New multi-target based therapeutics against an opportunistic superbug

Pseudomonas aeruginosa (P. aeruginosa) is one of the most opportunistic and challenging Gram-negative pathogens, often resistant to a number of widely used antibiotics even when combination therapies are administered, thereby joining the ranks of “superbugs”. Hence, there is a dearth of treatment options for community-acquired and nosocomial Pseudomonas infections due to several rapidly emerging multidrug-resistant phenotypes along with the complexity of rising resistance rates. Taken together, it is imperative to discover more potent drugs and novel therapeutic strategies to combat the infections caused by this superbug. It is recently highly appreciated that instead of inhibition of an individual target in the disease-associated network, modulating activity of multiple targets may be required to achieve optimal therapeutic benefit against robust pathogens. Multi-target therapeutics can be developed by two distinct approaches, a combination of two or more different agents to inhibit dissimilar targets or designing a multi-target specific single hybrid entity. The research presented here utilized both combinations based searches and designing hybrid compounds with pre-defined promiscuity methods for the discovery of novel therapeutics against the study pathogen. In the search for more effective strategies to combat the Pseudomonas infections, in vitro activities of antibiotics and natural dietary phytochemicals alone and in combination against P. aeruginosa strains were investigated using the microtitration checkerboard method, fractional inhibitory concentration (FIC) indices and time-kill analysis. Furthermore, to gain a new understanding of the molecular mechanism of synergistic interactions identified from the above study various susceptibility testing methods, confirmatory biochemical assays were performed and also validated using computational evaluation involving molecular docking studies. Additionally, expansion of the phytochemical-antibiotic interaction profiles were also carried out with various classes of antibiotics and phytochemicals to understand the interaction patterns by establishing a novel interaction network that provides a baseline to identify the mechanism of action of the phytochemicals and molecular mechanism of synergy. The above findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (antibiotics and phytochemicals). These combinations based searches aided in identifying novel synergistic phytochemical-antibiotic combinations, an alternate therapeutic option for the treatment of Pseudomonas infections. The second part of the project involved hypothesis driven multi-target drug design against P. aeruginosa and computational evaluation of the designed hybrid compounds using various computer-aided drug design methodologies combining homology modelling, physico-chemical, stereo-electronic properties predictions, molecular docking and dynamics simulations. Drug relevant physico-chemical properties and toxicity risks predicted using in silico based prediction toolkits, suggests that the designed hybrid compounds potentially qualify as suitable drug candidates. The stereo-electronic properties such as HOMO, LUMO and MEP maps of the hybrid compounds calculated using quantum chemical methods, correlate well with identified common pharmacophoric features required for the multi-site interactions. Docking and dynamics simulation studies reveal that the designed hybrid compounds have favourable binding affinity and stability in their respective binding-site cavities by forming strong hydrogen bonds and hydrophobic interactions with key active site residues. Significantly, the above results for the first time demonstrate an approach for rational design of multi-specificity hybrid scaffolds based on the structural optimization of phytochemicals/antibiotics. Looking forward the designed hybrid compounds could serve as a prospective lead in the antibacterial multi-target drug discovery.Doctor of Philosophy (MAE