Umesh Kalathiya

Structural and dynamic changes adopted by EmrE, multidrug transporter protein--Studies by molecular dynamics simulation.

EmrE protein transports positively charged aromatic drugs (xenobiotics) in exchange for two protons and thus provides bacteria resistance to variety of drugs. In order to understand how this protein may recognize ligands, the monomer and asymmetric apo-form of the EmrE dimer embedded in a heterogeneous phospholipid (POPE+POPG) membrane were studied by molecular dynamics simulations. Dimer is regarded as a functional form of the transporter, but to understand molecular aspects of its mode of action, a monomer was also included in our work. We analyzed hydrogen bonds which include inter- and intra-molecular interactions. Analyzing the long-lasting H-bond interactions, we found that water access to the internal transmembrane segments is regulated by residues with aromatic or basic side chains and fluctuating transmembrane helices. Our finding supports that GLU14 in EmrE apo-form is ready to interact or bind with substrate molecule. The analysis of distance center of masses and water entrance area indicate the feasibility of the dimer to undergo induced fit in order to accommodate a ligand. The results indicate that a binding pattern can be formed in the EmrE in such a way that GLU14 binds to the positively charged fragment of a substrate molecule, and other aromatic residues (i.e., TRP63 and TYR40) located in vicinity may accommodate other non-polar parts of substrate molecule. The results of our simulation also allow us to support experimentally testable hypotheses concerning functional inward-outward conformational changes of the protein.

Structure-based design and evaluation of novel N-phenyl-1H-indol-2-amine derivatives for fat mass and obesity-associated (FTO) protein inhibition

Fat mass and obesity-associated (FTO) protein contributes to non-syndromic human obesity which refers to excessive fat accumulation in human body and results in health risk. FTO protein has become a promising target for anti-obesity medicines as there is an immense need for the rational design of potent inhibitors to treat obesity. In our study, a new scaffold N-phenyl-1H-indol-2-amine was selected as a base for FTO protein inhibitors by applying scaffold hopping approach. Using this novel scaffold, different derivatives were designed by extending scaffold structure with potential functional groups. Molecular docking simulations were carried out by using two different docking algorithm implemented in CDOCKER (flexible docking) and AutoDock programs (rigid docking). Analyzing results of rigid and flexible docking, compound MU06 was selected based on different properties and predicted binding affinities for further analysis. Molecular dynamics simulation of FTO/MU06 complex was performed to characterize structure rationale and binding stability. Certainly, Arg96 and His231 residue of FTO protein showed stable interaction with inhibitor MU06 throughout the production dynamics phase. Three residues of FTO protein (Arg96, Asp233, and His231) were found common in making H-bond interactions with MU06 during molecular dynamics simulation and CDOCKER docking.

Molecular modeling and evaluation of novel dibenzopyrrole derivatives as telomerase inhibitors and potential drug for cancer therapy

During previous years, many studies on synthesis, as well as on anti-tumor, anti-inflammatory and anti-bacterial activities of the pyrazole derivatives have been described. Certain pyrazole derivatives exhibit important pharmacological activities and have proved to be useful template in drug research. Considering importance of pyrazole template, in current work the series of novel inhibitors were designed by replacing central ring of acridine with pyrazole ring. These heterocyclic compounds were proposed as a new potential base for telomerase inhibitors. Obtained dibenzopyrrole structure was used as a novel scaffold structure and extension of inhibitors was done by different functional groups. Docking of newly designed compounds in the telomerase active site (telomerase catalytic subunit TERT) was carried out. All dibenzopyrrole derivatives were evaluated by three docking programs: CDOCKER, Ligandfit docking (Scoring Functions) and AutoDock. Compound C_9g, C_9k and C_9l performed best in comparison to all designed inhibitors during the docking in all methods and in interaction analysis. Introduction of pyrazole and extension of dibenzopyrrole in compounds confirm that such compound may act as potential telomerase inhibitors.

