Computational and Biophysical Approaches to Identify Cell Wall-Associated Modulators in Salmonella enterica serovar Typhi

Abstract

An increase in the number of antibiotic-resistant bacterial pathogens, in recent times, has posed a great challenge for treating the affected patients. This has paved the way for the development and design of antibiotics against the previously less explored newer targets. Among these, peptidoglycan (PG) biosynthesis serves as a promising target for the design and development of novel drugs. The peptidoglycan cell wall synthesis in bacteria is essential for its viability. The enzyme class, Mur ligases, plays a key role in PG biosynthesis. Therefore, compounds with the ability to inhibit these enzymes (Mur ligase) can serve as potential candidates for developing small modulators. The enzyme, UDP-N-acetyl pyruvyl-glucosamine reductase (MurB), is essential for PG biosynthesis, a crucial part of the bacterial cell wall. The development of novel drugs to treat infections may thus focus on inhibiting MurB function. Understanding the mechanism of action of Mur B is central to developing efficient inhibitors. For the treatment of S. typhi infections, it is also critical to find therapeutic drugs that specifically target MurB. The enzyme Mur B from Salmonella enterica serovar Typhi (stMurB) was expressed and purified for biophysical characterization to gauge the molecular interactions and estimate thermodynamic stability, for determining attributes for possible therapeutic intervention. The thermal melting profile of MurB was monitored by circular dichroism (CD) and validated by performing differential scanning calorimetry (DSC). An in silico virtual screening of various natural inhibitors was conducted with modelled stMurB structure. The three top hits (quercetin, berberine, and scopoletin) obtained from in silico screening were validated for complex stability through molecular dynamics (MD) simulation. Further, fluorescence binding studies were undertaken for the selected natural inhibitors with stMurB alone and with its NADPH-bound form. The natural inhibitors, scopoletin and berberine, displayed lesser binding to stMurB compared to quercetin. Also, a stronger binding affinity was exhibited between quercetin and stMurB compared to NADPH and stMurB. Based on the above two findings, quercetin can be developed as an inhibitor of stMurB enzyme.

Keywords: Differential scanning calorimetry; Fluorescence binding; Molecular docking; Molecular dynamics simulation; Molecular modeling; Mur ligase; Natural compound Inhibitors.