Mechanistic insights into the conformational changes and alterations in residual communications due to the mutations in the pncA Gene of Mycobacterium tuberculosis: A computational perspective for effective therapeutic solutions.

Due to its emerging resistance to first-line anti-TB medications, tuberculosis (TB) is one of the most contagious illness in the world. According to reports, the effectiveness of treating TB is severely impacted by drug resistance, notably resistance caused by mutations in the pncA gene-encoded pyrazinamidase (PZase) to the front-line drug pyrazinamide (PZA). The present study focused on investigating the resistance mechanism caused by the mutations D12N, T47A, and H137R to better understand the structural and molecular events responsible for the resistance acquired by the pncA gene of Mycobacterium tuberculosis (MTB) at the structural level. Bioinformatics analysis predicted that all three mutations were deleterious and located near the active centre of the pncA, affecting its functional activity. Furthermore, molecular dynamics simulation (MDS) results established that mutations significantly reduced the structural stability and caused the rearrangement of FE2+ in the active centre of pncA. Moreover, essential dynamics analysis, including principal component analysis (PCA) and free energy landscape (FEL), concluded variations in the protein motion and decreased conformational space in the mutants. Additionally, the mutations potentially impacted the network topologies and altered the residual communications in the network. The complex simulation study results established the significant movement of the flap region from the active centre of mutant complexes, further supporting the flap region's significance in developing resistance to the PZA drug. This study advances our knowledge of the primary cause of the mechanism of PZA resistance and the structural dynamics of pncA mutants, which will help us to design new and potent chemical scaffolds to treat drug-resistant TB (DR-TB).

Biochemical and biophysical characterization of biosynthetic arginine decarboxylase from Thermus thermophilus.

The biosynthetic arginine decarboxylase in Thermus thermophilus is responsible for producing spermidine, a polyamine with numerous biological applications in humans. The arginine decarboxylase has significant applications in biotechnology industries, suggesting the need to evaluate its biochemical and biophysical characteristics at the molecular level. In this study, both in vitro and in silico methods were employed to investigate the structural and functional behavior of the arginine decarboxylase protein. In in vitro, MALDI-TOF, size exclusion, and assay studies were performed to examine the nature and activity of the protein. The MALDI-TOF analysis confirmed the purified protein as biosynthetic arginine decarboxylase. The assay results revealed that the Pyridoxal 5'-Phosphate (PLP) cofactor plays a crucial role in enhancing enzyme activity by producing agmatine (a by-product of spermidine). Further, optimum enzyme activity was observed at 50 °C, suggesting the extremophilic nature of the enzyme. Unlike other proteins, this enzyme displayed optimal activity at both acidic and basic pH, demonstrating its sensitivity to pH changes. Furthermore, the addition of divalent ions like Mg 2+ increased the rate of reaction. In in silico, structure modeling, and comparative molecular dynamics simulation studies were used to assess the protein stability and behavior at different pH and temperature conditions. The findings of this study could be applied to improve enzyme production in the industry.Communicated by Ramaswamy H. Sarma.

Computational insights into potential marine natural products as selective inhibitors of Mycobacterium tuberculosis InhA: A structure-based virtual screening study.

Several factors are associated with the emergence of drug resistance mechanisms, such as impermeable cell walls, gene mutations, and drug efflux systems. Consequently, bacteria acquire resistance, leading to a decrease in drug efficacy. A new and innovative strategy is required to combat drug resistance in tuberculosis (TB) effectively. Therefore, targeting the mycolic acid biosynthesis pathway, which is involved in synthesising mycolic acids (MAs), essential structural components responsible for mycobacterial pathogenicity, has garnered interest in TB research and the concept of drug resistance. In this context, InhA, which plays a crucial role in the fatty acid synthase-II (FAS-II) system of the MA biosynthetic pathway, was selected as a druggable target for screening investigation. To identify potential lead molecules against InhA, diverse marine natural products (MNPs) were collected from the comprehensive marine natural products database (CMNPD). Virtual screening studies aided in selecting potential lead molecules that best fit within the substrate-binding pocket (SBP) of InhA, forming crucial hydrogen bond interaction with the catalytic residue Tyr158. Three MNPs, CMNPD30814, CMNPD1702, and CMNPD27355, were chosen as prospective alternative molecules due to their favorable pharmacokinetic properties and lack of toxicity according to ProTox-II predictions. Additionally, improved reactivity of the MNPs was observed in the results of density functional theory (DFT) studies. Furthermore, comparative molecular dynamics simulation (MDS), principal component (PC)-based free energy landscape (FEL) analysis, and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) were employed to show enhanced structural stability, increased H-bond potential, and high binding affinity toward the target InhA. Moreover, the hot spot residues that contributed to the high binding energy profile and anchored the stability of the complexes were revealed with their individual interaction energy. The computational insights from this study provide potential avenues to combat TB through the multifaceted mode of action of these marine lead molecules, which can be further explored in future experimental investigations.

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.

Computational identification and molecular dynamics simulation of potential circularRNA derived peptide from gene expression profile of Rheumatoid arthritis, Alzheimer's disease, and Atrial fibrillation.