Molecular basis and quantitative assessment of TRF1 and TRF2 protein interactions with TIN2 and Apollo peptides

Shelterin is a six-protein complex (TRF1, TRF2, POT1, RAP1, TIN2, and TPP1) that also functions in smaller subsets in regulation and protection of human telomeres. Two closely related proteins, TRF1 and TRF2, make high-affinity contact directly with double-stranded telomeric DNA and serve as a molecular platform. Protein TIN2 binds to TRF1 and TRF2 dimer-forming domains, whereas Apollo makes interaction only with TRF2. To elucidate the molecular basis of these interactions, we employed molecular dynamics (MD) simulations of TRF1TRFH-TIN2TBM and TRF2TRFH-TIN2TBM/ApolloTBM complexes and of the isolated proteins. MD enabled a structural and dynamical comparison of protein–peptide complexes including H-bond interactions and interfacial residues that may regulate TRF protein binding to the given peptides, especially focusing on interactions described in crystallographic data. Residues with a selective function in both TRF1TRFH and TRF2TRFH and forming a stable hydrogen bond network with TIN2TBM or ApolloTBM peptides were traced. Our study revealed that TIN2TBM forms a well-defined binding mode with TRF1TRFH as compared to TRF2TRFH, and that the binding pocket of TIN2TBM is deeper for TRF2TRFH protein than ApolloTBM. The MD data provide a basis for the reinterpretation of mutational data obtained in crystallographic work for the TRF proteins. Together, the previously determined X-ray structure and our MD provide a detailed view of the TRF–peptide binding mode and the structure of TRF1/2 binding pockets. Particular TRF–peptide interactions are very specific for the formation of each protein–peptide complex, identifying TRF proteins as potential targets for the design of inhibitors/drugs modulating telomere machinery for anticancer therapy.

Identification of 1H-indene-(1,3,5,6)-tetrol derivatives as potent pancreatic lipase inhibitors using molecular docking and molecular dynamics approach

Pancreatic lipase is a potential therapeutic target to treat diet‐induced obesity in humans, as obesity‐related diseases continue to be a global problem. Despite intensive research on finding potential inhibitors, very few compounds have been introduced to clinical studies. In this work, new chemical scaffold 1H‐indene‐(1,3,5,6)‐tetrol was proposed using knowledge‐based approach, and 36 inhibitors were derived by modifying its functional groups at different positions in scaffold. To explore binding affinity and interactions of ligands with protein, CDOCKER and AutoDock programs were used for molecular docking studies. Analyzing results of rigid and flexible docking algorithms, inhibitors C_12, C_24, and C_36 were selected based on different properties and high predicted binding affinities for further analysis. These three inhibitors have different moieties placed at different functional groups in scaffold, and to characterize structural rationales for inhibitory activities of compounds, molecular dynamics simulations were performed (500 nSec). It has been shown through simulations that two structural fragments (indene and indole) in inhibitor can be treated as isosteric structures and their position at binding cleft can be replaced by each other. Taking into account these information, two lines of inhibitors can further be developed, each line based on a different core scaffold, that is, indene/indole.

SiMiSnoRNA: Collection of siRNA, miRNA, and snoRNA database for RNA interference / SiMiSnoRNA: RNA Interferansı için siRNA, miRNA ve snoRNA veritabanında depolanan siRNA, miRNA, and snoRNA koleksiyonları

