In Silico Screening to Identify Inhibitors of Growth Factor Receptor 2-Focal Adhesion Kinase Interaction for Therapeutic Treatment of Pathological Cardiac Hypertrophy.

The focal adhesion kinase-growth factor receptor 2 (FAK-Grb2) protein-protein interaction is implicated in pathogenesis of stress-induced cardiac hypertrophy. The focal adhesion targeting (FAT) domain of FAK unfolds to form a structural intermediate that interacts with a multibinding hot spot in the SH2 domain of Grb2. Disruption of the Grb2-FAT interaction is a therapeutic strategy for prevention of pathological cardiac hypertrophy. A pharmacophore was generated on the basis of structural and electrostatic properties of FAT bound to FAK using the Forge tool (Cresset). This pharmacophore was used as a query for Blaze server (Cresset) to screen a selectively enriched chemical library of 4,32,508 small molecules. The compounds selected were further filtered by hierarchical flexible docking approach using AutoDock v4. From the favorably docked compounds, five were selected on the basis of good adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties using SwissADME, MedChem Designer v.3, and MOLINSPIRATION. Stability of the binding mode of the inhibitors was further confirmed by molecular dynamic simulation study with AMBER v15 for a simulation time of 50 ns in aqueous environment. PM2307 was identified as the best inhibitor in terms of pharmacophoric features, dock score, and in silico ADMET analysis. The calculated binding affinity of PM2307 was better than that of the FAT-Grb2 complex as well as a previously reported small molecule inhibitor. PM2307 is also a quinolyl derivative sharing a similar scaffold with ofloxacin drugs, asserting its drug-like properties. Thus, it was proposed as a lead compound for development of drugs for pathological cardiac hypertrophy.

Modeling, dynamics and phosphoinositide binding of the pleckstrin homology domain of two novel PLCs: η1 and η2.

PH domains mediate interactions involved in cell signaling, intracellular membrane transport regulation and cytoskeleton organization. Some PH domains bind phosphoinositides with different affinity and specificity. The two novel PLCη (1 and 2) possess an N-terminal PH domain (PHη1 and PHη2 respectively) that has been implicated in membrane association and induction of PLC activity. Understanding of the structure and dynamics is crucial for future modulation of lipid-protein interactions in PHη1, PHη2 and other PH domains. Therefore, the three-dimensional structure of PHη1 and PHη2 was modeled using ITASSER and phosphoinositides (IP3 and IP4) were docked in the inferred binding site using HADDOCK server. Molecular Dynamics simulations of unliganded and phosphoinositide bound PHη1 and PHη2 were performed using AMBER14 to study the mechanism of interaction, and conformational dynamics in response to phosphoinositide binding. The binding affinity was predicted using Kdeep server. The models of PHη1 and PHη2 had a conserved structural core consisting of seven ß-strands and a C-terminal α-helix as seen in other PH domains. Sequence/structure analysis showed that phosphoinositide ligands bind PHη1 and PHη2 at the canonical binding site. Phosphoinositide binding induced movement of positively charged side chains towards the ligand, changes in the secondary structure especially at the ß5-ß6 loop and allosteric changes at the interface of ß1-ß2 and ß5-ß6 loops. Dynamics studies showed that the size of the binding site and differential affinity for IP3/IP4 binding is coordinated by the number, length, flexibility, secondary structure and allosteric interactions of the loops surrounding the phosphoinositide binding site.

Structure of focal adhesion kinase in healthy heart versus pathological cardiac hypertrophy: A modeling and simulation study.

Focal adhesion kinase (FAK) is required for signaling in the heart. S910 phosphorylated FAK is known to cause pathological cardiac hypertrophy. The switching of FAK between its inactive (-i), activated (-a) and hyperactive (-h) state is controlled by phosphorylation. FAK consists of three domains, namely: FERM, Kinase, and FAT joined by linkers L1 and L2. The structural basis of FAK phosphorylation and signaling to the downstream pathways is not understood. In this work, we carried out homology modeling and domain assembly of full length human iFAK and aFAK. 100 ns classical molecular dynamic simulations were performed using AMBER14 and effect of S910 phosphorylation on FAK was investigated. The iFAK model superposed on a small angel X-ray scattering (SAXS) derived model with RMSD of 1.18 Šfor 590 Cα atoms. aFAK showed S910 phosphorylation site in L2 shielded by FERM. S910 phosphorylation in hFAK led to its exposure accompanied by a large conformational change and exposing the previously buried Grb2 interaction site responsible for causing cardiac hypertrophy. The models of FAK are in agreement with diverse experimental data and observed differences in biological action. Understanding the structure activity relationships of FAK in response to phosphorylation is important for its future therapeutic modulation.

