Enabling early stage drug discovery targeting high molecular mass penicillin-binding proteins in Yersinia pestis

Abstract

Y. pestis is a G- facultative intracellular parasite, the causative agent of plague.The ability of this pathogen to acquire resistance, its potential as a biological weapon,high mortality caused by plague and its increasing prevalence, all highlight the need for investigating novel therapeutic strategies against this pathogen. It is possible that enzymes from the peptidoglycan biogenesis pathway, which are generally not addressed in therapy against plague, may present suitable drug targets. The rationale for studying selected penicillin-binding proteins (PBPs) in Y. pestis and a general introduction to this family of proteins, the history of β-lactam antibiotics and the organism of interest are given in Chapter one. High molecular mass (HMM) PBPs are well-established targets of β-lactam antibiotics. However, only in the past two decades has information about their structure started to emerge and the application of structure-based drug design against these proteins become possible. The aims of this project are provided detailing the proposed study of three HMM PBPs from Y. pestis (YpPBPs).Chapter two presents the experimental procedures used to investigate YpPBPs including cloning, gene expression and protein purification protocols along with crystallisation methods, data collection, data processing, structure solution and refinement. Protein-ligand interactions were investigated using several well-established biochemical techniques such as DSF, NMR and BLI as well as some alternative approaches such as ITM, single injection ITC and a nitrocefin hydrolysis assay.Production and crystallographic analyses of recombinant YpPBP3 are described inChapter three. Three structures of YpPBP3 were solved, refined and analysed. The interactions between a covalent ligand – carbenicillin, and the active site are outlined.Structures of apo and protein-ligand complex are compared and conformational changes discussed. This is followed by sections where interactions of YpPBP3 with selected β-lactams were investigated and a set of putative ligands was tested for binding this protein. In a similar fashion, Chapter four describes the production, crystallisation and structural analysis of YpPBP2. Two protein-ligand complexes featuring mecillinam and ertapenem were compared. Attempts to characterise interactions between YpPBP2 and selected β-lactams first provided ambiguous results, which together with crystallographic observations suggested loss of a covalent ligand. To investigate this phenomenon further, discs soaked in a chromogenic β-lactam, nitrocefin, were used. The test showed that YpPBP2, but not YpPBP3, could hydrolyse nitrocefin. Based on the results of this nitrocefin test, structural data, sequence comparison and literature analysis, a hypothesis was proposed explaining this unusual catalytic activity. In YpPBP2, an aspartic acid in a SxN/D motif might supply a proton to the ε-amino group of adjacent lysine, which in turn would act as a catalytic base to hydrolyse the ester bond. This explanation is consistent with the observed ability of compounds featuring strong polarising groups near the aspartate to inhibit YpPBP2 irreversibly and also the fact that YpPBP3, lacking the ability to remove β-lactams, features asparagine at this position, not aspartate. The thermal stability of YpPBP2 and 3 in the presence of selected β-lactams was compared. It was noted that compounds with opposing substitution profiles have different effects on YpPBPs. This observation is consistent with the fact that efficient β-lactam therapies involve two compounds with opposing substitution profiles where one compound is used as a β-lactamase inhibitor and the other targets HMM PBPs. Perhaps the success of these therapies can be partially explained by an effect on YpPBP2 and 3 homologues.Chapter five describes the production of recombinant YpPBP1b and extensive efforts dedicated to alleviating solubility issues. In-house solubility screening identified a buffer that would maintain YpPBP1b in solution. The protein was crystallised but no diffraction was obtained.A summary of the results achieved from this thesis work and suggestions for future work in this area are provided in Chapter six.The Appendix sections provide supplementary information for the thesis and details of other research undertaken during this Ph.D, which could not be further pursued due to time constraints.

Date of Award	2019
Original language	English
Awarding Institution	University of Dundee
Sponsors	Defence Science and Technology Laboratory
Supervisor	Bill Hunter (Supervisor)