Rajabrata Bhuyan

Modelling family 2 cystatins and their interaction with papain

Cystatins are extensively studied cysteine protease inhibitors, found in wide range of organisms with highly conserved structural folds. S-type of cystatins is well known for their abundance in saliva, high selectivity and poorer activity towards host cysteine proteases in comparison to their immediate ancestor cystatin C. Despite more than 90% sequence similarity, the members of this group show highly dissimilar binding affinity towards papain. Cystatin M/E is a potent inhibitor of legumain and papain like cysteine proteases and recognized for its involvement in skin barrier formation and potential role as a tumor suppressor gene. However, the structures of these proteins and their complexes with papain or legumain are still unknown. In the present study, we have employed computational methods to get insight into the interactions between papain and cystatins. Three-dimensional structures of the cystatins are generated by homology modelling, refined with molecular dynamics simulation, validated through numerous web servers and finally complexed with papain using ZDOCK algorithm in Discovery Studio. A high degree of shape complementarity is observed within the complexes, stabilized by numerous hydrogen bonds (HB) and hydrophobic interactions. Using interaction energy, HB and solvent accessible surface area analyses, we have identified a series of key residues that may be involved in papain–cystatin interaction. Differential approaches of cystatins towards papain are also noticed which are possibly responsible for diverse inhibitory activity within the group. These findings will improve our understanding of fundamental inhibitory mechanisms of cystatin and provide clues for further research.

An in silico structural insights into Plasmodium LytB protein and its inhibition

In most of the pathogenic organisms including Plasmodium falciparum, isoprenoids are synthesized via MEP (MethylErythritol 4-Phosphate) pathway. LytB is the last enzyme of this pathway which catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Since the MEP pathway is not used by humans, it represents an attractive target for the development of new anti-malarial compounds or inhibitors. Here a systematic in silico study has been conducted to get an insight into the structure of Plasmodium lytB as well as its affinities towards different inhibitors. We used comparative modeling technique to predict the three-dimensional (3D) structure of Plasmodium LytB taking Escherichia coli LytB protein (PDB ID: 3KE8) as template and the model was subsequently refined through molecular dynamics (MD) simulation. A large ligand data-set containing diphospate group was subjected for virtual screening against the target using GOLD 5.2 program. Considering the mode of binding and affinities, 17 leads were selected on basis of binding energies in comparison to its substrate HMBPP (Gold.Chemscore.DG: -20.9734 kcal/mol). Among them, five were discarded because of their inhibitory activity towards other human enzymes. The rest 12 potential leads carry all the properties of any “drug like” molecule and the knowledge of Plasmodium LytB-inhibitory mechanism which can provide valuable support for the anti-malarial-inhibitor design in future.

Probing binding mechanism of interleukin-6 and olokizumab: in silico design of potential lead antibodies for autoimmune and inflammatory diseases

Abstract Computer-aided antibody engineering has been successful in the design of new biologics for disease diagnosis and therapeutic interventions. Interleukin-6 (IL-6), a well-recognized drug target for various autoimmune and inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and psoriasis, was investigated in silico to design potential lead antibodies. Here, crystal structure of IL-6 along with monoclonal antibody olokizumab was explored to predict antigen–antibody (Ag − Ab)-interacting residues using DiscoTope, Paratome, and PyMOL. Tyr56, Tyr103 in heavy chain and Gly30, Ile31 in light chain of olokizumab were mutated with residues Ser, Thr, Tyr, Trp, and Phe. A set of 899 mutant macromolecules were designed, and binding affinity of these macromolecules to IL-6 was evaluated through Ag − Ab docking (ZDOCK, ClusPro, and Rosetta server), binding free-energy calculations using Molecular Mechanics/Poisson Boltzman Surface Area (MM/PBSA) method, and interaction energy estimation. In comparison to olokizumab, eight newly designed theoretical antibodies demonstrated better result in all assessments. Therefore, these newly designed macromolecules were proposed as potential lead antibodies to serve as a therapeutics option for IL-6-mediated diseases.

