Prospect of Anterior Gradient 2 homodimer inhibition via repurposing FDA-approved drugs using structure-based virtual screening.

Anterior Gradient 2 (AGR2) has recently been reported as a tumor biomarker in various cancers, i.e., breast, prostate and lung cancer. Predominantly, AGR2 exists as a homodimer via a dimerization domain (E60-K64); after it is self-dimerized, it helps FGF2 and VEGF to homo-dimerize and promotes the angiogenesis and the invasion of vascular endothelial cells and fibroblasts. Up till now, no small molecule has been discovered to inhibit the AGR2-AGR2 homodimer. Therefore, the present study was performed to prepare a validated 3D structure of AGR2 by homology modeling and discover a small molecule by screening the FDA-approved drugs library on AGR2 homodimer as a target protein. Thirteen different homology models of AGR2 were generated based on different templates which were narrowed down to 5 quality models sorted by their overall Z-scores. The top homology model based on PDB ID = 3PH9 was selected having the best Z-score and was further assessed by Verify-3D, ERRAT and RAMPAGE analysis. Structure-based virtual screening narrowed down the large library of FDA-approved drugs to ten potential AGR2-AGR2 homodimer inhibitors having FRED score lower than - 7.8 kcal/mol in which the top 5 drugs' binding stability was counter-validated by molecular dynamic simulation. To sum up, the present study prepared a validated 3D structure of AGR2 and, for the first time reported the discovery of 5 FDA-approved drugs to inhibit AGR2-AGR2 homodimer by using structure-based virtual screening. Moreover, the binding of the top 5 hits with AGR2 was also validated by molecular dynamic simulation. A validated 3D structure of Anterior Gradient 2 (AGR2) was prepared by homology modeling, which was used in virtual screening of FDA-approved drugs library for the discovery of prospective inhibitors of AGR2-AGR2 homodimer.

Mechanism of zinc ejection by disulfiram in nonstructural protein 5A.

Hepatitis C virus (HCV) is a notorious member of the Flaviviridae family of enveloped, positive-strand RNA viruses. Non-structural protein 5A (NS5A) plays a key role in HCV replication and assembly. NS5A is a multi-domain protein which includes an N-terminal amphipathic membrane anchoring alpha helix, a highly structured domain-1, and two intrinsically disordered domains 2-3. The highly structured domain-1 contains a zinc finger (Zf)-site, and binding of zinc stabilizes the overall structure, while ejection of this zinc from the Zf-site destabilizes the overall structure. Therefore, NS5A is an attractive target for anti-HCV therapy by disulfiram, through ejection of zinc from the Zf-site. However, the zinc ejection mechanism is poorly understood. To disclose this mechanism based on three different states, A-state (NS5A protein), B-state (NS5A + Zn), and C-state (NS5A + Zn + disulfiram), we have performed molecular dynamics (MD) simulation in tandem with DFT calculations in the current study. The MD results indicate that disulfiram triggers Zn ejection from the Zf-site predominantly through altering the overall conformation ensemble. On the other hand, the DFT assessment demonstrates that the Zn adopts a tetrahedral configuration at the Zf-site with four Cys residues, which indicates a stable protein structure morphology. Disulfiram binding induces major conformational changes at the Zf-site, introduces new interactions of Cys39 with disulfiram, and further weakens the interaction of this residue with Zn, causing ejection of zinc from the Zf-site. The proposed mechanism elucidates the therapeutic potential of disulfiram and offers theoretical guidance for the advancement of drug candidates.

A computational subtractive genome analysis for the characterization of novel drug targets in Klebsiella pneumonia strain PittNDM01.