Structural basis of focal adhesion targeting domain-mediated signaling in cardiac hypertrophy.

The focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) exists in monomeric closed (c) or arm exchanged (ae) dimeric state. FAT interaction with Grb2 necessitates an intermediate open (o) state that interacts with Grb2 and activates signaling pathways leading to pathological cardiac hypertrophy. Targeted molecular dynamics (TMD) simulation was carried out in order to capture the structure of the intermediate formed by opening of Helix1 (H1) from monomeric cFAT leading to the formation of monomeric aeFAT. During TMD, H1 separated from the four helices bundle of cFAT, completely unfolded and performed a full turn before folding back to a helix inclined at an acute angle to the helical bundle in aeFAT. The entire transition can be described in six distinct intermediate structural stages. The most significant correlation of H1 motion was observed with Loop3 (L3) and is the likely reason for the complete disruption of the FAT interaction with paxillin during the transition. High-affinity analogs of the paxillin LD4 region can be a promising strategy to drive the equilibrium towards cFAT, thus antagonizing FAT-Grb2 association. During transition, the overall shift in orientation of all the four helices rejects paxillin binding and approves Grb2 association. Exposure and ß-turn conformation of the YENV motif (residues 925-928) in oFAT-facilitated phosphorylation and Grb2 binding. Docking, MD simulation and conservation analysis of oFAT-Grb2 complex provided insight into the structural determinants of binding and specificity. Our work provides a structural basis for pharmacological modulation of dynamic conformational changes and interactions of FAT.

Modeling of Plasmodium falciparum Telomerase Reverse Transcriptase Ternary Complex: Repurposing of Nucleoside Analog Inhibitors.

The Plasmodium falciparum telomerase reverse transcriptase (PfTERT) is a ribonucleoprotein that assists the maintenance of the telomeric ends of chromosomes by reverse transcription of its own RNA subunit. It represents an attractive therapeutic target for eradication of the plasmodial parasite at the asexual liver stage. Automated modeling using MUSTER and knowledge-based techniques were used to obtain a three-dimensional model of the active site of reverse transcriptase domain of PfTERT, which is responsible for catalyzing the addition of incoming dNTPs to the growing DNA strand in presence of divalent magnesium ions. Further, the ternary complex of the active site of PfTERT bound to a DNA-RNA duplex was also modeled using Haddock server and represents the functional form of the enzyme. Initially, established nucleoside analog inhibitors of PfTERT, AZTTP, and ddGTP were docked in the modeled binding site of the PfTERT ternary complex using AutoDock v4.2. Subsequently, docking studies were carried out with 14 approved nucleoside analog inhibitors. Docking studies predicted that floxuridine, gemcitabine, stavudine, and vidarabine have high affinity for the PfTERT ternary complex. Further analysis on the basis of known side effects led us to propose repositioning of vidarabine as a suitable drug candidate for inhibition of PfTERT.

Integrative analysis of ocular complications in atherosclerosis unveils pathway convergence and crosstalk.

Atherosclerosis is a life-threatening disease and a major cause of mortalities worldwide. While many of the atherosclerotic sequelae are reflected as microvascular effects in the eye, the molecular mechanisms of their development is not yet known. In this study, we employed a systems biology approach to unveil the most significant events and key molecular mediators of ophthalmic sequelae caused by atherosclerosis. Literature mining was used to identify the proteins involved in both atherosclerosis and ophthalmic diseases. A protein-protein interaction (PPI) network was prepared using the literature-mined seed nodes. Network topological analysis was carried out using Cytoscape, while network nodes were annotated using database for annotation, visualization and integrated discovery in order to identify the most enriched pathways and processes. Network analysis revealed that mitogen-activated protein kinase 1 (MAPK1) and protein kinase C occur with highest betweenness centrality, degree and closeness centrality, thus reflecting their functional importance to the network. Our analysis shows that atherosclerosis-associated ophthalmic complications are caused by the convergence of neurotrophin signaling pathways, multiple immune response pathways and focal adhesion pathway on the MAPK signaling pathway. The PPI network shares features with vasoregression, a process underlying multiple vascular eye diseases. Our study presents a first clear and composite picture of the components and crosstalk of the main pathways of atherosclerosis-induced ocular diseases. The hub bottleneck nodes highlight the presence of molecules important for mediating the ophthalmic complications of atherosclerosis and contain five established drug targets for future therapeutic modulation efforts.