Computational identification and analysis of deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) in the human POR gene: a structural and functional impact.

Cytochrome P450 oxidoreductase (POR) protein is essential for steroidogenesis, and POR gene mutations are frequently associated with P450 Oxidoreductase Deficiency (PORD), a disorder of hormone production. To our knowledge, no previous attempt has been made to identify and analyze the deleterious/pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) in the human POR gene through an extensive computational approach. Computational algorithms and tools were employed to identify, characterize, and validate the pathogenic SNPs associated with certain diseases. To begin with, all the high-confidence SNPs were collected, and their structural and functional impacts on the protein structures were explored. The results of various in silico analyses affirm that the A287P and R457H variants of POR could destabilize the interactions between the amino acids and the hydrogen bond networks, resulting in functional deviations of POR. The literature study further confirms that the pathogenic mutations (A287P and R457H) are associated with the onset of PORD. Molecular dynamics simulations (MDS) and essential dynamics (ED) studies characterized the structural consequences of prioritized deleterious mutations, representing the structural destabilization that might disrupt POR biological function. The identified deleterious mutations at the cofactor's binding domains might interfere with the essential interactions between the protein and cofactors, thus inhibiting POR catalytic activity. The consolidated insights from the computational analyses can be used to predict potential deleterious mutants and understand the disease's pathological basis and the molecular mechanism of drug metabolism for the application of personalized medication. HIGHLIGHTSNADPH cytochrome P450 oxidoreductase (POR) mutations are associated with a broad spectrum of human diseasesIdentified and analyzed the most deleterious nsSNPs of POR through the sequence and structure-based prediction toolsInvestigated the structural and functional impacts of the most significant mutations (A287P and R457H) associated with PORDMolecular dynamics and PCA-based FEL analysis were utilized to probe the mutation-induced structural alterations in PORCommunicated by Ramaswamy H. Sarma.

Design of multi-epitope based vaccine against Mycobacterium tuberculosis: a subtractive proteomics and reverse vaccinology based immunoinformatics approach.

Tuberculosis is an airborne transmissible disease caused by Mycobacterium tuberculosis that infects millions of lives worldwide. There is still no single comprehensive therapy or preventative available for the lethal illness. Currently, the available vaccine, BCG is ineffectual in preventing the prophylactic adult pulmonary TB and reactivation of latent tuberculosis. Therefore, this investigation was intended to design a new multi-epitope vaccine that can address the existing problems. The subtractive proteomics approach was implemented to prioritize essential, virulence, druggable, and antigenic proteins as suitable vaccine candidates. Furthermore, a reverse vaccinology-based immunoinformatics technique was employed to identify potential B-cell, helper T lymphocytes (HTL), and cytotoxic T lymphocytes (CTL) epitopes from the target proteins. Immune-stimulating adjuvant, linkers, and PADRE (Pan HLA-DR epitopes) amino acid sequences along with the selected epitopes were used to construct a chimeric multi-epitope vaccine. The molecular docking and normal mode analysis (NMA) were carried out to evaluate the binding mode of the designed vaccine with different immunogenic receptors (MHC-I, MHC-II, and Tlr4). In addition, the MD simulation, followed by essential dynamics study and MMPBSA analysis, was carried out to understand the dynamics and stability of the complexes. In-silico cloning was accomplished using E.coli as an expression system to express the designed vaccine successfully. Finally, the immune simulation study has foreseen that our designed vaccine could induce a significant immune response by elevation of different immunoglobulins in the host. However, there is an imperative need for the experimental validation of the designed vaccine in animal models to confer effectiveness and safety.HIGHLIGHTSMulti-epitope based vaccine was designed against Mycobacterium tuberculosis using subtractive proteomics and Immunoinformatics approach.The vaccine was found to be antigenic, non-allergenic, immunogenic, and stable based on in-silico prediction.Population coverage analysis of the proposed vaccine predicts an effective response in the world population.The molecular docking, MD simulation, and MM-PBSA study confirm the stable interaction of the vaccine with immunogenic receptors.In silico cloning and immune simulation of the vaccine demonstrated its successful expression in E.coli and induction of immune response in the host. Communicated by Ramaswamy H. Sarma.

