Computational studies on Membrane Proteins (bovine CNGA1 & mouse TSPO)

Around thirty percent of total proteins are present in the membrane and play an important role to communicate intracellular and extracellular region. Their presence in the membrane is one of the limiting steps to determine protein structure and to understand their mechanisms. Hence bioinformatics techniques and computational tools play an important role to overcome these issues in characterizing the structural/functional mechanism of membrane proteins. In this thesis, I have developed and used state of the art computational techniques applied to two different pharmaceutically relevant membrane proteins, Cyclic nucleotide-gated channels (CNG) and translocator membrane protein (TSPO). CNG ion channels are embedded into the neuronal membrane. Till date, the structure and their gating mechanism are subject to interest. Different approaches like electrophysiology, single molecule force spectroscopy, biophysics, etc. have been employed to study these channels. Here I studied the gating mechanism of the CNGA1 ion channel by use of homology modeling and coarse-grained molecular dynamics. TSPO is a key biomarker for the diagnostics of inflammation in the brain. Limited Structural and functional information available on mammalian TSPOs homodimers. Computational studies suggested that the NMR-solved structure is not prone to dimer formation and is not stable in a membrane environment and has been an object of vivid criticism. To address this issue we use homology modeling technique and molecular dynamics approach. Principle results are: 1. I have successfully created homology models for CNGA1 homotetramer and performed coarse-grained simulation in the presence and absence of cGMP molecule and developed the coarse-grained force-field parameters for cGMP. 2. I have proposed a new model of the functionally relevant dimeric form of mTSPO. The model is fully consistent with solid-state NMR spectral data. Our predictions provide for the first time structural insights on this pharmaceutically important target fully consistent with experimental data. 3. During these studies, and in order to optimize the preparation of the systems it was necessary to develop an automated tool for creating the input files for doing coarse-grained simulations. These tools are shared with the community through a publically available online web-server that simplifies the task of generating input files which help in performing simulation and retrieving the result data for small simulations. The web-server, MERMAID is available at MERMAID (http://molsim.sci.univr.it/mangesh/). The application of novel computational approaches in this thesis allowed me to characterize extensively both systems by offering a rational to a huge amount of experimental data on biological relevant systems

MERMAID: dedicated web server to prepare and run coarse-grained membrane protein dynamics

Atomistic molecular dynamics simulations of membrane proteins have been shown to be extremely useful for characterizing the molecular features underlying their function, but require high computational power, limiting the understanding of complex events in membrane proteins, e.g. ion channels gating, GPCRs activation. To overcome this issue, it has been shown that coarse-grained approaches, although requiring less computational power, are still capable of correctly describing molecular events underlying big conformational changes in biological systems. Here, we present the Martini coarse-grained membrane protein dynamics (MERMAID), a publicly available web interface that allows the user to prepare and run coarse-grained molecular dynamics (CGMD) simulations and to analyse the trajectories

3D-QSAR and molecular docking analysis of (4-piperidinyl)-piperazines as acetyl-CoA carboxylases inhibitors

Acetyl-CoA carboxylase (ACC) is a crucial metabolic enzyme, which plays a vital role in fatty acid metabolism and obesity induced type 2 diabetes. Herein, we have performed 3D-QSAR and molecular docking analysis on a novel series of (4-piperidinyl)-piperazines to design potent ACC inhibitors. This study correlates the ACC inhibitory activities of 68 (4-piperidinyl)-piperazine derivatives with several stereo-chemical parameters representing steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor fields. The CoMFA and CoMSIA models exhibited excellent rncv2 values of 0.974 and 0.985, and rcv2 values of 0.671 and 0.693, respectively. CoMFA predicted rpred2 of 0.910 and CoMSIA predicted rpred2 of 0.963 showed that the predicted values were in good agreement with experimental values. Glide5.5 program was used to explore the binding mode of inhibitors inside the active site of ACC. We have accordingly designed novel ACC inhibitors by utilising the LeapFrog and predicted with excellent inhibitory activity in the developed models

Neuroprotective Potential of Peroxisome Proliferator Activated Receptor-α Agonist in Cognitive Impairment in Parkinson’s Disease: Behavioral, Biochemical, and PBPK Profile

Parkinson’s disease (PD) is a common neurodegenerative disorder affecting 1% of the population by the age of 65 years and 4-5% of the population by the age of 85 years. PD affects functional capabilities of the patient by producing motor symptoms and nonmotor symptoms. Apart from this, it is also associated with a higher risk of cognitive impairment that may lead to memory loss, confusion, and decreased attention span. In this study, we have investigated the effect of fenofibrate, a PPAR-α agonist in cognitive impairment model in PD. Bilateral intranigral administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (100 µg/1 µL/side) produced significant cognitive dysfunctions. Fenofibrate treatment at 10, 30, and 100 mg/kg for twenty-five days was found to be neuroprotective and improved cognitive impairment in MPTP-induced PD model as evident from behavioral, biochemical (MDA, GSH, TNF-α, and IL-6), immunohistochemistry (TH), and DNA fragmentation (TUNEL positive cells) studies. Further, physiologically based pharmacokinetic (PBPK) modeling study was performed using GastroPlus to characterize the kinetics of fenofibric acid in the brain. A good agreement was found between pharmacokinetic parameters obtained from the actual and simulated plasma concentration-time profiles of fenofibric acid. Results of this study suggest that PPAR-α agonist (fenofibrate) is neuroprotective in PD-induced cognitive impairment

Structural Prediction of the Dimeric Form of the Mammalian Translocator Membrane Protein TSPO: A Key Target for Brain Diagnostics

Positron emission tomography (PET) radioligands targeting the human translocatormembrane protein (TSPO) are broadly used for the investigations of neuroinflammatory conditionsassociated with neurological disorders. Structural information on the mammalian proteinhomodimers—the suggested functional state of the protein—is limited to a solid-state nuclearmagnetic resonance (NMR) study and to a model based on the previously-deposited solution NMRstructure of the monomeric mouse protein. Computational studies performed here suggest thatthe NMR-solved structure in the presence of detergents is not prone to dimer formation and isfurthermore unstable in its native membrane environment. We, therefore, propose a new modelof the functionally-relevant dimeric form of the mouse protein, based on a prokaryotic homologue.The model, fully consistent with solid-state NMR data, is very different from the previous predictions.Hence, it provides, for the first time, structural insights into this pharmaceutically-important targetwhich are fully consistent with experimental data