Discovery of new inhibitors of bacterial peptidoglycan biosynthesis enzymes MurA and MurB

Nowadays, we are in a transition era where it is evident that therapy of bacterial infections is not a trivial task, understood by modern medicine and supplied with abundant medicines. Emergence of bacterial resistance, globalisation and access to modern medicinal practice together with a decreasing output of antibacterial discovery pipeline, projects a major concern for the future. In the light of multidrug-resistant bacteria, bacteria resistant to the last line of defence drugs and resulting decrease in the efficacy of antibiotics, future research programmes in antibacterial drug design demand ultimate respect and financial engagement from industrial and scientific communities. This postdoctoral scientific project will contribute to the antibacterial drug research by exploring MurA transferase and MurB reductase enzymes as therapeutic targets in antibacterial drug discovery. Both enzymes are present at the intracellular steps in the biosynthesis of  UDP-N-acetylmuramyl-pentapeptide – a key building block for the peptidoglycan biosynthesis and bacterial cell-wall construction and are essential for bacterial cell viability. Studied enzymes do not have mammalian homologues and are regarded as one of the prominent targets in antibacterial drug research, sadly with limited scientific data available. Proposed project will use rational and computer-assisted drug design methodologies of ligand-based, structure-based and fragment-based drug design with bioinformatics to identify novel inhibitors of MurA and MurB enzymes. Design phase of the compounds will also be supported by extensive molecular dynamics studies. As the project emphasis will be on the antibacterial drug design, in house in vitro antibacterial testing will be emphasized from the early stages of design all along the project timeframe with supporting toxicological testing. Special attention will be also given to pharmacokinetic properties with CACO and PAMPA permeability testing, all in order to obtain compounds with potential for further in-depth pharmacological evaluation and further development. As no clear mode-of-action is reported for new reported inhibitors of Mur enzymes with antibacterial activity, this project will try to gain an insight in mode-of-action by focusing on collection of commercial compounds with development and organic synthesis of MurA and MurB inhibitors suitable for such studies. As the task is highly interdisciplinary in nature, research will be performed in collaboration with international research network of partners. Ultimate goal of the project is development of MurA and MurB inhibitors with antibacterial activity and an insight into the mode-of-action where inhibition of studied enzyme can be linked to the antibacterial activity.

Relevance to the development of science of the proposed research Project is justified in multiple key areas and key scientific questions that need further elaboration. Antibacterial drug discovery is a substantial and one of the most important fields in medicinal chemistry. Proposed Project is expected to contribute to the discovery of novel antibacterials with new mechanisms of action. During the research Project, multiple compounds and compound series will be evaluated for their in vitro antimicrobial activity with the concomitant support of modern drug design methodologies and bioinformatics. This agglomerate approach of design on the isolated therapeutic target with consideration of in vitro antibacterial activity evaluation could mediate the identification of compounds with antibacterial properties that are suitable for further mode of action studies. Upon identification of suitable inhibitors with antibacterial activity, this Project will attempt to provide mode of action study considering the data on mode of action for Mur enzyme inhibitors with antibacterial activity are greatly needed. The results of the Project could have further ramifications in: firstly, the study of bacterial cell wall permeability and secondly, in the study of bacterial resistance. Former scientific problem is a formidable hurdle where bacterial cell wall permeability often still relies on empirical data and limited libraries of defined molecular scaffolds. With the emphasis on antibacterial activity evaluation and usage of modern bioinformatics approaches, an insight into this problem will be provided. Latter ubiquitous phenomenon of bacterial resistance surpassed the scientific literature to become an international joint effort to understand, control, react appropriately and be responsible towards usage of antibacterial drugs. Project could provide new molecular tools to study this phenomenon and provide compounds with a good potential for development towards novel antibacterial drugs. All along the Project research cycle a multitude of drug discovery tools will be employed and close collaboration within research group and with Slovene and international partners will be utilised: Rational design of enzyme inhibitors Computer-assisted drug discovery (in silico drug design: structure-based drug design, de-novo design of enzyme inhibitors, combinatorial library design, virtual screening) Fragment-based drug design Development and optimisation of new organic synthetic methods and synthetic protocols Optimization of hits towards lead compounds Development of simple and rapid methods for biochemical evaluation of enzyme inhibitors Development of new in silico tools for drug discovery

From 1990s until now, there has been a marked decrease of industrial involvement in antibacterial drug discovery programmes. Major pharmaceutical companies have abandoned the antibiotic discovery field in favour of other therapeutic targets, mainly due to poor financial returns and deficient scientific data. It is therefore imperative, that academia scientific community progresses the field and provides the groundwork for focused research on novel antibacterials. Results of the proposed Project could also facilitate the establishment of small research companies that implement technology transfer and pathway towards practical application of underlining scientific contribution. In context of Slovenian economy, where pharmaceutical industry is a single biggest contributor, joint venture between academia and Slovenian pharmaceutical industry could assure firm economic growth and establishment of creative-collaborative milieu. Historically, antibacterial research programmes played a key role in establishment of Slovenian pharmaceutical industry, but the focus has recently shifted towards generics. Proposed Project therefore not only enables the research on compounds of pharmaceutical interest, but also provides socio­economic input in the form of academia-industry dialog and training of professionals in the interdisciplinary fields of chemistry, medicinal chemistry, biochemistry, theoretical-chemistry and bioinformatics. The Project leader works in the research group of prof. Stanislav Gobec, a group with a prominent Slovenian and international contributions to the field of medicinal chemistry. Researchers involved in the prepared Project will gain competences for autonomous creative work and will contribute to increasing international recognition of Slovenian pharmaceutical chemistry research and pharmaceutical industry. Furthermore, all actions of the proposed project will be taken in the light of cultivating scientific excellence that constitutes the basis for Slovenian national identity in the world and advancing the field of novel antibacterial research. Antibacterial drugs with novel mechanisms of action are in desperate need and would enable new therapeutic approaches towards complex infections with resistant bacteria and facilitate the control of emerging bacterial resistance.