The Salmonella typhi Cell Division Activator Protein StCAP Impacts Pathogenesis by Influencing Critical Molecular Events.

Conserved molecular signatures in multidrug-resistant Salmonella typhi can serve as novel therapeutic targets for mitigation of infection. In this regard, we present the S. typhi cell division activator protein (StCAP) as a conserved target across S. typhi variants. From in silico and fluorimetric assessments, we found that StCAP is a DNA-binding protein. Replacement of the identified DNA-interacting residue Arg34 of StCAP with Ala34 showed a dramatic (15-fold) increase in Kd value compared to the wild type (Kd 546 nm) as well as a decrease in thermal stability (10 °C shift). Out of the two screened molecules against the DNA-binding pocket of StCAP, eltrombopag, and nilotinib, the former displayed better binding. Eltrombopag inhibited the stand-alone S. typhi culture with an IC50 of 38 µM. The effect was much more pronounced on THP-1-derived macrophages (T1Mac) infected with S. typhi where colony formation was severely hindered with IC50 reduced further to 10 µM. Apoptotic protease activating factor1 (Apaf1), a key molecule for intrinsic apoptosis, was identified as an StCAP-interacting partner by pull-down assay against T1Mac. Further, StCAP-transfected T1Mac showed a significant increase in LC3 II (autophagy marker) expression and downregulation of caspase 3 protein. From these experiments, we conclude that StCAP provides a crucial survival advantage to S. typhi during infection, thereby making it a potent alternative therapeutic target.

Spike Protein Mutation-Induced Changes in the Kinetic and Thermodynamic Behavior of Its Receptor Binding Domains Explain Their Higher Propensity to Attain Open States in SARS-CoV-2 Variants of Concern.

Spike (S) protein opening in SARS-CoV-2 controls the accessibility of its receptor binding domains (RBDs) to host receptors and immune recognition. Along the evolution of SARS-CoV-2 to its variants of concern (VOC)-alpha, beta, gamma, delta, and omicron-their S proteins showed a higher propensity to attain open states. Deciphering how mutations in S protein can shape its conformational dynamics will contribute to the understanding of viral host tropism. Here using microsecond-scale multiple molecular dynamics simulations (MDS), we provide insights into the kinetic and thermodynamic contributions of these mutations to RBD opening pathways in S proteins of SARS-CoV-2 VOCs. Mutational effects were analyzed using atomistic (i) equilibrium MDS of closed and open states of S proteins and (ii) nonequilibrium MDS for closed-to-open transitions. In MDS of closed or open states, RBDs in S proteins of VOCs showed lower thermodynamic stability with higher kinetic fluctuations, compared to S proteins of ancestral SARS-CoV-2. For closed-to-open transitions in S proteins of VOCs, we observed apparently faster RBD opening with a 1.5-2-fold decrease in the thermodynamic free-energy barrier (ΔGclosed→open). Saturation mutagenesis studies highlighted S protein mutations that may control its conformational dynamics and presentation to host receptors.

Evolutionarily conserved heat shock protein, HtpX, as an adjunct target against antibiotic-resistant Neisseria gonorrhoeae.

The emergence of multiple drug resistance and extreme drug resistance pathogens necessitates the continuous evaluation of the pathogenic genome to identify conserved molecular targets and their respective inhibitors. In this study, we mapped the global mutational landscape of Neisseria gonorrhoeae (an intracellular pathogen notoriously known to cause the sexually transmitted disease gonorrhoea). We identified highly variable amino acid positions in the antibiotic target genes like the penA, ponA, 23s rRNA, rpoB, gyrA, parC, mtrR and porB. Some variations are directly reported to confer resistance to the currently used front-line drugs like ceftriaxone, cefixime, azithromycin and ciprofloxacin. Further, by whole genome comparison and Shannon entropy analysis, we identified a completely conserved protein HtpX in the drug-resistant as well as susceptible isolates of N. gonorrhoeae (NgHtpX). Comparison with the only available information of Escherichia coli HtpX suggested it to be a transmembrane metalloprotease having a role in stress response. The critical zinc-binding residue of NgHtpX was mapped to E141. By applying composite high throughput screening followed by MD simulations, we identified pemirolast and thalidomide as high-energy binding ligands of NgHtpX. Following cloning and expression of the purified metal-binding domain of NgHtpX (NgHtpXd), its Zn2+ -binding (Kd = 0.4 µM) and drug-binding (pemirolast, Kd = 3.47 µM; and thalidomide, Kd = 1.04 µM) potentials were determined using in-vitro fluorescence quenching experiment. When tested on N. gonorrhoeae cultures, both the ligands imposed a dose-dependent reduction in viability. Overall, our results provide high entropy positions in the targets of presently used antibiotics, which can be further explored to understand the AMR mechanism. Additionally, HtpX and its specific inhibitors identified can be utilised effectively in managing gonococcal infections.