Pyridine appended 2-hydrazinylthiazole derivatives: design, synthesis, in vitro and in silico antimycobacterial studies.

An array of pyridine appended 2-hydrazinylthiazole derivatives has been synthesized to discover novel chemotherapeutic agents for Mycobacterium tuberculosis (Mtb). The drug-likeness of pyridine appended 2-hydrazinylthiazole derivatives was validated using the Lipinski and Veber rules. The designed thiazole molecules have been synthesized through Hantzsch thiazole methodologies. The in vitro antimycobacterial studies have been conducted using Luciferase reporter phage (LRP) assay. Out of thirty pyridine appended 2-hydrazinylthiazole derivatives, the compounds 2b, 3b, 5b, and 8b have exhibited good antimycobacterial activity against Mtb, an H37Rv strain with the minimum inhibitory concentration in the range of 6.40-7.14 µM. In addition, in vitro cytotoxicity of active molecules has been observed against Human Embryonic Kidney Cell lines (HEK293t) using MTT assay. The compounds 3b and 8b are nontoxic and their cell viability is 87% and 96.71% respectively. The in silico analyses of the pyridine appended 2-hydrazinylthiazole derivatives have been studied to find the mode of binding of the active compounds with KasA protein of Mtb. The active compounds showed a strong binding score (-5.27 to -6.23 kcal mol-1).

An integrated computational investigation to unveil the structural impacts of mutation on the InhA structural gene of Mycobacterium tuberculosis.

Growing concern about the difficulty in diagnosis and treatments of drug-resistant tuberculosis falls under the major global health issues. There is an urgent need for finding novel strategies to develop drugs or bioactive molecules against the global threat of Mycobacterium tuberculosis (MTB). Isoniazid (INH) is a front line drug against tuberculosis; it primarily targets the enoyl-acyl carrier protein reductase (InhA), a potent drug target in the mycolic acid pathway of MTB. To gain deeper insight into the impact of INH resistant mutation and its influence on the structural dynamics of InhA, combined conformational dynamics and residue interaction network (RIN) studies were performed. The molecular dynamics investigation provided a hint about the structural changes altering protein activity. The principal component analysis (PCA) based free energy landscape plot highlighted the highest stable part of wild-type (WT) and mutant structures. Intriguingly, the mutation at the 78th position of InhA from its native residue valine to alanine increases the structural stability with higher NADH binding affinity. The MM-PBSA based binding energy calculations confirm that electrostatic interactions played a critical role in the binding of NADH at the binding site of InhA. The calculated binding energy score, as well as potential hydrogen bonds and salt bridge networks, proved the strong binding of mutant InhA as compared to WT. Further, the mutation potentially altered the protein network topology, thereby subsequently affected the landscape of NADH binding. The present study is an attempt to understand the structural and functional impact associated with a drug-resistant mutation (V78A) thus it will be helpful in designing potent inhibitors against drug-resistant tuberculosis.

Insight into the structural and functional analysis of the impact of missense mutation on cytochrome P450 oxidoreductase.

Cytochrome P450 oxidoreductase (POR) is a steroidogenic and drug-metabolizing enzyme. It helps in the NADPH dependent transfer of electrons to cytochrome P450 (CYP) enzymes for their biological activity. In this study, we employed integrative computational approaches to decipher the impact of proline to leucine missense mutation at position 384 (P384L) in the connecting/hinge domain region which is essential for the catalytic activity of POR. Analysis of protein stability using DUET, MUpro, CUPSAT, I-Mutant2.0, iStable and SAAFEC servers predicted that mutation might alter the structural stability of POR. The significant conformational changes induced by the mutation to the POR structure were analyzed by long-range molecular dynamics simulation. The results revealed that missense mutation decreased the conformational stability of POR as compared to wild type (WT). The PCA based FEL analysis described the mutant-specific conformational alterations and dominant motions essential for the biological activity of POR. The LIGPLOT interaction analysis showed the different binding architecture of FMN, FAD, and NADPH as a result of mutation. The increased number of hydrogen bonds in the FEL conformation of WT proved the strong binding of cofactors in the binding pocket as compared to the mutant. The porcupine plot analysis associated with cross-correlation analysis depicted the high-intensity flexible motion exhibited by functionally important FAD and NADPH binding domain regions. The computational findings unravel the impact of mutation at the structural level, which could be helpful in understanding the molecular mechanism of drug metabolism.

