| 1. |
- Abhinand, P. A., et al.
(författare)
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Insights on the structural perturbations in human MTHFR Ala222Val mutant by protein modeling and molecular dynamics
- 2016
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Ingår i: Journal of Biomolecular Structure and Dynamics. - : Informa UK Limited. - 0739-1102 .- 1538-0254. ; 34:4, s. 892-905
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Tidskriftsartikel (refereegranskat)abstract
- Methylenetetrahydrofolate reductase (MTHFR) protein catalyzes the only biochemical reaction which produces methyltetrahydrofolate, the active form of folic acid essential for several molecular functions. The Ala222Val polymorphism of human MTHFR encodes a thermolabile protein associated with increased risk of neural tube defects and cardiovascular disease. Experimental studies have shown that the mutation does not affect the kinetic properties of MTHFR, but inactivates the protein by increasing flavin adenine dinucleotide (FAD) loss. The lack of completely solved crystal structure of MTHFR is an impediment in understanding the structural perturbations caused by the Ala222Val mutation; computational modeling provides a suitable alternative. The three-dimensional structure of human MTHFR protein was obtained through homology modeling, by taking the MTHFR structures from Escherichia coli and Thermus thermophilus as templates. Subsequently, the modeled structure was docked with FAD using Glide, which revealed a very good binding affinity, authenticated by a Glide XP score of-10.3983 (kcal mol-1). The MTHFR was mutated by changing Alanine 222 to Valine. The wild-type MTHFR-FAD complex and the Ala222Val mutant MTHFR-FAD complex were subjected to molecular dynamics simulation over 50 ns period. The average difference in backbone root mean square deviation (RMSD) between wild and mutant variant was found to be ~.11 Å. The greater degree of fluctuations in the mutant protein translates to increased conformational stability as a result of mutation. The FAD-binding ability of the mutant MTHFR was also found to be significantly lowered as a result of decreased protein grip caused by increased conformational flexibility. The study provides insights into the Ala222Val mutation of human MTHFR that induces major conformational changes in the tertiary structure, causing a significant reduction in the FAD-binding affinity.
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| 2. |
- Bhakat, Soumendranath
(författare)
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Effect of T68A/N126Y mutations on the conformational and ligand binding landscape of Coxsackievirus B3 3C protease.
- 2015
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Ingår i: Molecular BioSystems. - : Royal Society of Chemistry (RSC). - 1742-2051 .- 1742-206X. ; 11:8, s. 2303-2311
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Tidskriftsartikel (refereegranskat)abstract
- 3C protease of Coxsackievirus B3 (CVB3) plays an essential role in the viral replication cycle, and therefore, emerged as an attractive therapeutic target for the treatment of human diseases caused by CVB3 infection. In this study, we report the first account of the molecular impact of the T68A/N126Y double mutant (MutantBound) using an integrated computational approach. Molecular dynamics simulation and post-dynamics binding free energy, principal component analysis (PCA), hydrogen bond occupancy, SASA, Rg and RMSF confirm that T68A/N126Y instigated an increased conformational flexibility due to the loss of intra- and inter-molecular hydrogen bond interactions and other prominent binding forces, which led to a decreased protease grip on the ligand (). The double mutations triggered a distortion orientation of in the active site and decreases the binding energy, ΔGbind (∼3 kcal mol(-1)), compared to the wild type (WildBound). The van der Waals and electrostatic energy contributions coming from residues 68 and 126 are lower for MutantBound when compared with WildBound. In addition, variation in the overall enzyme motion as evident from the PCA, distorted hydrogen bonding network and loss of protein-ligand interactions resulted in a loss of inhibitor efficiency. The comprehensive molecular insight gained from this study should be of great importance in understanding the drug resistance against CVB3 3C protease; also, it will assist in the designing of novel Coxsackievirus B3 inhibitors with high ligand efficacy on resistant strains.
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| 3. |
- Bhakat, Soumendranath, et al.
