Submission
| Title: | Adjuvants for the Potentiation of the Activity of -Lactam Antibiotics Against Methicillin-Resistant Staphylococcus aureus |
| Presenter: | Caitlyn Thomas |
| Institution: | University of Notre Dame |
| Authors: | Caitlyn A. Thomas, Choon Kim, Amr M. El-Araby, Biruk T. Birhanu, Mayland Chang, Shahriar Mobashery, Department of Chemistry and Biochemistry, University of Notre Dame |
Abstract
| Background/Significance/Rationale: | Methicillin-resistant Staphylococcus aureus (MRSA) causes serious infections in humans and emerged in response to treatment with second-generation penicillins. A common resistance mechanism is by the functions of the bla and mec operons, which encode a -lactam sensor/signal transducer protein BlaR/MecR, a gene repressor BlaI/MecI and a resistant determinant BlaZ or PBP2a. BlaR/MecR is responsible for sensing a -lactam and transducing a signal to its cytoplasmic domain. Without a -lactam, blaZ (gene encoding serine–lactamase BlaZ) is down-regulated by the transcriptional repressor BlaI. Upon -lactam binding to the sensor domain of BlaR (BlaR-SD), BlaI is hydrolyzed and allows for transcription of blaZ. |
| Methods: | A fluorescence-reporter assay was used to screen a compound library. Minimum inhibitory concentration experiments were used to determine the activity of compounds against various MSSA and MRSA strains. These methods were used to determine the mechanism of inhibition of compound 1: nano-differential scanning fluorimetry (nanoDSF); surface plasmon resonance (SPR); PBP anti-sense experiments; and scanning electron microscopy (SEM). In preparation for animal experiments a time-kill assay was performed. |
| Results/Findings: | A fluorescence-reporter assay identified 80 compounds from a 1,974-compound library as potential antibiotic adjuvants. We performed assays for potentiation of the activity of oxacillin against MSSA and MRSA strains. Twenty-four compounds showed promising potentiating ability (2- to 4,096-fold decrease in MIC). Seven compounds exhibited melting temperature shifts using nanoDSF, suggesting binding to BlaR-SD. SPR determined compound 1 has a binding affinity of 31 M to BlaR-SD. Growth curves showing hypersusceptibility of S. aureus penicillin-binding proteins (PBP) antisense strains show Compound 1 targets PBP2 and PBP2a. SEM images showed severe disruption in the S. aureus cell wall. The time-kill assay showed 3-log reduction in bacterial count when N315 MRSA was treated with compound 1 and oxacillin. |
| Conclusions/Discussion: | In conclusion, compound 1 targets BlaR-SD, PBP2, and PBP2a, which restores S. aureus susceptibility to treatment by oxacillin. |
| Translational/Human Health Impact: | There are currently few antibiotics available in the clinic capable of treating MRSA infections. These findings open new treatment options for patients infected with MRSA. |