Molecular dynamics of Kv1.3 ion channel and structural basis of its inhibition by scorpion toxin-OSK1 derivatives

Kv1.3 is one of the widely distributed Shaker type voltage gated potassium channel which performs the outward flow of K(+) ions in excitable cells. In immunological synapse, Kv1.3 plays a pivotal role in antigen dependent activation and proliferation of lymphocytes along with the KCa3.1. The up-regulation of Kv1.3 leads to several T-cell-mediated autoimmune diseases, hence considered as an attractive pharmacological drug target. Here, we have employed molecular modeling, docking and simulation techniques to examine the dynamical properties of Kv1.3 in both open and closed state conformation embedded in DPPC membrane as well as its modes of inhibition against the popularly known scorpion venom OSK1 and its three mutant analogues. The Kv1.3 in open conformation took comparatively more time to get stabilized than the closed state. Both conformations ascertain their stability and the transition between closed to active states is more consistent with the paddle model of channel gating. The binding modes of channel-toxin complexes are well established by identifying strongly interacting amino acids lining at their polar surfaces. Our findings suggest that, two mutant derivatives OSK1-K16,D20 & OSK1-P12,K16,D20 have increased inhibitory potency against Kv1.3. We also pointed out some particular residues responsible for binding of OSK1 with Kv1.3 over other Shaker-type ion channels. We believe that the insights came from Kv1.3-OSK1 interaction will be valuable in pharmacological studies for strategic development of both potent and selective therapeutic drugs against T-cell-mediated autoimmune diseases.

Conformational dynamics of shaker-type Kv1.1 ion channel in open, closed, and two mutated states.

The dynamic properties of shaker-type Kv1.1 ion channel in its open, closed, & two mutated (E325D & V408A) states embedded in DPPC membrane have been investigated using all-atom force field-based MD simulation. Here, we represent the detailed channel stability, gating environment of charge-carrying residues, salt bridge interaction among the voltage-sensing domains (VSDs), movement of S4 helix, and ion conduction of pore. At positive potential, the S4 helix undergoes lateral fluctuations in accordance with their gating motions found in every model. During transition from closed to active state conformation, charged residues of S4 move “up” across the membrane with an average tilt angle difference of 24°, which is more consistent with the paddle model of channel gating. The E325D mutation at C-terminal end of S4–S5 helical linker leads the channel to a rapid activated state by pushing the gating charge residues upward beside the VSDs resulting in more prominent tilt of S4. Similarly in V408A mutant model, disruption of hydrophobic gate at S6 C-terminal end takes place, which causes the violation of channel-active conformation by bringing the C-terminal end of S4 to its corresponding resting state. The ion permeation is observed only in open-state conformation.

Dynamics and modulation studies of human voltage gated Kv1.5 channel

The voltage gated Kv1.5 channels conduct the ultrarapid delayed rectifier current (IKur) and play critical role in repolarization of action potential duration. It is the most rapidly activated channel and has very little or no inactivated states. In human cardiac cells, these channels are expressed more extensively in atrial myocytes than ventricle. From the evidences of its localization and functions, Kv1.5 has been declared a selective drug target for the treatment of atrial fibrillation (AF). In this present study, we have tried to identify the rapidly activating property of Kv1.5 and studied its mode of inhibition using molecular modeling, docking, and simulation techniques. Channel in open conformation is found to be stabilized quickly within the dipalmitoylphosphatidylcholine membrane, whereas most of the secondary structure elements were lost in closed state conformation. The obvious reason behind its ultra-rapid property is possibly due to the amino acid alteration in S4–S5 linker; the replacement of Lysine by Glutamine and vice versa. The popular published drugs as well as newly identified lead molecules were able to inhibit the Kv1.5 in a very similar pattern, mainly through the nonpolar interactions, and formed sable complexes. V512 is found as the main contributor for the interaction along with the other important residues such as V505, I508, A509, V512, P513, and V516. Furthermore, two screened novel compounds show surprisingly better inhibitory potency and can be considered for the future perspective of antiarrhythmic survey.