Structural insight into conformational dynamics of non-active site mutations in KasA: A Mycobacterium tuberculosis target protein.

The enzyme ß-Ketoacyl ACP synthase I (KasA) is a potent drug target in mycolic acid pathway of Mycobacterium tuberculosis (Mtb). In the present study, we investigated the structural dynamics of wild-type (WT) and mutants KasA (D66N, G269S, G312S, and F413L) in both monomer and dimer form to provide insight into protein structural stability. To gain better understanding of structural flexibility of KasA, combined molecular dynamics and essential dynamics were employed to analyze the conformational changes induced by non-active site mutations. The results confirm that non-active site mutations lower the structural stability in dimer KasA as compared to WT. The protein network topology and close residue interactions of WT and mutant residues of KasA have been predicted through residue interaction network analysis (RIN). Non-active site mutations distort RIN architecture and subsequently affect the drug binding landscape. T-pad associated with mode vector analysis comprehensively pronounces the structural impact caused by non-active site mutations. It also identified the critical fluctuating residues present in the gate segment (GS) region (115-147). The non-active site mutations altered the structural stability of the mutant protein structures, and these mutations may be a cause for the resistance mechanism of KasA against anti-tuberculosis drugs. Further, it is observed that dimer mutant KasA proteins display much more structural flexibility than WT at the ligand binding site which is evident from the binding site analysis and hydrogen bond interaction patterns. This study provides a better understanding of the structural dynamic behaviour of KasA mutants, thereby facilitating the need to find a novel and potent inhibitor against Mtb.

Purification, Characterization of Alkaline Cold Active Lipase from Acinetobacter radioresistens PR8 and Development of a New Zymography Method for Lipase Detection.

BACKGROUND: Bacterial lipases find so many industrial applications. Therefore, new source of lipase suitable for industrial conditions is always required. Lipase zymography methods use costly chromogenic substrates and indicator dyes and are few in numbers. OBJECTIVE: The objectives of this work include lipase purification and its characterization from Acinetobacter radioresiens PR8 and development of new zymography method for lipase detection. METHOD: The lipase was purified using conventional method and cation exchange chromatography and it was characterized biochemically and analytically. Based on these characterization new in-gel lipase zymography method was developed. RESULTS: In this present work, an alkalophilic lipase producing bacterium was isolated from soil; screened for extracellular lipase activity and identified to be Acinetobacter radioresistens PR8 (Genbank accession ID: MF073322). Enzyme production kinetics showed maximum production (4.16 U/ml at pH 9) of enzyme after 72 h. The lipase activity was found to be highest in olive oil (1% v/v; 8.1 U/ml). Low molecular weight (27 kDa) alkaline (pH 9) cold active (20 °C) lipase was purified from Acinetobacter radioresistens PR8. Lipase was characterized using PMF, FT-IR and its high conformational stability (Transition temperature: 122.3 °C) was attributed from its DSC spectrum. The importance of magnesium and sodium ions for enhancing lipase activity was obtained from flux balance analysis. CONCLUSION: Based on the lipase activating role of Mn2+ and Na+ ions, optimum temperature, pH with no chromogenic substrates and indicator dyes, a new in gel zymography method for lipase detection was developed.

Molecular interaction of 2,4-diacetylphloroglucinol (DAPG) with human serum albumin (HSA): The spectroscopic, calorimetric and computational investigation.