The emergence of carbapenem-resistant Klebsiella Pneumoniae had been reported previously, which needs rapid attention. Currently, Pittsburgh University Hospital reported a new strain of carbapenem-resistant Klebsiella pneumoniae that was co-producing OXA-232 and NDM-1 named as PittNDM01. This strain is resistant to almost all beta-lactam antibiotics such as Carbapenem as well as to fluoroquinolones and aminoglycosides. Globally, failure to the wide-spread pathogenic strains had been observed due to the increased and antibiotic resistance, which leads to less antimicrobial drug efficacy. Since last decades, computational genomic approaches have been introduced to fight against resistant pathogens, which is an advanced approach for novel drug targets investigation. The current study emphasizes the utilization of the available genomic and proteomic data of Klebsiella pneumoniae PittNDM01 for the identification of novel drug targets for future drug developments. Comparative genomic analysis and molecular biological tools were applied, results in observing 582 non-human homologous-essential proteins of Klebsiella pneumoniae. Among the total 582 proteins, 66 were closely related to the pathogen-specific pathway. Out of all 66-targeted proteins, ten non-homologous essential proteins were found to have druggability potential. The subcellular localization of these proteins revealed; 6 proteins in the cytoplasm, 2 in the inner membrane, and one each in periplasmic space and outer membrane. All the above 10 proteins were compared to the proteins sequences of gut flora to eliminate the homologous proteins. In total, 6-novel non-human and non-gut flora essential drug targets of Klebsiella pneumoniae PittNDM01 strain were identified. Further, the 3D structures of the identified drug target proteins were developed, and protein-protein interaction network analysis was performed to know the functional annotation of the desire proteins. Therefore, these non-homologous essential targets ensure the survival of the pathogen and hence can be targeted for drug discovery.

Gain-of-Function SHP2 E76Q Mutant Rescuing Autoinhibition Mechanism Associated with Juvenile Myelomonocytic Leukemia.

Juvenile myelomonocytic leukemia (JMML) is an invasive myeloproliferative neoplasm and is a childhood disease with very high clinical lethality. The SHP2 is encoded by the PTPN11 gene, which is a nonreceptor (pY)-phosphatase and mutation causes JMML. The structural hierarchy of SHP2 includes protein tyrosine phosphatase domain (PTP) and Src-homology 2 domain (N-SH2 and C-SH2). Somatic mutation (E76Q) in the interface of SH2-PTP domain is the most commonly identified mutation found in up to 35% of patients with JMML. The mechanism of this mutant associated with JMML is poorly understood. Here, molecular dynamics simulation was performed on wild-type and mutant (E76Q) of SHP2 to explore the precise impact of gain-of-function on PTP's activity. Consequently, such impact rescues the SHP2 protein from autoinhibition state through losing the interface interactions of Q256/F7 and S502/Q76 or weakening interactions of Q256/R4, Q510/G60, and Q506/A72 between N-SH2 and PTP domains. The consequences of these interactions further relieve the D'E loop away from the PTP catalytic site. The following study would provide a mechanistic insight for better understanding of how individual SHP2 mutations alter the PTP's activity at the atomic level.

Predicting Multi-Interfacial Binding Mechanisms of NLRP3 and ASC Pyrin Domains in Inflammasome Activation.

NLRP3-PYD inflammasome activates an inflammatory pathway in response to a wide variety of cell damage or infections. Dysregulated NLRP3 inflammatory signaling has many chronic inflammatory and autoimmune disorders. NLRP3 and ASC have a PYD, a superfamily member of the Death Domain, which plays a key role in inflammatory assembly. The ASC interacts with NLRP3 through a homotypic PYD and recruits the procaspase-1 through a homotypic caspase recruitment domain interaction. Here, we used several computational approaches to reveal the interactions of the NLRP3 and ASC PYD domains that lead to the activation of the inflammasome complex. We have characterized ASC and NLRP3-PYD intermolecular interactions by protein-protein docking, and further molecular dynamics (MD) simulations were conducted to evaluate the stability of NLRP3/ASC-PYD complex. Subsequently, we have identified several residues that stabilize the NLRP3/ASC-PYD complex in different faces (i.e., Face-1 to Face-4). The research framework offers new insights into the molecular mechanisms of inflammasome and apoptosis signaling as well as the ease of the drug discovery process.