Abstract Objective: The discovery of sequence specific gene silencing which occurs due to the presence of double- stranded RNAs has considerable impact on biology, revealing an unknown level of regulation of gene expression. This process is known as RNA interference (RNAi) or RNA silencing in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecule. Two types of small RNA molecules-small interfering RNA (siRNA) and microRNA (miRNA) are central to RNA interference. Therefore, SiMiSnoRNA database has been developed to focus on the efficient storage of RNA interferences or small RNA sequences and qualitative analysis of its structures across variety of organisms. Methods: SiMiSnoRNA database is developed using WAMP server which implements Apache, MySQL, and PHPas principal components. Results: A flexible web-based search engine is developed to obtain fast access to specific small RNA sequence information. Conclusion: BLAST search analysis within SiMiSnoRNA enables users to compare their own query sequence data with SiMiSnoRNA database to retrieve related information. To facilitate data consistency, publicly available information from NCBI has been integratedinto database that can be conveniently used for research on the experimental and molecular biology. Özet Amaç: Çift sarmallı RNAların varlığı nedeniyle gerçekleşen sekansa özel gen sessizliğinin keşfi biyoloji biliminde büyük etki yarattı, böylelikle gen ekspresyonunun düzenlendiği, daha önce bilinmeyen yeni bir düzey açığa çıktı. Bu proses, RNA moleküllerinin, belli mRNA molekülünün yıkımına yol açarak gen ekspresyonunu engellediği RNA enterferansı (RNAi) veya RNA sessizleştirmesi olarak biliniyor. RNA enterferansının merkezinde iki tip RNA molekülü bulunuyor; küçük enterferans RNA (siRNA) ve mikroRNA (miRNA). Bu nedenle, RNA enterferansları ve küçük RNA sekanslarının etkin biçimde depolanmasına ve çeşitli organizmalardaki yapısının nicelikli analizine odaklanan SiMiSnoRNA veritabanı oluşturuldu. Metod: SiMiSnoRNA veritabanı Apache, MySQL, ve PHP kullanılan bir WAMP server üzerinde oluşturuldu. Bulgular: Belli küçük RNA sekansları ile ilgili bilgiye hızlı erişim sağlayabilmek için esnek ve web tabanlı bir arama motoru geliştirildi. Sonuç: SiMiSnoRNA sisteminin BLAST arama motoru sayesinde kullanıcılar, hakkında bilgi almak istedikleri sekans verisini girerek SiMiSnoRNA veritabanında arama yapabilecek. Veri doğrulamasını kolaylaştırmak için NCBI’daki kamuya açık bilgi veri tabanına entegre edilmiştir ve deneysel ve moleküler biyoloji dallarında araştırmalar için de kolaylıkla kullanılabilir

Extracting functional groups of ALLINI to design derivatives of FDA‐approved drugs: Inhibition of HIV‐1 integrase

HIV‐1 integrase (IN) is crucial for integration of viral DNA into the host genome and a promising target in development of antiretroviral inhibitors. In this work, six new compounds were designed by linking the structures of two different class of HIV‐1 IN inhibitors (active site binders and allosteric IN inhibitors (ALLINIs)). Among newly designed compounds, INRAT10b was found most potent HIV‐1 IN inhibitor considering different docking results. To further validate protein–ligand interactions obtained from dockings, molecular dynamics simulations were performed for inhibitor raltegravir and INRAT10b placed either at active site or allosteric site of HIV‐1 IN (monomer or dimer). Results suggest that both raltegravir and INRAT10b were interacting with residue Gln62, Gly140, Ile141, and Ser147. However, INRAT10b interacts better with high H‐bond occupancy, which can explain the strong binding affinity of INRAT10b than raltegravir with the HIV‐1 IN protein. Subdomains rearrangements in HIV‐1 IN suggest that the C‐terminal and catalytic core domains develop their closeness in the presence of ligand. More significantly, the newly designed derivatives represent novel compounds targeting catalytic site and C‐terminal (protein–protein interaction) domains simultaneously. And we also propose INRAT10b as a promising lead compound for the development of potent HIV‐1 IN inhibitors.