Drug molecule interaction with human serum albumin (HSA) affects the distribution and elimination of the drug. The compound, 2,4-diacetylphloroglucinol (DAPG) has been known for its antimicrobial, antiviral, antihelminthic and anticancer properties. However, its interaction with HSA is not yet reported. In this study, the interaction between HSA and DAPG was investigated through steady-state fluorescence, time-resolved fluorescence (TRF), circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy, isothermal titration calorimetry (ITC), molecular docking and molecular dynamics simulation (MDS). Fluorescence spectroscopy results showed the strong quenching of intrinsic fluorescence of HSA due to interaction with DAPG, through dynamic quenching mechanism. The compound bound to HSA with reversible and moderate affinity which explained its easy diffusion from circulatory system to target tissue. The thermodynamic parameters from fluorescence spectroscopic data clearly revealed the contribution of hydrophobic forces but, the role of hydrogen bonds was not negligible according to the ITC studies. The interaction was exothermic and spontaneous in nature. Binding with DAPG reduced the helical content of protein suggesting the unfolding of HSA. Site marker fluorescence experiments revealed the change in binding constant of DAPG in the presence of site I (warfarin) but not site II marker (ibuprofen) which confirmed that the DAPG bound to site I. ITC experiments also supported this as site I marker could not bind to HSA-DAPG complex while site II marker was accommodated in the complex. In silico studies further showed the lowest binding affinity and more stability of DAPG in site I than in site II. Thus the data presented in this study confirms the binding of DAPG to the site I of HSA which may help in further understanding of pharmacokinetic properties of DAPG.

One-Pot Synthesis of Densely Substituted Pyrazolo[3,4-b]-4,7-dihydropyridines.

We have achieved a facile synthesis of a combinatorial library of densely substituted pyrazolo[3,4-b]-4,7-dihydropyridines- the mimics of antigenital wart drug podophyllotoxin-from 5-aminopyrazoles and 4-(methylthio) 4H-chromenes. The C(4) pyrazolyl 4H-chromenes, which also possess structural features of podophyllotoxin, were isolable intermediates in the two-step, one-pot condensation. The condensation took place in a one-pot, multicomponent manner when 3-oxo-3-phenylpropanenitriles, hydrazine (precursors for 5-aminopyrazoles) and 4-(methylthio)-4H-chromenes were heated in refluxing ethanol. The condensation, however, stops at 4H-chromene stage when methyl hydrazine or phenylhydrazine were employed. Our findings offer an opportunity for synthesis of a combinatorial library of podophyllotoxin mimics, thus paving the way for discovery of lead candidates for cancer treatment.

Structural insights into tumor-specific chaperoning activity of gamma synuclein in protecting estrogen receptor alpha 36 and its role in tamoxifen resistance in breast cancer.

Gamma synuclein (γSyn), a tumor-specific molecular chaperone, protects Hsp90 client proteins like ERα36 and stimulates rapid membrane-initiated estrogen signalling in breast cancer cells. However, the structural perspectives of this tumor-specific chaperone function of γSyn remains unclear. Hence, in this present work, we studied the conformational dynamics of ERα36 in the absence and presence of Hsp90 and γSyn. Results indicate that in a chaperone-free state, ERα36 undergoes an inter-domain movement and exposes the hydrophobic patch of residues that are responsible for binding with ubiquitin. However, independent of Hsp90, γSyn, by establishing transient interactions, prevents interdomain movement, unveils the co-activator binding groove, masks the ubiquitin-binding residues and maintains 'open' pocket conformation of LBD. By doing so, γSyn effectively protects ERα36 from degradation and maintains its functional state like Hsp90 based chaperoning machinery but independent of ATP. Our studies also show that the γSyn protected conformation of ERα36 can effectively bind with both estradiol (E2) and 4-hydroxy tamoxifen (4-OHT). Although they exhibit unique binding modes, they maintained the functionally active conformation of ERα36. Interestingly, the molecular dynamics simulation studies showed that 4-OHT, like γSyn, prevented the interdomain movements, primes the co-activator binding groove of ERα36 for complexation with downstream signalling proteins and this mechanism explains its agonist activity and associated anti-estrogen resistance observed in the presence of ERα36. The observed differences in the chaperoning mechanism of γSyn sheds light on its selectivity over Hsp90 in cancer cells, for promoting rapid protection of crucial oncogenic proteins. Based on our findings, we speculate that the compounds, which can hamper association of γSyn with ERα36 and/or can arrest ERα36 in an ubiquitin binding state, would be promising alternatives for treating ERα36 expressed breast carcinomas.

