The OXA-class of β-lactamases. A structural view on antibiotic resistance

Lund, Bjarte Aarmo

dc.contributor.advisor	Leiros, Hanna-Kirsti Schrøder
dc.contributor.author	Lund, Bjarte Aarmo
dc.date.accessioned	2017-09-13T11:01:32Z
dc.date.available	2017-09-13T11:01:32Z
dc.date.issued	2017-09-01
dc.description.abstract	Antibiotic resistance is a topic that concerns everyone, and by 2050 deaths due to antibiotic resistant bacteria may surpass number of deaths due to cancer. The OXA-class of antibiotic resistance enzymes is a formidable threat, but has not received the same attention as other resistance enzymes. The goal of the project was to understand antibiotic resistance enzymes at an atomic scale and to develop molecules that may inactivate OXA enzymes responsible for antibiotic resistance. We studied the OXA-class of antibiotic resistance enzymes, which makes bacteria resistant to important antibiotics including the carbapenem meropenem. The main method was protein crystallography. In order to identify new inhibitors, molecules that disrupts the OXA-48 enzyme activity, we screened a library of 490 small molecules by combining biophysical and biochemical methods. Based on three-dimensional structural information from protein and inhibitor interactions, over 50 new compounds were synthesized, and we characterized inhibitor properties towards OXA-48. We determined more than 40 complexes of OXA-48 bound to new compounds. We also enzymatically characterized the new OXA-436 enzyme, and determined three-dimensional structures of OXA-181 and OXA-245. Results from these studies have expanded our knowledge on how OXA-class enzymes contribute to antibiotic resistance crisis, and our work on developing new compounds, active as inhibitors against OXA-48 lays a foundation for new inhibitor drugs, and understanding of antibiotic resistance at the atomic level.	en_US
dc.description.doctoraltype	ph.d.	en_US
dc.description.popularabstract	Antibiotic resistance at an atomic level: Antibiotic resistant bacteria is killing thousands of people every day. These bacteria were originally sensitive to antibiotics, but have acquired genes coding for biological machinery, enzymes, capable of destroying antibiotics. We wanted to understand more about how these enzymes work and how to stop them. Penicillins are the most commonly used antibiotics, because they are effective against a broad range of bacteria and have a good safety profile. However, enzymes called β-lactamases break down these antibiotics by opening the central β-lactam ring of the antibiotics. The genes encoding the β-lactamases spread easily between bacteria, and have been identified in bacteria causing human diseases such as Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. The broad-spectrum antibiotic carbapenems, belong to the β-lactam group of antibiotics, and are used as last resort drug by medical doctors, when other antibiotics are ineffective or the bacteria which are causing the infection is unknown. OXA-48 is one of the most frightening β-lactamases because it is capable of breaking down carbapenems. There is currently only one approved drug-combination efficient against OXA-48. OXA-48 belongs to a special class of β-lactamase enzymes, which have unique properties compared to other enzymes. Two OXA-48 enzymes will stick together and form an enzyme dimer. In general enzymes wrap around the substrate molecules, and the interacting parts of the enzyme is called the active site. For OXA-48 a CO2 molecule is incorporated and bound to one amino acid in the active site and this modification is uncommon for enzymes. In order to understand more about how OXA-48 works at an atomic level we used powerful X-ray beams at particle accelerators to obtain three dimensional structures from protein crystals. Protein crystals are similar to salt crystals, but they are usually microscopic (smaller than 1 mm in length) and very hard to grow. One efficient