Purification and characterization of the antibacterial peptidase lysostaphin from Staphylococcus simulans: Adverse influence of Zn2+ on bacteriolytic activity.

Lysostaphin, a bacteriolytic toxin from Staphylococcus simulans, is a Zn2+-dependent endopeptidase that cleaves pentaglycine cross-bridges found in peptidoglycan of certain Staphylococci. Here, we have investigated a critical influence of Zn2+ ions on lysostaphin-induced bioactivity. Initially, we succeeded in producing a large amount with high purity of the 28-kDa His-tagged mature lysostaphin via soluble expression in Escherichia coli and subsequent purification via immobilized-Ni2+ affinity chromatography (IMAC). The purified monomeric bacteriocin exhibited concentration-dependent bioactivity against S. aureus and its methicillin-resistant strain through cell-wall hydrolysis rather than membrane perturbation. Following pre-incubation of the purified lysostaphin with exogenous Zn2+, a marked inhibition in staphylolytic activity was observed. When the pre-mixture was exposed to 1,10-phenanthroline (PNT, a Zn2+-chelator), the adverse effect of the exogenous Zn2+ on bioactivity was greatly decreased. Conversely, lysostaphin pre-treated with excess PNT retained relatively high bioactivity, indicating ineffective chelation of PNT to detach the catalytic Zn2+ from the active-site pocket. Structural analysis of the lysostaphin-catalytic domain together with amino acid sequence alignments of lysostaphin-like endopeptidases revealed a potential extraneous Zn2+-binding site found in close proximity to the Zn2+-coordinating active site. Overall our results provide more insights into an adverse influence of exogenous Zn2+ ions on staphylolytic activity of the purified Zn2+-dependent endopeptidase lysostaphin, implicating the presence of an extraneous inhibitory metal-binding site.

Identifying plant-derived antiviral alkaloids as dual inhibitors of SARS-CoV-2 main protease and spike glycoprotein through computational screening.

COVID-19 is currently considered the ninth-deadliest pandemic, spreading through direct or indirect contact with infected individuals. It has imposed a consistent strain on both the financial and healthcare resources of many countries. To address this challenge, there is a pressing need for the development of new potential therapeutic agents for the treatment of this disease. To identify potential antiviral agents as novel dual inhibitors of SARS-CoV-2, we retrieved 404 alkaloids from 12 selected medicinal antiviral plants and virtually screened them against the renowned catalytic sites and favorable interacting residues of two essential proteins of SARS-CoV-2, namely, the main protease and spike glycoprotein. Based on docking scores, 12 metabolites with dual inhibitory potential were subjected to drug-likeness, bioactivity scores, and drug-like ability analyses. These analyses included the ligand-receptor stability and interactions at the potential active sites of target proteins, which were analyzed and confirmed through molecular dynamic simulations of the three lead metabolites. We also conducted a detailed binding free energy analysis of pivotal SARS-CoV-2 protein inhibitors using molecular mechanics techniques to reveal their interaction dynamics and stability. Overall, our results demonstrated that 12 alkaloids, namely, adouetine Y, evodiamide C, ergosine, hayatinine, (+)-homoaromoline, isatithioetherin C, N,alpha-L-rhamnopyranosyl vincosamide, pelosine, reserpine, toddalidimerine, toddayanis, and zanthocadinanine, are shortlisted as metabolites based on their interactions with target proteins. All 12 lead metabolites exhibited a higher unbound fraction and therefore greater distribution compared with the standards. Particularly, adouetine Y demonstrated high docking scores but exhibited a nonspontaneous binding profile. In contrast, ergosine and evodiamide C showed favorable binding interactions and superior stability in molecular dynamics simulations. Ergosine demonstrated exceptional performance in several key pharmaceutical metrics. Pharmacokinetic evaluations revealed that ergosine exhibited pronounced bioactivity, good absorption, and optimal bioavailability. Additionally, it was predicted not to cause skin sensitivity and was found to be non-hepatotoxic. Importantly, ergosine and evodiamide C emerged as superior drug candidates for dual inhibition of SARS-CoV-2 due to their strong binding affinity and drug-like ability, comparable to known inhibitors like N3 and molnupiravir. This study is limited by its in silico nature and demands the need for future in vitro and in vivo studies to confirm these findings.

Exploration of nonlinear optical enhancement in acceptor-π-donor indacenodithiophene based derivatives via structural variations: a DFT approach.

Herein, a series of indacenodithiophene-based derivatives (TNPD1-TNPD6) were designed having D-π-A architecture via end capped acceptor modulation of a reference molecule (TNPR) to investigate nonlinear optical (NLO) behavior. Quantum chemical calculations were accomplished to examine electronic, structural and optical properties utilizing a density functional theory (DFT) approach at M06 functional with 6-311G(d,p) basis set. For this, natural bond orbitals (NBOs), density of states (DOS), frontier molecular orbitals (FMOs), transition density matrix (TDM) and non-linear optical (NLO) analyses were performed for TNPR and TNPD1-TNPD6. The structural modifications revealed a significant electronic contribution in tuning the HOMOs and LUMOs of the derivatives with lowered energy gaps and wider absorption spectra. FMOs findings revealed that compound TNPD5 was found with the lowest energy gap (1.692 eV) and with the highest softness (0.591 eV-1) among the derivatives. Furthermore, a UV-Vis study also disclosed that maximum absorption (λmax = 852.242 nm) was exhibited by TNPD5 in chloroform solvent. All the derivatives exhibited significant NLO results; in particular, TNPD5 showed the highest first hyper-polarizability (ßtot = 4.653 × 10-27 esu) and second hyper-polarizability (γtot = 9.472 × 10-32 esu). These DFT findings revealed that the end-capped substituents play a key role in enhancing the NLO response of these push-pull chromophores and the studied derivatives can be utilized as efficient NLO materials.