a series of benzofuran derivatives were tested for their ability to act as STING agonists, induce IFN-I and inhibit viral replication. Compounds were firstly evaluated in a gene reporter assay measuring luciferase activity driven by the human IFN-β promoter in cells expressing exogenous STING (HEK293T). Seven of them were able to induce IFN-β transcription while no induction of the IFN promoter was observed in the presence of a mutated and inactive STING, showing specific protein-ligand interaction. Docking studies were performed to predict their putative binding mode. The best hit compounds were then tested on human coronavirus 229E replication in BEAS-2B and MRC-5 cells and three derivatives showed EC50 values in the μM range. Such compounds were also tested on SARS-CoV-2 replication in BEAS-2B cells and in Calu-3 showing they can inhibit SARS-CoV-2 replication at nanomolar concentrations. To further confirm their IFN- dependent antiviral activity, compounds were tested to verify their effect on phospho-IRF3 nuclear localization, that was found to be induced by benzofuran derivatives, and SARS-CoV-2 replication in Vero E6 cells, lacking IFN production, founding them to be inactive. In conclusion, we identified benzofurans as STING- dependent immunostimulatory compounds and host-targeting inhibitors of coronaviruses representing a novel chemical scaffold for the development of broad-spectrum antivirals

Beyond structural genomics: computational approaches for the identification of ligand binding sites in protein structures

t Structural genomics projects have revealed structures for a large number of proteins of unknown function. Understanding the interactions between these proteins and their ligands would provide an initial step in their functional characterization. Binding site identification methods are a fast and cost-effective way to facilitate the characterization of functionally important protein regions. In this review we describe our recently developed methods for binding site identification in the context of existing methods. The advantage of energy-based approaches is emphasized, since they provide flexibility in the identifi- cation and characterization of different types of binding site

SWI/SNF subunit BAF155 N-terminus structure informs the impact of cancer-associated mutations and reveals a potential drug binding site.

SWI/SNF (BAF) chromatin remodelling complexes are key regulators of gene expression programs, and attractive drug targets for cancer therapies. Here we show that the N-terminus of the BAF155/SMARCC1 subunit contains a putative DNA-binding MarR-like domain, a chromodomain and a BRCT domain that are interconnected to each other to form a distinct module. In this structure the chromodomain makes interdomain interactions and has lost its canonical function to bind to methylated lysines. The structure provides new insights into the missense mutations that target this module in cancer. This study also reveals two adjacent, highly-conserved pockets in a cleft between the domains that form a potential binding site, which can be targeted with small molecules, offering a new strategy to target SWI/SNF complexes

Characterisation of the binding of dihydro-alpha-lipoic acid to fibrinogen and the effects on fibrinogen oxidation and fibrin formation

A reduced form of the alpha-lipoic acid, dihydro-alpha-lipoic acid (DHLA) is a potent, naturally occurring antioxidant which can be consumed as food constituent or as supplement at doses up to 600 mg/day. DHLA has inhibitory effect on coagulation as it can reduce concentrations of some coagulation factors. In this study, a direct interaction between DHLA and fibrinogen, the main protein in coagulation, is described. Binding constant for DHLA/fibrinogen complex is of moderate strength (104) and interaction probably occurs in D regions of fibrinogen, as shown by docking simulations. Fibrinogen stability remains the same with only marginal structural changes in its secondary structure favouring more ordered molecular organisation upon DHLA binding. Fibrinogen with bound DHLA forms fibrin with thicker fibers, as measured by coagulation assay and is protected from oxidation to certain extent. Obtained results support beneficial effects of DHLA on fibrinogen and consequently on coagulation process, suggesting that DHLA supplementation may be indicated for persons with an increased risk of developing thrombotic complications, particularly those whose fibrin is characterised by increased oxidative modification and formation of thinner and less porous fibers. Also, DHLA in complex with fibrinogen can be located at site of injury where it may exert antioxidant effects.This is the peer-reviewed version of the article: International Journal of Biological Macromolecules, 2020, 147, 319-325, doi: [https://dx.doi.org/10.1016/j.ijbiomac.2020.01.098]The published version: [http://cer.ihtm.bg.ac.rs/handle/123456789/3378

Insights into the molecular mechanism of allostery in Hsp70s

Hsp70s chaperone an amazing number and variety of cellular protein folding processes. Key to their versatility is the recognition of a short degenerate sequence motif, present in practically all polypeptides, and a bidirectional allosteric intramolecular regulation mechanism linking their N-terminal nucleotide binding domain (NBD) and their C-terminal polypeptide substrate binding domain (SBD). Through this interdomain communication ATP binding to the NBD and ATP hydrolysis control the affinity of the SBD for polypeptide substrates and substrate binding to the SBD triggers ATP hydrolysis. Genetic screens for defective variants of Hsp70s and systematic analysis of available structures of the isolated domains revealed some residues involved in allosteric control. Recent elucidation of the crystal structure of the Hsp70 homolog DnaK in the ATP bound open conformation as well as numerous NMR and mutagenesis studies bring us closer to an understanding of the communication between NBD and SBD. In this review we will discuss our current view of the allosteric control mechanism of Hsp70 chaperones

In silico assessment of potential druggable pockets on the surface of α1-Antitrypsin conformers

The search for druggable pockets on the surface of a protein is often performed on a single conformer, treated as a rigid body. Transient druggable pockets may be missed in this approach. Here, we describe a methodology for systematic in silico analysis of surface clefts across multiple conformers of the metastable protein α1-antitrypsin (A1AT). Pathological mutations disturb the conformational landscape of A1AT, triggering polymerisation that leads to emphysema and hepatic cirrhosis. Computational screens for small molecule inhibitors of polymerisation have generally focused on one major druggable site visible in all crystal structures of native A1AT. In an alternative approach, we scan all surface clefts observed in crystal structures of A1AT and in 100 computationally produced conformers, mimicking the native solution ensemble. We assess the persistence, variability and druggability of these pockets. Finally, we employ molecular docking using publicly available libraries of small molecules to explore scaffold preferences for each site. Our approach identifies a number of novel target sites for drug design. In particular one transient site shows favourable characteristics for druggability due to high enclosure and hydrophobicity. Hits against this and other druggable sites achieve docking scores corresponding to a Kd in the µM–nM range, comparing favourably with a recently identified promising lead. Preliminary ThermoFluor studies support the docking predictions. In conclusion, our strategy shows considerable promise compared with the conventional single pocket/single conformer approach to in silico screening. Our best-scoring ligands warrant further experimental investigation