Polypharmacological repurposing approach identifies approved drugs as potential inhibitors of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tb), the causative pathogen of tuberculosis (TB) remains the leading cause of death from single infectious agent. Furthermore, its evolution to multi-drug resistant (MDR) and extremely drug-resistant (XDR) strains necessitate de novo identification of drug-targets/candidates or to repurpose existing drugs against known targets through drug repurposing. Repurposing of drugs has gained traction recently where orphan drugs are exploited for new indications. In the current study, we have combined drug repurposing with polypharmacological targeting approach to modulate structure-function of multiple proteins in M. tb. Based on previously established essentiality of genes in M. tb, four proteins implicated in acceleration of protein folding (PpiB), chaperone assisted protein folding (MoxR1), microbial replication (RipA) and host immune modulation (S-adenosyl dependent methyltransferase, sMTase) were selected. Genetic diversity analyses in target proteins showed accumulation of mutations outside respective substrate/drug binding sites. Using a composite receptor-template based screening method followed by molecular dynamics simulations, we have identified potential candidates from FDA approved drugs database; Anidulafungin (anti-fungal), Azilsartan (anti-hypertensive) and Degarelix (anti-cancer). Isothermal titration calorimetric analyses showed that the drugs can bind with high affinity to target proteins and interfere with known protein-protein interaction of MoxR1 and RipA. Cell based inhibitory assays of these drugs against M. tb (H37Ra) culture indicates their potential to interfere with pathogen growth and replication. Topographic assessment of drug-treated bacteria showed induction of morphological aberrations in M. tb. The approved candidates may also serve as scaffolds for optimization to future anti-mycobacterial agents which can target MDR strains of M. tb.

Deoxyelephantopin-a novel PPARγ agonist regresses pressure overload-induced cardiac fibrosis via IL-6/STAT-3 pathway in crosstalk with PKCδ.

Pathological cardiac hypertrophy is associated with ventricular fibrosis leading to heart failure. The use of thiazolidinediones as Peroxisome Proliferator-Activated Receptor-gamma (PPARγ)-modulating anti-hypertrophic therapeutics has been restricted due to major side-effects. The present study aims to evaluate the anti-fibrotic potential of a novel PPARγ agonist, deoxyelephantopin (DEP) in cardiac hypertrophy. AngiotensinII treatment in vitro and renal artery ligation in vivo were performed to mimic pressure overload-induced cardiac hypertrophy. Myocardial fibrosis was evaluated by Masson's trichrome staining and hydroxyproline assay. Our results showed that DEP treatment significantly improves the echocardiographic parameters by ameliorating ventricular fibrosis without any bystander damage to other major organs. Following molecular docking, all-atomistic molecular dynamics simulation, reverse transcription-polymerase chain reaction and immunoblot analyses, we established DEP as a PPARγ agonist stably interacting with the ligand-binding domain of PPARγ. DEP specifically downregulated the Signal Transducer and Activator of Transcription (STAT)-3-mediated collagen gene expression in a PPARγ-dependent manner, as confirmed by PPARγ silencing and site-directed mutagenesis of DEP-interacting PPARγ residues. Although DEP impaired STAT-3 activation, it did not have any effect on the upstream Interleukin (IL)-6 level implying possible crosstalk of the IL-6/STAT-3 axis with other signaling mediators. Mechanistically, DEP increased the binding of PPARγ with Protein Kinase C-delta (PKCδ) which impeded the membrane translocation and activation of PKCδ, downregulating STAT-3 phosphorylation and resultant fibrosis. This study, therefore, for the first time demonstrates DEP as a novel cardioprotective PPARγ agonist. The therapeutic potential of DEP as an anti-fibrotic remedy can be exploited against hypertrophic heart failure in the future.

Energetics of Spike Protein Opening of SARS-CoV-1 and SARS-CoV-2 and Its Variants of Concern: Implications in Host Receptor Scanning and Transmission.

The receptor binding domain(s) (RBD) of spike (S) proteins of SARS-CoV-1 and SARS-CoV-2 (severe acute respiratory syndrome coronavirus) undergoes closed to open transition to engage with host ACE2 receptors. In this study, using multi atomistic (equilibrium) and targeted (non-equilibrium) molecular dynamics simulations, we have compared energetics of RBD opening pathways in full-length (modeled from cryo-EM structures) S proteins of SARS-CoV-1 and SARS-CoV-2. Our data indicate that amino acid variations at the RBD interaction interface can culminate into distinct free energy landscapes of RBD opening in these S proteins. We further report that mutations in the S protein of SARS-CoV-2 variants of concern can reduce the protein-protein interaction affinity of RBD(s) with its neighboring domains and could favor its opening to access ACE2 receptors. The findings can also aid in predicting the impact of future mutations on the rate of S protein opening for rapid host receptor scanning.