(författare)
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Flap Dynamics in Pepsin-Like Aspartic Proteases : A Computational Perspective Using Plasmepsin-II and BACE-1 as Model Systems
- 2022
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Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 62:4, s. 914-926
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Tidskriftsartikel (refereegranskat)abstract
- The flexibility of β hairpin structure known as the flap plays a key role in catalytic activity and substrate intake in pepsin-like aspartic proteases. Most of these enzymes share structural and sequential similarity. In this study, we have used apo Plm-II and BACE-1 as model systems. In the apo form of the proteases, a conserved tyrosine residue in the flap region remains in a dynamic equilibrium between the normal and flipped states through rotation of the χ1 and χ2 angles. Independent MD simulations of Plm-II and BACE-1 remained stuck either in the normal or flipped state. Metadynamics simulations using side-chain torsion angles (χ1 and χ2 of tyrosine) as collective variables sampled the transition between the normal and flipped states. Qualitatively, the two states were predicted to be equally populated. The normal and flipped states were stabilized by H-bond interactions to a tryptophan residue and to the catalytic aspartate, respectively. Further, mutation of tyrosine to an amino-acid with smaller side-chain, such as alanine, reduced the flexibility of the flap and resulted in a flap collapse (flap loses flexibility and remains stuck in a particular state). This is in accordance with previous experimental studies, which showed that mutation to alanine resulted in loss of activity in pepsin-like aspartic proteases. Our results suggest that the ring flipping associated with the tyrosine side-chain is the key order parameter that governs flap dynamics and opening of the binding pocket in most pepsin-like aspartic proteases.
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| 4. |
- Bhakat, Soumendranath
(författare)
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Molecular Recognition and Conformational Dynamics in Macromolecules
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Computational methods gained a widespread use in drug discovery. Understanding conformational dynamics of protein and mechanisms of protein-ligand binding are two major areas in drug discovery. Molecular dynamics (MD) simulation have been routinely used to study conformational dynamics of protein and mechanisms of protein-ligand binding. In classical MD simulation, the system often remains stuck in a local free energy minimum for a long time. Hence, conformational changes associated with long timescales (e.g. loop motion, ligand binding/unbinding etc.) are beyond reach of classical MD simulation. Metadynamics is an enhanced sampling method which deposits bias along some chosen reaction coordinate and forces the system to escape local minimum thus, allows better sampling of the conformational space. In this thesis, I have used MD and metadynamics to study protein-ligand binding andconformational dynamics of globular proteins. We found that the presence of trapped water in the binding site of the protein plays a key role ligand binding. Further, we found that the side-chains of binding site residues and flexibility ofligands play a key role in the protein-ligand binding. We also studied how rotation of tyrosine dictates conformational dynamics in a class of protein known as pepsin-like aspartic protease. We found that apo protease remains in adynamic equilibrium between normal and flipped states due to rotation of tyrosine side-chain. Conformational dynamics also plays a crucial role in hydrogen exchange via solvent penetration. Local fluctuations in protein breaks the hydrogen bond interactions involving backbone amides which allows solvent penetration. We defined this metastable state as broken state. In the broken state, the backbone amide forms hydrogen bond interaction with water molecule. Using molecular dynamics and metadynamics we predicted free energy difference between the broken and ground state (backbone amide remains hydrogen bonded with neighboring residue) in a small globular protein.