DB Dehydrogenase: an online integrated structural database on enzyme dehydrogenase

Dehydrogenase enzymes are almost inevitable for metabolic processes. Shortage or malfunctioning of dehydrogenases often leads to several acute diseases like cancers, retinal diseases, diabetes mellitus, Alzheimer, hepatitis B & C etc. With advancement in modern-day research, huge amount of sequential, structural and functional data are generated everyday and widens the gap between structural attributes and its functional understanding. DB Dehydrogenase is an effort to relate the functionalities of dehydrogenase with its structures. It is a completely web-based structural database, covering almost all dehydrogenases [~150 enzyme classes, ~1200 entries from ~160 organisms] whose structures are known. It is created by extracting and integrating various online resources to provide the true and reliable data and implemented by MySQL relational database through user friendly web interfaces using CGI Perl. Flexible search options are there for data extraction and exploration. To summarize, sequence, structure, function of all dehydrogenases in one place along with the necessary option of cross-referencing; this database will be utile for researchers to carry out further work in this field. Availability The database is available for free at http://www.bifku.in/DBD/

Exploration and validation of diphosphate-based Plasmodium LytB inhibitors using computational approaches

LytB or IspH is an indispensable enzyme and a suitable drug target of Plasmodium falciparum that participate in isoprenoid biosynthesis of nonmevalonate pathway (MEP). Recently, we have investigated the structural dynamics of Plasmodium LytB and proposed some novel diphosphate‐based inhibitors using molecular modeling and docking studies. Here, we have tried to characterize those previously screened molecules by quantitative structure activity relationships and pharmacophore‐based analyses, as well as validated the dynamics of their interactions with LytB protein. Five total compounds having PubChem CID 516, 125696, 165275, 448012, and 9921431 were predicted with significant inhibitory activity by quantitative structure activity relationships and pharmacophore models. Again, the molecular dynamics simulation results showed that these five compounds are able to form stable complexes with the receptor through many direct and water mediated interactions. The binding free energies calculated by Poisson‐Boltzmann surface area method resulted within the range between −99.77 and − 43.74 kcal/mol, which favoured their profound inhibitory affinity. Residues of LytB like His41, His74, Ser222, Ser223, and Asn224 in LytB were the main protagonists in contributing the majority of interaction energies to the ligands. Finally, the ADMET, toxicity, and drug‐likeness scores also affirm these compounds to be considered for further development of new antimalarial inhibitor in the future.

Role of catechins on ET-1 induced stimulation of PLD and NADPH oxidase activities in pulmonary smooth muscle cells: Determination of the probable mechanism by molecular docking studies

The treatment of human pulmonary artery smooth muscle cells with ET-1 stimulates the activity of PLD and NADPH oxidase, but this stimulation is inhibited by pretreatment with bosentan (ET-1 receptor antagonist), FIPI (PLD inhibitor), apocynin (NADPH oxidase inhibitor), and EGCG and ECG (catechins having a galloyl group), but not EGC and EC (catechins devoid of a galloyl group). Herein, using molecular docking analyses based on our biochemical studies, we determined the probable mechanism by which the catechins containing a galloyl group inhibit the stimulation of PLD activity induced by ET-1. The ET-1-induced stimulation of PLD activity was inhibited by SecinH3 (inhibitor of cytohesin). Arf6 and cytohesin-1 are associated in the cell membrane, which is not inhibited by the catechins during ET-1 treatment of the cells. However, EGCG and ECG inhibited the binding of GTPγS with Arf6, even in the presence of cytohesin-1. The molecular docking analyses revealed that the catechins containing a galloyl group (EGCG and ECG) with cytohesin-1-Arf6GDP, but not the catechins without a galloyl group (EGC and EC), prevent GDP-GTP exchange in Arf6, which seems to be an important mechanism for inhibiting the activation of PLD induced by ET-1, and subsequently increases the activity of NADPH oxidase.