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|Title:||Rational design and development of moenomycin A : derived fluorescent biosensors for screening of peptidoglycan glycosyltransferase inhibitors||Authors:||Yuan, Jian||Advisors:||Wong, K. Y. (ABCT)||Keywords:||Biosensors.||Issue Date:||2016||Publisher:||The Hong Kong Polytechnic University||Abstract:||Antibacterial resistance prevents the effective treatment of an ever-increasing number of pathogenic bacteria infections, which has caused a serious threat to human health across the world. To combat the growing problem of antibiotic resistance, there is an urgent need to develop novel antibiotics with new chemotypes or acting on novel targets. The peptidoglycan glycosyltransferase (PGT), which catalyzes the essential process of lipid II (the single unit of peptidoglycan) polymerization, is a promising target for new antibiotic development as a result of the following two reasons. Firstly, PGT is located at the outside of bacterial cell membrane which makes it easy to access to drugs. The second reason is that PGT is highly conservative in both antibiotic sensitive and resistant strains. PGT as an antibacterial target has been studied for several decades. However, there is no antimicrobial drugs targeting PGT used clinically. Moenomycin A (Moe A), a natural product isolated from several strains of Streptomyces, is the only well proven PGT inhibitor by binding to the active sites of PGT. However, Moe A does not show antibacterial activity in human due to its poor bioavailability. Moe A is currently only used as a growth promoter in animal feed. One of the major reasons for the slow progress on development of PGT inhibitors is lack of efficient assays to evaluate potential drug candidates. In recent years, several crystal structures of complexes of PBPs with Moe A and lipid II unveiled the mysterious process of transglycosylation in peptidoglycan biosynthesis. This new development has prompted researchers to develop new assays for PGT inhibitor screening to facilitate novel antibiotic discovery. In this project, a fluorescent biosensing system for PGT inhibitor screening was constructed based on photoinduced electron transfer (PET).
In constructing the biosensor, three fluorescent Moe A derivatives (F-n-Moe A, n = 2, 3 and 4) were synthesized by attaching fluorescein-5-isothiocyanate isomer I (FITC) to Moe A. Furthermore, five amino acid residues near the active site (Q161, H162, D199, Y210 and D241) of the S. aureus PBP2 glycosyltransferase domain were muted to tryptophan, a fluorescence quencher of fluorescein. The interaction between Moe A and PGT mutants as analyzed by surface plasmon resonance (SPR) spectroscopy showed that the mutations did not significantly alter the binding affinity. Fluorescent measurement showed that the D199W and D241W mutants can quench the fluorescence of the three probes at different levels. Moreover, addition of free Moe A can subsequently restore the fluorescence intensity of the quenched probes. When the fluorescein-labelled Moe A binds to the active site of PGT domain, the nearby tryptophan residue can interact with and quench the fluorophore group of the probes through a PET process if the linkers have appropriate lengths. When free Moe A was added into the system, it competitively binds to the same pocket of the probes and the fluorescent quenching effect was relieved once the probes were displaced by Moe A and the fluorescence intensity of the probes was restored. Among different combinations of mutants and probes, three probe / mutant pairs, namely F-3-Moe A / D241W, F-4-Moe A / D199W and F-4-Moe A / D241W, showed the best fluorescence quenching efficiency. While Moe A was added into the system, the fluorescence intensity of probes were restored up to 90 % of the original level, whereas no change in fluorescence signal was observed when the antibiotics ampicillin and kanamycin were added as negative controls. In order to further validate the biosensing system, the change in fluorescence of the F-4-Moe A / D199W pair with two small molecule PGT inhibitors 14 and 16 reported in literature were also studied. While compound 16 did not induce any observable fluorescence change for the F-4-Moe A / D199W pair, compound 14 was able to restore its fluorescence. Compared to fluorescence polarization based bioassay, the PET-based bioassay has the advantage that the enzyme can be immobilized on a surface during the screening process. The success in the construction of this PET-based biosensor will allow the future development of high-throughput screening of PGT inhibitors using microfluidic chip-based devices.
|Description:||PolyU Library Call No.: [THS] LG51 .H577P ABCT 2016 Yuan
xii, 173 pages :color illustrations
|URI:||http://hdl.handle.net/10397/60382||Rights:||All rights reserved.|
|Appears in Collections:||Thesis|
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