High Throughput Pipeline for Structure Based Drug Design, and Antibiotic Resistance Enzymes in particular

When we are sick from a bacterial infection and need to take antibiotics, we wish for the antibiotic to reduce the infection or kill the microbes. However, when bacteria are resistant to the drug effect, the treatment becomes a challenge. The spread and number of antibiotic resistant bacteria are increasing dramatically, and treatment of what used to be a normal infection suddenly becomes hard or even impossible. This project is entitled: 'High Throughput Pipeline for Structure Based Drug Design, and Antibiotic Resistance Enzymes in particular', and we focus on enzymes making bacteria resistant to beta-lactam drugs, namely the enzymes beta-lactamases. We use X-ray crystallography, Surface Plasmon Resonance (SPR), microbiology and protein assays in our studies. This part of the project work in close collaboration with another FRIMEDBIO project, with other scientist UiT The Arctic University of Norway and The University Hospital of North Norway (UNN). By using SPR the scientist in this project has measured the binging strength of about 500 fragment to 3 different lactamase enzymes. This has generated a lot of data, and 20 fragments were identified to bind with higher affinity for one lactamase, and 40 fragments are found to bind to another lactamase. These hits are followed further, and 7 complex structures with fragments bound to protein are achieved. To obtain these results, we have collected >100 X-ray data sets because it is hard to get the fragments to bind to a protein. We have developed a new method for obtaining a complex protein structure. This new method have been applied several times and the success rate of obtain complex structures have been increased a lot. The method is published and called DMSO-free co-crystallization. Both the project leader (Leiros) and the scientist hired in this project (Christopeit) were both heavily involved in organizing the 52nd Norwegian Biochemical Society (NBS) winter meeting in Tromsø 21.-24.1.2016 as Committee Leader and as Exhibitor and Provider Contact. The NBS meeting is an important arena for biotechnology research in Norway and in particular there were two excellent talks from two companies who make money using biotechnology. The scientist in this project worked at the European Synchrotron Radiation Facility (ESRF) for 6 weeks, he learnt a lot more protein crystallography and did successful experiments on protein in solution using «Small angle X-ray scattering (SAXS)». We have also contributed with lectures at Norsk Biokjemisk Selskap (NBS) seminars at Ås and in Oslo, given popular science lectures to high school students, and contributed to school visits in the NorStruct lab. The most important contribution for this project was: i) the use of the Surface Plasmon Resonance (SPR) technology to screen fragments binding antibiotic resistant enzymes where fragment analogues are now synthetized in the organic chemistry lab in Tromsø for improves potency, and ii) the development of the new method '?DMSO-free co-crystallization' since this method gave several inhibitor-protein complexes used in further in a structure based drug design project. The project has been filmed published on You tube a presentation on the LacZymes research project (https://www.youtube.com/watch?v=NT1ksFkPOHs&feature=youtu.be), Another YouTube film of Professor Karen Bush, is from her visit to Tromsø and as 'My occupation as a chemist' https://www.youtube.com/watch?v=19rmBVU_v_4&t=17s&index=4&list=PL77367A2AADC6EB4A.

High-resolution crystal structures of proteins reveal insights into function and enable rational drug and inhibitor design. Complex structures are even more valuable as specific interaction details, hydrogen binding donors and acceptors, residue determina nts and binding pockets are explored. But, to obtain a complex by soaking or co-crystalizing of drug/inhibitor result in many crystals soaked for different time, concentrations or solvent, thus each crystal is unique and must be tested preferable in a Hig h Throughput Pipeline. To obtain this we seek: i) >20 general cryo conditions optimized for drug/inhibitor solubility and ii) an efficient use of MX beam lines in order to rapidly detect binding. We will use the clinical relevant metallo-beta-lactamases VIM-2, VIM-7, AIM-1, GIM-1 and NDM-1 involved in antibiotic resistance against the most commonly used antibiotic beta-lactams. For these proteins the crystallization conditions are known, inhibitors are from new synthetized inhibitors, from marine extrac ts, or an in-house compound library that will be extended, and the drugs are commercially available. A High Throughput Pipeline for crystal structure evaluation is crucial for the scientific success, obtaining a complex with drug or inhibitor that will elucidate the pharmacophore with the beta-lactam drug, that will prolong the therapeutic lifetime of the drug, since the drug resistance caused by metallo-beta-lactamases are inactivated by the inhibitor. The ultimate goal is an inhibitor co-administered with the beta-lactam drug, which will prolong the life time of the drug, since the drug resistance caused by the metallo-beta-lactamases are inactivated by the inhibitor.