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Antimicrobial and Resistance-Modifying Activities of LY2183240 Regioisomers

De Resende, Pedro Ernesto; (2017) Antimicrobial and Resistance-Modifying Activities of LY2183240 Regioisomers. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The rapid development of antimicrobial resistance over the past three decades represents a critical public health threat and urgent challenge. In this context, in order to establish lead compounds or identify novel modes of action one approach is to re-investigate drugs that affect eukaryotic processes for antimicrobial activity, particularly if their drug targets have homologies with bacterial proteins. In this study, regioisomers of LY2183240, a potent inhibitor of anandamide transport and fatty acid amide hydrolase were selected after a small screening study. The 2,5-LY2183240 regioisomer was shown to possess potent antimicrobial activity selective towards certain Gram-positive bacteria, which included Staphylococcus aureus and Bacillus subtilis, but not Enterococcus faecalis or Streptococcus pneumoniae. Conversely, the 1,5-LY2183240-regioisomer had no anti-bacterial activity strongly implicating the position of the carbamoyl on the tetrazole in the structure-activity relationship of the molecule. Investigation of the mechanism of antimicrobial activity suggested that while 2,5- LY2183240 had bacteriostatic activity this was probably not due to inhibition of protein or teichoic acid synthesis. Nevertheless, this activity may be related to the inhibition of bacterial fatty acid synthesis. Supporting this hypothesis, addition of exogenous fatty acids within Tween 80 was able to compromise 2-5-LY2183240 anti-staphylococcal activity. Based on the spectrum of activity and known redundancies at each of the steps in the fatty acid synthesis pathway, the most likely target was deduced to be FabI. However, characterization of a 2,5-LY2183240- resistant mutant revealed no alteration to the deduced amino acid sequence of FabI or relevant changes to FabI protein expression, an observation confirmed by analysis of the fabI promoter region and western blot studies. Since resistance to 2,5- LY2183240 could be mediated through non-target related factors such as drug efflux or drug entry into the cell, FabI could not be ruled out as a potential target. In addition, whole cell protein profiling revealed the 2,5-LY2183240-mutant to have differences in the expressions of several proteins, suggesting resistance may occur through a more global effect. Due to the promiscuous nature of LY2183240, the possibility of having multiple targets in S. aureus that collectively exhibit a bacteriostatic effect cannot be ruled out. This study also showed that the LY2183240 regioisomers are specific inhibitors of class C β-lactamases with optimum inhibitory activity dependent upon the position of the carbonyl on the tetrazole heterocyclic group (Ki values of 1.8 and 2.45 μM for 1,5- and 2,5-LY2183240, respectively). Molecular modelling suggested that LY2183240 regioisomers bind within the catalytic site of class C β-lactamases, interacting with some of the same residues, including Tyr150, Lys315 and Thr316, used by the substrate nitrocefin and β-lactamase inhibitors. The results substantiate the competitive inhibition model determined by enzyme kinetic studies. Furthermore, mass spectrometry data revealed non-covalent interactions between AmpC β- lactamase and LY2183240 regioisomers, which together with the non-reversible inhibitory activity, may indicate that both regioisomers present a high affinity towards this cephalosporinase and dissociate very slowly from the enzyme. LY2183240 may prove useful as a chemical scaffold for the development of novel and highly selective inhibitors of class C β-lactamases.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Antimicrobial and Resistance-Modifying Activities of LY2183240 Regioisomers
Event: UCL
Open access status: An open access version is available from UCL Discovery
Language: English
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10039061
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