Combating Antibiotic Resistance - Development of Inhibitors against Antibiotic Resistance Enzymes

When you have a bacterial infection and you take antibiotics, you always want the antibiotic to reduce or kill the microbes that cause the infection. But, when the bacteria are resistant to the antibiotic effect, the treatment becomes difficult. The development and spread of antibiotic resistant bacteria is a serious threat to our modern health system, and what used to be "normal" infections suddenly becomes treating, and can even cause deaths. This project is named "Combating Antibiotic Resistance" and we focus on the enzymes causing antibiotic resistance, and more specifically the beta-lactamases that inactivated penicillin-like antibiotics. The techniques we use are: X-ray crystallography, microbiology, enzyme assays, and protein inhibitor studies. We are one technician, one PhD student and 50% of the scientist who is also the projectleader. This last years we have in collaboration with Umeå University screened 14 000 different compounds and tested their ability to block the activity of the metallo-beta-lactamase enzyme VIM-2. VIM-2 is a clinically relevant drug target, and we have found around 12 very interesting hits, where we will continue to do additional experiments and also explore the chemical structures further. We have published one large paper on one enzyme with 8 point mutations, and this enzyme causes bacterial resistance in a clinical isolate. These mutants and wild type are studied at the atomic level, by determining the three dimensional structure by X-ray crystallography, we have done enzyme assay in solution, and tested all mutants in the bacteria Escherichia coli against 12 different beta-lactam (penicillin-like) antibiotics. The protein expression level in the different mutants is also investigated. The PhD candidate has done many inhibition studies of pure enzyme and new synthetized potential inhibitors. The results show that some molecules are efficient as inhibitors, others have poor solubility while others have no effect. The best inhibitions are taken further and tested against clinical isolates at the microbiology lab at the University Hospital of North Norway, UNN. Again, some inhibitor molecules are better than others. For the best ones, we study these further by looking at the chemical structure, investigating the functional groups, and make more inhibitor compounds. All compounds tested are made specifically for this project by organic chemists at UiT The Arctic University of Norway, Tromsø or at the University of Oslo. We are trying to express some of our enzymes with isotopic labelling (15-N and/or 13-C) to do NMR studies on protein - inhibitor interactions, but so far we are struggling with incorporation of the isotopes. We had a talk and posters at Norwegian Biochemical Society Contact Meeting (NSB) at (Svalbard, February 2015; Tromsø, January 2016). We have had talks to yearly ?chemistry conference? for high school students on antibiotic resistance and chemistry. The PhD student and the technician have been at the yearly BioStruct conference (Jægtvolden, August 2015 and August 2016) for the National graduate school in structural biology. We have also made a huge poster and had an exhibition at Tromsø museum (Nov 2014- April 2015), which was a glass ?cabinet? with a poster (3.5 x 1 meters), X-ray crystallography equipment, bottles with solutions, promoting X-ray crystallography, and our search for inhibitors against enzymes involved in antibiotic resistance using X-ray crystallography. The PhD student has been active at the Science days promoting chemistry and science to the public, which was a great success.

This research proposal describes the complementary and synergistic activities of three research groups with national functions: NorStruct and MabCent at the University of Tromsø (UiT), and the National Reference Centre for Detection of Antimicrobial Resis tance at the University Hospital of North Norway/Research Group for Host-Microbe Interactions, UiT to meet a global clinical need. Herein, we outline a systematic and unique approach for discovering and fully characterizing inhibitors against two key grou ps of bacterial enzymes responsible for antibiotic resistance in clinically important bacterial pathogens; namely, metallo-beta-lactamases and Nim-reductases. This multi-disciplinary collaboration involves structural biologists, medicinal chemists, medi cal microbiologists and experts on therapeutics. The project has a clear vision of developing lead compounds attractive for biotechnology and pharmaceutical enterprises to bring forward towards the market and thereby create additional growth in these emer ging businesses.