Computational approaches screening DNA aptamers against conserved outer membrane protein W of Vibrio cholerae O1- an investigation expanding the potential for point-of-care detection with aptasensors.

Foodborne outbreaks urge public health domain to upgrade diagnosis by means of simpler, quicker, and more affordable pathogen detection methods. A molecular recognition probe against an analyte of interest makes up a biosensor, along with a method for turning the recognition event into a quantifiable signal. Single-stranded DNA or RNA aptamers are promising bio-recognition molecules for a range of targets, including a wide range of non-nucleic acid targets with which they are highly specific and affine. In the proposed study, 40 DNA aptamers were screened and analyzed interactions using in-silico SELEX procedures, which can selectively interact with active sites at the extracellular region of the Outer membrane Protein W (OmpW) of Vibrio Cholerae. Multiple modeling techniques, like protein structural prediction with I-TASSER, aptamer structural modeling using M-fold, RNA composer, protein-DNA docking using HADDOCK, and large-scale (500 ns) molecular dynamics simulations through GROMACS have been employed. Out of 40, six aptamers having lowest free energy were docked against the predicted active site at the extracellular region of OmpW. VBAPT4-OmpW and VBAPT17-OmpW, the two highest-scoring Aptamer-Protein complexes, were chosen for molecular dynamics simulations. VBAPT4-OmpW is quite unable to attain its structural local minima after 500 ns. But VBAPT17-OmpW is showing great stability and is not destructive even after 500 ns. RMSF, DSSP, PCA, and Essential Dynamics all provided additional confirmation. Current findings, combined with the fabrication of biosensor devices, could pave the way for an innovative pathogen detection platform with high sensitivity, along with an effective and low-impact curative strategy for corresponding diseases.Communicated by Ramaswamy H. Sarma.

A Comprehensive in vitro and in silico Assessment on Inhibition of CYP51B and Ergosterol Biosynthesis by Eugenol in Rhizopus oryzae.

Mucormycosis, also known as Zygomycosis, is a disease caused by invasive fungi, predominantly Rhizopus species belonging to the Order of Mucorales. Seeing from the chemistry perspective, heterocyclic compounds with an "azole" moiety are widely employed as antifungal agent for minimising the effect of mucormycosis as a prescribed treatment. These azoles serve as non-competitive inhibitors of fungal CYP51B by predominantly binding to its heme moiety, rendering its inhibition. However, long-term usage and abuse of azoles as antifungal medicines has resulted in drug resistance among certain fungal pathogens. Hence, there is an unmet need to find alternative therapeutic compounds. In present study, we used various in vitro tests to investigate the antifungal activity of eugenol against R. oryzae/R. arrhizus, including ergosterol quantification to test inhibition of ergosterol production mediated antifungal action. The minimum inhibitory concentration (MIC) value obtained for eugenol was 512 µg/ml with reduced ergosterol concentration of 77.11 ± 3.25% at MIC/2 concentration. Further, the molecular interactions of eugenol with fungal CYP51B were meticulously studied making use of proteomics in silico study including molecular docking and molecular dynamics simulations that showed eugenol to be strongly interacting with heme in an identical fashion to that shown by azole drugs (in this case, clotrimazole was evaluated). This is the first of a kind study showing the simulation study of eugenol with CYP51B of fungi. This inhibition results in ergosterol synthesis and is also studied and compared with keeping clotrimazole as a reference.

Unravelling the antifungal mode of action of curcumin by potential inhibition of CYP51B: A computational study validated in vitro on mucormycosis agent, Rhizopus oryzae.

Like human, fungi too are known to share lot of structural similarities amongst their CYPs (Cytochrome P450 super family of enzymes) which allows antifungal 'azole' compounds to interact with CYPs of human. Clotrimazole, an 'azole' antifungal drug, is a known inhibitor of fungal CYP named CYP51B. Curcumin, a phytochemical obtained from Curcuma longa has the ability to interact with several different human CYPs to induce inhibition. The sequence and the structural similarities amongst both human and fungal CYPs suggest a strong possibility for curcumin to interact with fungal CYP51B to behave like an antifungal agent. To test this hypothesis a study was designed involving mucormycosis agent, Rhizopus oryzae. The ability of curcumin to interact with fungal CYP51B was analysed computationally through molecular docking, MM-GBSA and Molecular Dynamics (MD) simulation assessment. Further, interaction profile for fungal CYP51B-curcumin was compared with human CYP3A4-curcumin, as there are published evidence describing curcumin as an inhibitor of human CYPs. Additionally, to validate in silico findings, an in vitro assay was performed to examine the antifungal potentials of curcumin on the R. oryzae. Conclusive results allow us to determine a plausible mode of action of curcumin to act as an antifungal against a mucormycosis agent.