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| 5. |
- Bhakat, Soumendranath
(författare)
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Pepsin-like aspartic proteases (PAPs) as model systems for combining biomolecular simulation with biophysical experiments
- 2021
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Ingår i: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 11:18, s. 11026-11047
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Forskningsöversikt (refereegranskat)abstract
- Pepsin-like aspartic proteases (PAPs) are a class of aspartic proteases which shares tremendous structural similarity with human pepsin. One of the key structural features of PAPs is the presence of a β-hairpin motif otherwise known as flap. The biological function of the PAPs is highly dependent on the conformational dynamics of the flap region. In apo PAPs, the conformational dynamics of the flap is dominated by the rotational degrees of freedom associated withχ1 andχ2 angles of conserved Tyr (or Phe in some cases). However it is plausible that dihedral order parameters associated with several other residues might play crucial roles in the conformational dynamics of apo PAPs. Due to their size, complexities associated with conformational dynamics and clinical significance (drug targets for malaria, Alzheimer's diseaseetc.), PAPs provide a challenging testing ground for computational and experimental methods focusing on understanding conformational dynamics and molecular recognition in biomolecules. The opening of the flap region is necessary to accommodate substrate/ligand in the active site of the PAPs. The BIG challenge is to gain atomistic details into how reversible ligand binding/unbinding (molecular recognition) affects the conformational dynamics. Recent reports of kinetics (Ki,Kd) and thermodynamic parameters (ΔH,TΔS, and ΔG) associated with macro-cyclic ligands bound to BACE1 (belongs to PAP family) provide a perfect challenge (how to deal with big ligands with multiple torsional angles and select optimum order parameters to study reversible ligand binding/unbinding) for computational methods to predict binding free energies and kinetics beyond typical test systemse.g.benzamide-trypsin. In this work, i reviewed several order parameters which were proposed to capture the conformational dynamics and molecular recognition in PAPs. I further highlighted how machine learning methods can be used as order parameters in the context of PAPs. I then proposed some open ideas and challenges in the context of molecular simulation and put forward my case on how biophysical experimentse.g.NMR, time-resolved FRETetc.can be used in conjunction with biomolecular simulation to gain complete atomistic insights into the conformational dynamics of PAPs.
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| 6. |
- Bhakat, Soumendranath, et al.
(författare)
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Prediction of binding poses to FXR using multi-targeted docking combined with molecular dynamics and enhanced sampling
- 2018
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Ingår i: Journal of Computer-Aided Molecular Design. - : Springer Science and Business Media LLC. - 0920-654X .- 1573-4951. ; 32:1, s. 59-73
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Tidskriftsartikel (refereegranskat)abstract
- Advanced molecular docking methods often aim at capturing the flexibility of the protein upon binding to the ligand. In this study, we investigate whether instead a simple rigid docking method can be applied, if combined with multiple target structures to model the backbone flexibility and molecular dynamics simulations to model the sidechain and ligand flexibility. The methods are tested for the binding of 35 ligands to FXR as part of the first stage of the Drug Design Data Resource (D3R) Grand Challenge 2 blind challenge. The results show that the multiple-target docking protocol performs surprisingly well, with correct poses found for 21 of the ligands. MD simulations started on the docked structures are remarkably stable, but show almost no tendency of refining the structure closer to the experimentally found binding pose. Reconnaissance metadynamics enhances the exploration of new binding poses, but additional collective variables involving the protein are needed to exploit the full potential of the method.
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| 7. |
- Bhakat, Soumendranath, et al.
(författare)
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Reaching beyond HIV/HCV: nelfinavir as a potential starting point for broad-spectrum protease inhibitors against dengue and chikungunya virus
- 2015
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Ingår i: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 5:104, s. 85938-85949
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Tidskriftsartikel (refereegranskat)abstract
- Drug repurposing or re-profiling has become an effective strategy to identify novel indications for already-approved drugs. In this study, peptidomimetic FDA-approved HIV/HCV inhibitors were explored for their potential to be repurposed for the inhibition of the replication of dengue (DENV) and chikungunya virus (CHIKV) by targeting the NS2B-NS3 and NSP2 protease, respectively. MM/GBSA-based binding free energy results put nelfinavir forward as a potential inhibitor of both dengue and chikungunya virus, which subsequently was further explored in a virus-cell-based assay for both viruses. Nelfinavir showed modest antiviral activity against CHIKV (EC50 = 14 +/- 1 mu M and a selectivity index of 1.6) and was slightly more active against DENV-2 (EC50 = 3.5 +/- 0.4 mu M and a selectivity index of 4.6). Even though the antiviral potency was limited, the fact that some activity was observed in these assays made it worthwhile exploring the potential and properties of nelfinavir as a stepping-stone compound: a more detailed computational analysis was performed to understand the binding mode, interaction, hydrogen bond distance, occupancy and minimum pharmacophoric features. The comprehensive data set that resulted from these analyses may prove to be useful for the development of novel DENV and CHIKV protease inhibitors.
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| 8. |
- Bhakat, Soumendranath, et al.