Structural perspective of ARHI mediated inhibition of STAT3 signaling: an insight into the inactive to active transition of ARHI and its interaction with STAT3 and importinß.

ARHI, a putative tumor suppressor protein with unique 32 amino acid extension in the N-terminal region, differs from oncogenes Ras and Rap, negatively regulates STAT3 signaling and inhibits the migration of ovarian cancer cells. ARHI associates directly with STAT3, also forms complex with importinß, and prevents formation of RanGTPase-importinß complex, which is essential for transporting STAT3 into the nucleus. Hence, the structural aspects pertaining to ARHI mediated inhibition of STAT3 translocation can provide hints on the regulation of STAT3 signaling mechanism. Accordingly, in the present study, the structure of ARHI was predicted and its transition from inactive to active state studied using MD simulations and free energy landscape analysis. The transition of ARHI is marked by the movement of switch I region towards γ-phosphate of GTP, in addition, the hydrophobic interaction between N-terminal helix and switch II region of ARHI accounts for its low intrinsic GTPase activity. Further, the protein-protein interaction studies reveal that the residues of N-terminal helix, effector domain, P-loop and G box motif of ARHI actively form polar and non-polar interaction with NTD of STAT3 and make them compact thereby rendering STAT3 inaccessible for Ran-importinß mediated translocation. On the other hand, ARHI competes with RanGTPase and interacts with importinß via basic-acidic patch interaction, which leads to inhibition of STAT3 translocation. The interacting residues involved for this structural mechanism would be instrumental in designing inhibitors for STAT3, which mimics ARHI thereby leading to the suppression of cancer cell growth.

Exploring the structural features of Aspartate Trans Carbamoylase (TtATCase) from Thermus thermophilus HB8 through in silico approaches: a potential drug target for inborn error of pyrimidine metabolism.

Enzymes involved in the pyrimidine biosynthesis pathway have become an important target for the pharmacological intervention. One among those enzymes, Aspartate Trans Carbamoylase (ATCase), catalyses the condensation of aspartate and carbamoyl phosphate to form N-carbamoyl-l-aspartate and inorganic phosphate. The present study provides the molecular insights into the enzyme ATCase. The three-dimensional structure of ATCase from Thermus thermophilus HB8 was modeled based on the crystal structure of ATCase in Pyrococcus abyssi (PDB ID:1ML4). Molecular dynamics simulation was performed to identify the conformational stability of TtATCase with and without its ligand complexes. Based on the pharmacokinetic properties and the glide-docking scores of ligands from four databases (Maybridge, Binding, Asinex and Technology for Organic Synthesis (TOS laboratory) for the screening of ligands, we identified four potential ligand molecules for TtATCase. From the molecular docking results, we proposed that the residues Thr53, Arg104, and Gln219 are consistently involved in strong hydrogen-bonding interactions and play a vital role in the TtATCase activity. From the results of molecular dynamics simulation, the ligand molecules are found to bind appropriately to the target enzyme. However, the structure of TtATCase needs to be determined experimentally to confirm this.

Molecular dynamic simulations of the tubulin-human gamma synuclein complex: structural insight into the regulatory mechanism involved in inducing resistance against Taxol.