In-silico design of an immunoinformatics based multi-epitope vaccine against Leishmania donovani.

BACKGROUND: Visceral Leishmaniasis (VL) is a fatal vector-borne parasitic disorder occurring mainly in tropical and subtropical regions. VL falls under the category of neglected tropical diseases with growing drug resistance and lacking a licensed vaccine. Conventional vaccine synthesis techniques are often very laborious and challenging. With the advancement of bioinformatics and its application in immunology, it is now more convenient to design multi-epitope vaccines comprising predicted immuno-dominant epitopes of multiple antigenic proteins. We have chosen four antigenic proteins of Leishmania donovani and identified their T-cell and B-cell epitopes, utilizing those for in-silico chimeric vaccine designing. The various physicochemical characteristics of the vaccine have been explored and the tertiary structure of the chimeric construct is predicted to perform docking studies and molecular dynamics simulations. RESULTS: The vaccine construct is generated by joining the epitopes with specific linkers. The predicted tertiary structure of the vaccine has been found to be valid and docking studies reveal the construct shows a high affinity towards the TLR-4 receptor. Population coverage analysis shows the vaccine can be effective on the majority of the world population. In-silico immune simulation studies confirms the vaccine to raise a pro-inflammatory response with the proliferation of activated T and B cells. In-silico codon optimization and cloning of the vaccine nucleic acid sequence have also been achieved in the pET28a vector. CONCLUSION: The above bioinformatics data support that the construct may act as a potential vaccine. Further wet lab synthesis of the vaccine and in vivo works has to be undertaken in animal model to confirm vaccine potency.

Antimicrobial-resistant Neisseria gonorrhoeae can be targeted using inhibitors against evolutionary conservedl-asparaginase.

The emergence of multiple drug-resistant "super gonorrhoea" complicates the management and treatment of Neisseria gonorrhoeae infections due to the progressive accumulation of mutations in the biological targets of frontline antimicrobials. Continuous evaluation and reporting of newer molecular targets and their inhibitors are necessary. Here, we present l-asparaginase of N. gonorrhoeae (NgA) as a new molecular target based on structure-based high-throughput screening, molecular dynamics(MD) simulations, and validation by biophysical, biochemical, and cell viability assays. We observed that the NgA is evolutionarily conserved in both the drug-resistant and susceptible strains of N. gonorrhoeae, indicating its importance in the growth and survival of the pathogen. Three Food and Drug Administration-approved drugs, pemirolast, thalidomide, and decitabine, were identified as potential inhibitors of NgA using high-throughput screening. The binding energies of the drugs with NgA were -20.14, -19.67, and -16.47 kcal/mol, respectively, compared to -6.82 ± 1.46 for enzyme-substrate l-Asn, as obtained through MD simulations. Subsequently, fluorescence quenching and differential scanning calorimetry experiments validated the in silico data. The observance of inhibition of NgA activity at micromolar drug concentrations further strengthened our findings. Conclusive evidence came from the cell viability assays where these drugs were found to impede the growth of N. gonorrhoeae culture effectively. Thus, our study establishes l-asparaginase as a new molecular target against gonococcal infections. From this study, we propose that targeting of NgA can be explored to control N. gonorrhoeae infections in combination therapy.

Structure and Sequence Based Analysis of Pullulanases: Understanding Dual Catalytic Mechanism.

BACKGROUND: Starch processing requires a combination of enzymes with other chemical and physical processes, which increases cost and time. Enzymes used in these processes have a characteristic (α/ß)8 barrel domain architecture, although, show variable activity. Pullulanase type 1 and isoamylase act on α-1-6 linkage, amylase on α-1-4 linkage whereas pullulanase type 2 acts on both α-1-6, and α-1-4 linkages of starch. OBJECTIVE: This article focusses on elucidating the importance of sequence and structural-based differences in pullulanase, that may lead to its dual catalytic nature. METHODS: Initially, sequences and structures of pullulanase type 1, pullulanase type 2, amylase and isoamylase were retrieved from the database (NCBI and PDB). Homology modelling using SWISS-MODEL and PHYRE2 was carried out for predicting the structure of the enzymes with unavailable structures. Further, the modelled structures were validated using ANOLEA, Verify 3D and PROCHECK, structures with high confidence value were selected and used for analysis. Finally, the selected structures were compared by using PDBefold, and their domain alignment and analysis was performed manually using Pymol. RESULTS: Modelled structures of pullulanase and isoamylase were validated and selected based on the confidence score. Comparative analysis of complete structures low similarity between the enzymes, although, domain analysis showed good similarity. Moreover, alignment of catalytic site residues showed high similarities with the change in orientation of critical site residues (HIS 242, ASP 347 and GLN 375). CONCLUSION: The change in orientation of active site residues along with the absence or presence of few residues might play a crucial role in imparting dual functionality.