(författare)
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Resolving the problem of trapped water in binding cavities : prediction of host–guest binding free energies in the SAMPL5 challenge by funnel metadynamics
- 2017
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Ingår i: Journal of Computer-Aided Molecular Design. - : Springer Science and Business Media LLC. - 0920-654X .- 1573-4951. ; , s. 119-132
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Tidskriftsartikel (refereegranskat)abstract
- The funnel metadynamics method enables rigorous calculation of the potential of mean force along an arbitrary binding path and thereby evaluation of the absolute binding free energy. A problem of such physical paths is that the mechanism characterizing the binding process is not always obvious. In particular, it might involve reorganization of the solvent in the binding site, which is not easily captured with a few geometrically defined collective variables that can be used for biasing. In this paper, we propose and test a simple method to resolve this trapped-water problem by dividing the process into an artificial host-desolvation step and an actual binding step. We show that, under certain circumstances, the contribution from the desolvation step can be calculated without introducing further statistical errors. We apply the method to the problem of predicting host–guest binding free energies in the SAMPL5 blind challenge, using two octa-acid hosts and six guest molecules. For one of the hosts, well-converged results are obtained and the prediction of relative binding free energies is the best among all the SAMPL5 submissions. For the other host, which has a narrower binding pocket, the statistical uncertainties are slightly higher; longer simulations would therefore be needed to obtain conclusive results.
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| 9. |
- Kumalo, Hezekiel M, et al.
(författare)
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Heat Shock Protein 90 (Hsp90) as Anti-cancer Target for Drug Discovery: An Ample Computational Perspective.
- 2015
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Ingår i: Chemical Biology and Drug Design. - : Wiley. - 1747-0285 .- 1747-0277. ; 86:5, s. 1131-1160
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Forskningsöversikt (refereegranskat)abstract
- There are over 100 different types of cancer, and each is classified based on the type of cell that is initially affected. If left untreated, cancer can result in serious health problems and eventually death. Recently the paradigm of cancer chemotherapy has evolved to use a combination approach, which involves the use of multiple drugs each of which targets an individual protein. Inhibition of heat shock protein 90 (Hsp90) is one of the novel key cancer targets. Because of its ability to target several signaling pathways, Hsp90 inhibition emerged as a useful strategy to treat a wide variety of cancers. Molecular modeling approaches and methodologies have become "close counterparts" to experiments in drug design and discovery workflows. A wide-range of molecular modeling approaches have been developed, each of which has different objectives and outcomes. In this review, we provide an up-to-date systematic overview on the different computational models implemented towards the design of Hsp90 inhibitors as anti-cancer agents. Although this is the main emphasis of this review, different topics such as; background and current statistics of cancer, different anti-cancer targets including Hsp90, the structure and function of Hsp90 from an experimental perspective e.g. X-ray and NMR are also addressed in this report. To the best of our knowledge, this review is the first account, which comprehensively outlines various molecular modeling efforts directed towards identification of anti-cancer drugs targeting Hsp90. We believe that the information, methods and perspectives highlighted in this report would assist researchers in the discovery of potential anti-cancer agents. This article is protected by copyright. All rights reserved.
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| 10. |
- Mahanti, Mukul, et al.
(författare)
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Flap Dynamics in Aspartic Proteases : A Computational Perspective
- 2016
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Ingår i: Chemical Biology and Drug Design. - : Wiley. - 1747-0285 .- 1747-0277. ; 88:2, s. 159-177
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Forskningsöversikt (refereegranskat)abstract
- Recent advances in biochemistry and drug design have placed proteases as one of the critical target groups for developing novel small-molecule inhibitors. Among all proteases, aspartic proteases have gained significant attention due to their role in HIV/AIDS, malaria, Alzheimer's disease, etc. The binding cleft is covered by one or two β-hairpins (flaps) which need to be opened before a ligand can bind. After binding, the flaps close to retain the ligand in the active site. Development of computational tools has improved our understanding of flap dynamics and its role in ligand recognition. In the past decade, several computational approaches, for example molecular dynamics (MD) simulations, coarse-grained simulations, replica-exchange molecular dynamics (REMD) and metadynamics, have been used to understand flap dynamics and conformational motions associated with flap movements. This review is intended to summarize the computational progress towards understanding the flap dynamics of proteases and to be a reference for future studies in this field.
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