way killing bacteria expressing an OXA-48 gene would combining a known antibiotic with a new OXA-48 β-lactamase inhibitor. Inhibitors are molecules that decrease the enzyme activity. From library of 500 molecules we identified molecules that could replace the antibiotics in the active site of OXA-48, thus reduce the enzyme activity. Based on these small molecules we built bigger molecules to make a potent OXA-48 inhibitor. We used x-ray crystallography to understand how the molecules bind in the enzyme, and determined 44 complexes structures of OXA-48 bound to different inhibitors. We have demonstrated that our inhibitors decrease the OXA-48 activity enzyme; however, work is still needed to make the compounds active in bacteria e.g. to cross the protective bacterial membrane. We also characterized OXA-163, OXA-181, OXA-245 and OXA-436, which are OXA-48 like, in order to understand the great diversity seen among the OXA enzymes. We were the first to determine the crystal structure of both OXA-181 and OXA-245. Results from these studies have increased our understanding of OXA-48 like antibiotic resistance enzymes. Our novel inhibitors may also contribute to new drugs in the future, and as building blocks for new projects.	en_US
dc.description.sponsorship	Forskerskolen BioStruct	en_US
dc.description	The papers II-VI of this thesis are not available in Munin. <br> <br> Paper II: Lund, B. A., Christopeit, T., Guttormsen, Y., Bayer, A., Leiros, H. K. S.: “Screening and Design of Inhibitor Scaffolds for the Antibiotic Resistance Oxacillinase-48 (OXA-48) through Surface Plasmon Resonance Screening”. Available in <a href=http://dx.doi.org/10.1021/acs.jmedchem.6b00660> J. Med. Chem. 2016, 59, 5542−5554. </a> <br> <br> Paper III: Ahkter, S., Lund, B. A., Lange, M., Ismael, A., Isaksson, J., Christopeit, T., Leiros, H. K. S., Bayer, A.:“A focused fragment library targeting the antibiotic resistance enzyme - Oxacillinase-48: synthesis, structural evaluation and inhibitor design”. (Manuscript). <br> <br> Paper IV: Lund, B. A., Thomassen, A. M., Carlsen, T. J. O., Leiros, H. K. S.: “Structure activity and thermostability investigations of OXA-163, OXA-181 and OXA-245, using biochemical, crystal structures and differential scanning calorimetry analysis”. (Manuscript). <br> <br> Paper V: Samuelsen, Ø., Hansen, F., Aasnæs, B., Hasman, H., Lund, B. A., Leiros, H. K. S., Lilje, B., Janice, J., Jakobsen, L., Littauer, P., Søes, L. M., Holzknecht, B. J., Andersen, L. P., Stegger, M., Andersen, P. S., Hammerum, A. M.: “Dissemination and Characteristics of a Novel Plasmid-Encoded Carbapenem-Hydrolyzing Class D b-Lactamase, OXA-436 from Four Patients Involving Six Different Hospitals in Denmark”. (Manuscript). <br> <br> Paper VI: Lund, B. A., Thomassen, A. M., Nesheim, B., Carlsen, T. J. O., Isaksson, J., Christopeit, T., Leiros, H. K. S.: “Structural basis for OXA-48 dimerization”. (Manuscript).	en_US
dc.identifier.uri	https://hdl.handle.net/10037/11441
dc.language.iso	eng	en_US
dc.publisher	UiT Norges arktiske universitet	en_US
dc.publisher	UiT The Arctic University of Norway	en_US
dc.rights.accessRights	openAccess	en_US
dc.rights.holder	Copyright 2017 The Author(s)
dc.rights.uri	https://creativecommons.org/licenses/by-nc-sa/3.0	en_US
dc.rights	Attribution-NonCommercial-ShareAlike 3.0 Unported (CC BY-NC-SA 3.0)	en_US
dc.subject	VDP::Matematikk og Naturvitenskap: 400::Kjemi: 440::Fysikalsk kjemi: 443	en_US
dc.subject	VDP::Mathematics and natural science: 400::Chemistry: 440::Physical chemistry: 443	en_US
dc.subject	VDP::Matematikk og Naturvitenskap: 400::Basale biofag: 470::Biokjemi: 476	en_US
dc.subject	VDP::Mathematics and natural science: 400::Basic biosciences: 470::Biochemistry: 476	en_US
dc.subject	Strukturkjemi	en_US
dc.subject	Structural biology	en_US
dc.title	The OXA-class of β-lactamases. A structural view on antibiotic resistance	en_US
dc.type	Doctoral thesis	en_US
dc.type	Doktorgradsavhandling	en_US