Members of the synuclein family (α, ß and γ synucleins) are intrinsically disordered in nature and play a crucial role in the progression of various neurodegenerative disorders and cancers. The association of γSyn with both BubR1 as well as microtubule subunits renders resistance against various anti-cancer drugs. However, the structural aspects underlying drug resistance have not been explored. In this study, the mechanism involved in the association between γSyn and microtubule subunits (αßTub) was investigated and the results reveal a strong interaction between γSyn and the tail regions of αßTub. Complexation of γSyn induces conformational rearrangements in the nucleotide binding loops (NBL), interdomain and tail regions of both α and ßTub. Moreover, in ßTub, the massive displacement observed in M and S loops significantly alters the binding site of microtubule targeting drugs like Taxol. The resulting weak association between Taxol and ßTub of the γSyn-αßTub complex was confirmed by molecular dynamic simulation studies. In addition, the effect of Taxol on NBL, M and S loops of αßTub, is reversed in the presence of γSyn. These results clearly indicate that the presence of γSyn annulled the allosteric regulation imposed by Taxol on the αßTub complex as well as preventing the binding of microtubule targeting drugs, which eventually leads to the development of resistance against these drugs in cancer cells.

Structural insights into interacting mechanism of ID1 protein with an antagonist ID1/3-PA7 and agonist ETS-1 in treatment of ovarian cancer: molecular docking and dynamics studies.

Among the many abnormally expressed proteins in ovarian cancer, the prominent cancer in women, ID1 (inhibitors of DNA binding protein 1) is a potential one among other several targets. Interaction of ID1 with ETS-1 (transcriptional activator of p16(INK4a)) suppresses the transcription of p16(INK4a) and causes abnormal cell proliferation. A peptide aptamer (ID1/3-PA7) has been designed to prevent this interaction and thereby leading to the transcription of p16(INK4a). However, the structural basis behind the molecular interaction of ID1 with ETS-1 (agonist) and ID1/3-PA7 (antagonist) is poorly understood. In order to understand this structural recognition and their interaction mechanism, in silico methods were used. From this interaction analysis, the residues of ETS-1 involved in interaction with the p16(INK4a) promoter were found to be targeted by ID1. Subsequently, ETS-1 binding residues of ID1 were found to be targeted by its aptamer- ID1/3-PA7. These results suggest that both ETS-1 and ID1/3-PA7 binds at the same region harbored by the residues-H97, D100, R103, D104, L107, A144, C145, D149, D150 and C154 of ID1. All these observations correlate with the experimental reports, suggesting that the identified residues might play a crucial role in promulgating the oncogenic effects of ID1. In silico alanine scanning mutagenesis also confirms the role of identified hot spot residues in p16(INK4a) regulation. Finally, the molecular dynamic simulation studies reveal the prolonged stability of the aforementioned interacting complexes. The obtained results throw light on the structure and residues of ID1 involved in transcriptional regulation of p16(INK4a).

Molecular dynamics simulation of the Staphylococcus aureus YsxC protein: molecular insights into ribosome assembly and allosteric inhibition of the protein.

YsxC from Staphylococcus aureus is a member of the GTPase protein family, and is involved in the ribosomal assembly and stability of this microorganism through its interactions with the L17, S2 and S10 ribosomal proteins. Inhibition of its interactions with L17, S2, S10 and the ß' subunit of RNA polymerase influences ribosomal assembly, which may affect the growth of the microorganism. This makes YsxC a novel target for the design of inhibitors to treat the disease caused by S. aureus. Understanding the interaction mechanism between YsxC and its partners would aid in the identification of potential catalytic residues, which could then be targeted to inhibit its function. Accordingly, in the present study, an in silico analysis of the interactions between YsxC and L17, S2 and S10 was performed, and the potential residues involved in these interactions were identified. Based on the simulation results, a possible mechanism for the interactions between these proteins was also proposed. Finally, six ligands from among a library of 81,000 chemical molecules were found to interact with parts of the G2 and switch II regions of the YsxC protein. Moreover, their interactions with the YsxC protein were observed to provoke changes at its GTP-binding site, which suggests that the binding of these ligands leads to a reduction in GTPase activity, and they were also found to affect the interactions of YsxC with its partners. This observation indicates that the proposed interacting site of YsxC may act as an allosteric site, and disrupting interactions at this site might lead to novel allosteric inhibition of the YsxC protein.