The two most serious global health challenges confronting human society today are autoimmune disorders (AIDs) and neurological diseases (NDs), both of which shorten people's lives and worsen the situation. Despite their extensive impact, statistics show that AIDs is associated with a higher risk of ND. Circular RNAs (circRNAs) are critical in several illnesses and disorders, especially AID and ND. Therefore, the present study focused on understanding the underlying causes of the pathophysiology of diseases such as AID and ND through in silico-based research. In order to determine how circRNAs are related to various disease pathways, this study examined the gene expression data sets for Rheumatoid arthritis (RA), Alzheimer's disease (AD), and atrial fibrillation (AF). Our study identified and analyzed two circRNAs, their respective host genes (DHTKD1 and RAN) and their related miRNAs, which could serve as potential markers for treating disorders like myotonic dystrophy type 1, spinocerebellar ataxia and fragile X syndrome. Further, the circRNA-derived peptide was identified and analysed with the molecular dynamics simulation (MDS) followed by a principal component (PC) based free energy landscape (FEL) profile. The computational results obtained here provide a basis for the development of therapeutics against AD, RA and AF. Moreover, further functional studies are needed to validate their role in disease aetiology and to provide a detailed understanding of their association with AID and ND.Communicated by Ramaswamy H. Sarma.

HighlightsSignificant circular RNAs associated with autoimmune disorders and neurological diseases are identifiedIdentified circular RNAs and their host genes (DHTKD1 and RAN) impacted multiple disease pathwaysThe stability of circularRNA-derived peptide was checked with MD simulation followed by PCA-based FEL analysisThe potential of circular RNAs as biomarkers or therapeutic targets for human diseases was highlighted.

Differential gene expression analysis combined with molecular dynamics simulation study to elucidate the novel potential biomarker involved in pulmonary TB.

Tuberculosis (TB) is a lethal multisystem disease that attacks the lungs' first line of defense. A substantial threat to public health and a primary cause of death is pulmonary TB. This study aimed to identify and investigate the probable differentially expressed genes (DEGs) primarily involved in Pulmonary TB. Accordingly, three independent gene expression data sets, numbered GSE139825, GSE139871, and GSE54992, were utilized for this purpose. The identified DEGs were used for bioinformatics-based analysis, including physical gene interaction, Gene Ontology (GO), network analysis and pathway studies using the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG). The computational analysis predicted that TNFAIP6 is the significant DEG in the gene expression profiling of TB datasets. According to gene ontology analysis, TNFAIP6 is also essential in injury and inflammation. Further, TNFA1P6 is strongly linked to arsenic poisoning, evident from the results of NetworkAnalyst, a comprehensive and interactive platform for gene expression profiling via network visual analytics. As a result, the TNFAIP6 gene was ultimately chosen as a candidate DEG and subsequently employed for in silico structural characterization studies. The tertiary structure of TNFAIP6 was modelled using the ROBETTA server, followed by validation with SAVES and ProSA webserver. Additionally, structural dynamic studies, including molecular dynamics simulation (MDS) and essential dynamics analysis, including principal component (PC) based free energy landscape (FEL) analysis, was used for checking the stability of TNFAIP6 models. The dynamics result established the structural rigidity of modelled TNFAIP6 through RMSD, RMSF and RoG results. The FEL analysis revealed the restricted conformational flexibility of TNFAIP6 by displaying a single minimum energy basin in the contour plot. The comprehensive computational analysis established that TNFAIP6 could serve as a viable biomarker to assess the severity of pulmonary TB.

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.

Molecular docking and simulation studies of gustatory receptor of Aedes aegypti: A potent drug target to distract host-seeking behaviour in mosquitoes.

BACKGROUND & OBJECTIVES: It is well reported that exhaled CO 2 and skin odour from human being assist female mosquitoes to locate human host. Basically, the receptors for this activity are expressed in cpA neurons. In both Aedes aegypti and Anopheles gambiae, this CO 2-sensitive olfactory neuron detects myriad number of chemicals present in human skin. Therefore, manipulation of gustatory receptors housing these neurons may serve as important targets for behavioural intervention. The study was aimed towards virtual screening of small molecules in the analyzed conserved active site residues of gustatory receptor and molecular dynamics simulation study of optimum protein-ligand complex to identify a suitable lead molecule for distracting host-seeking behaviour of mosquitoes. METHODS: The conserved residue analysis of gustatory receptor (GR) of Ae. aegypti and An. gambiae was performed. The structure of GR protein from Ae. aegypti was modeled and validated, and then molecular docking was performed to screen 2903 small molecules against the predicted active residues of GR. Further, simulation studies were also carried out to prove protein-ligand stability. RESULTS: The glutamine 154 residue of GR was found to be highly conserved in Ae. aegypti and An. gambiae. Docking results indicated that the dodecanoic acid, 1,2,3-propanetriyl ester (dynasan 112) was interacting with this residue, as it showed better LibDock score than previously reported ethyl acetate used as mosquito repellant. Simulation studies indicated the structural instability of GR protein in docked form with dynasan 112 suggesting its involvement in structural changes. Based on the interaction energies and stability, this compound has been proposed to be used in mosquitoes' repellant. INTERPRETATION & CONCLUSION: A novel effective odorant acting as inhibitor of GR is proposed based on its stability, docking score, interactions and RMSD, considering ethyl pyruvate as a standard inhibitor. Host preference and host-seeking ability of mosquito vectors play key roles in disease transmission, a clear understanding of these aspects is essential for preventing the spread of the disease.

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.