The structurally similar TRFH domain of TRF1 and TRF2 dimers shows distinct behaviour towards TIN2

The telomere repeat binding-factor 1 and 2 (TRF1 and TRF2) proteins of the shelterin complex bind to duplex telomeric DNA as homodimers, and the homodimerization is mediated by their TRFH (TRF-homology) domains. We performed molecular dynamic (MD) simulations of the dimer forms of TRF1TRFH and TRF2TRFH in the presence/absence of the TIN2TBM (TIN2, TRF-interacting nuclear protein 2, TBM, TRF-binding motif) peptide. The MD results suggest that TIN2TBM is necessary to ensure the stability of TRF1TRFH homodimer but not the TRF2TRFH homodimer. In TRF1-TIN2-TRF2 complex, the peptide enhances the protein-protein interactions to yield a stable heterodimer. Both monomers in TRF1TRFH homodimer interact almost equally with the peptide, whereas in TRF2TRFH homodimer, monomer TRF2TRFH(M1) exhibits more dominant interactions than the TRF2TRFH(M2). The common residues of TRF1/2TRFH(M1) that form interactions with TIN2TBM in all peptide-bound systems originate from the H3 (helix) and L3 (loop) regions. Additionally, in the homodimer systems, residues of TRF1/2TRFH(M2) also interact with the peptide. The residue pair E71-K213 is responsible for different conformations of TRF1TRFH homodimers; specifically, this residue pair enhances the protein-peptide/protein interactions in peptide-bound/unbound systems, respectively. TRF1TRFH and TRF2TRFH proteins have a conserved but different interface responsible for the protein-protein/peptide interactions that exist in the corresponding dimers.

Comparative molecular dynamics study of dimeric and monomeric forms of HIV-1 protease in ligand bound and unbound state

Human immunodeficiency virus type 1 protease is a viral-encoded enzyme and it is essential for replication and assembly of the virus. Inactivation of HIV-1 protease causes production of immature, noninfectious viral particles and thus HIV-1 protease is an attractive target in anti-AIDS drug design. In our current work, we performed molecular dynamics (MD) calculations (500 ns) for two different ligands (COM5 - designed in our previous study, and Darunavir) and made effort to understand dynamics behaviour of our designed compound COM5. An apo form of HIV-1 protease as monomer and dimer form was also studied in order to analyze response of protein to the ligand. MD results suggest that presence of ligand in hinders the stability of HIV-1 protease and one monomer from dimer systems is dominant on other monomer in terms of interaction made with ligands. We were able to trace functional residues as well as continuous motion of opening and closing (clapping) of flap region in HIV-1 protease (apo form) during entire 1000 ns of MD simulation. COM5 showed almost similar behaviour towards HIV-1 protease enzyme as Darunavir and propose as promising lead compound for the development of new inhibitor for HIV-1 protease.

Molecular basis and potential activity of HIV-1 reverse transcriptase towards triethylamine based compounds

Reverse transcriptase (RT) inhibitors are currently used to treat human immunodeficiency virus (HIV)‐1 infections. In this work, novel triethylamine derivatives were designed and studied by rigid and flexible docking and molecular dynamics (MD) approaches. An apo form of HIV‐1 RT was also studied by MD simulation to analyze comparative response of protein in ligand‐bound and ligand‐unbound forms. Among newly designed HIV‐1 RT inhibitors, compound HIV104 was the most potent inhibitor considering different docking results. Molecular docking results were further validated by MD simulations of an HIV‐1 RT/HIV104 complex using two independent software (Discovery Studio Client 3.1 and GROMACS) to perform comparative analysis. Results suggest that hydroxyl and carboxyl groups present at –R1 position in compounds favored strong H‐bond contacts as well as good interaction energy profile. Our MD results are consistent with the observations that conformational dynamics between the thumb and finger subdomains of HIV‐1 RT controls its dynamics on substrate binding and subsequent activity. MD studies of HIV‐1 RT/HIV104 provide insight into interrelatedness of residue scale interactions and global conformational change and also hint at the complex nature of allosteric inhibition. Thus, the results obtained from this study facilitate the design of potent HIV‐1 RT inhibitors.