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Description : Review on Discovery of an antivirulence compound that reverses ß-lactam resistance in MRSA

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Published on : Nov 25, 2020 | Views : 2065 | Downloads : 5

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By: Ahmed Abouelghar 201600649 Ashrakat Hamam 2016010201 Supervised by: Dr. Reem Khidr Discovery of an antivirulence compound that reverses β-lactam resistance in MRSA Omar M. El-Halfawy, Tomasz L. Czarny, Ronald S. Flannagan, Jonathan Day, José Carlos Bozelli Jr., Robert C. Kuiack, Ahmed Salim, Philip Eckert, Richard M. Epand, Martin J. McGavin, Michael G. Organ, David E. Heinrichs & Eric D. Brown
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Introduction Community and nosocomial S. aureus acquired infections are the most common type of infections worldwide and a leading cause of mortality. The last approved newly discovered class of antibiotics was discovered more than 30 years ago. Two new promising approaches that are under investigation now are antibiotic adjuvants and antivirulence. The aim of this work was to find a drug that has both antibiotic adjuvant and antivirulence activities.
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Intro Targocil is an inhibitor of TarG, a wall teichoic acid (WTA) flippase. WTA are Gram-positive cell wall phosphate-rich polymers and they play a role β-lactams resistance and s. auras virulence. GraR is a is the response regulator of GraRS two component regulatory system. It regulates the expression of many genes related to cell envelope stress response. Of these genes are the mprF and dltABCD that encodes enzymes that increases the positive charge of the cell surface by adding lysine to the membrane phospholipids and d-alanine to WTAs respectively.
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References Results, High-throughput screening and hit prioritization: -544 primary hits. -9 secondary hits. -MAC-545496 and MAC-533252 showed high potency. -MAC-545496 was protarized. A general schematic of the screening workflow. Screen of synergism with the cefuroxime at 1/8 MIC (64 μg/mL) and the primary hits at 10 μM concentration.
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References Results, Mode of action determination: Nebraska transposon mutant library (NTML) screen.
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References Results, Mode of action determination: Checkerboard assays of MAC-545496 and cefuroxime against the graR::Tn mutant Checkerboard assays of MAC-545496 and cefuroxime against a spontaneous resistant Mutant (G217* GraR)
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References Results, Mode of action determination: Dose–response curve of MAC-545496 on the expression of mprF following luciferase activity in the wild-type S. aureus USA300
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References Results: Effect of MAC-545496 on other bacteria: In this study MAC545496 has not tested only on S. aureus, but also in other gram positive bacteria. It was found that the drug does not synergize with B-lactams (cefuroxime) against three of S. epidermidis isolates and has no effect on vancomycin. Fig.4: MAC-545496 does not alter the antibiotic susceptibility of other bacteria
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Fig.5:Alignment of the ribbon structures of USA300 GraR full length (Green) and its S. epidermidis NIH04008 homolog (grey, left panel) or the USA300 GraR truncated protein detected in the MAC-545496 resistant mutants (Red, right panel) References Results However, there is a high (91%) similarity between GraXRS VraFG systems based on aligning both of the proteins as shown in figure below.
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References Results: MAC-545496 inhibits biofilm formation: MAC-545496 was found only to inhibit the citrate induced biofilm formation in wild type and this is based on the concentration of the drug as shown in figure 6. Fig.6: MAC-545496 inhibits S. aureus USA300 biofilm formation
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References Results: In cellulo and in vivo efficacy as monotherapy: It was found that 20 µgml−1 concentration of MAC-545496 was enough to completely inhibit the S. aureus replication in macrophages. Fig.8: MAC-545496 abrogates intracellular survival of S. aureus USA300.
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References Results: In cellulo and in vivo efficacy as monotherapy: Using MAC-545496 as in vivo treatment for MRSA infected Galleria mellonella larvae, it was successful. Fig.9: MAC-545496 attenuates virulence in vivo. .
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References Results: Drug Solubility Problem MAC-545496 has poor aqueous solubility which hindered its systematic testing in a mammalian model. However, it was reported that GraR inhibition is efficient against MRSA infection in mouse model[21]. MAC-545496 was tested in human serum and it was inhibited totally, as it was able to show some synergy with cefuroxime against S. aureus USA300. Another efforts were directed to the synthesis of the drug to increase its potency without the loss of nitro and chloro-pyridinyl group as they play a major role in its activity. There alot of efforts directed to increase the solubility of the drug to be tested in mammalian models as it represent a viable target for monotherapy against MRSA .
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References Methodology 1.Strains and reagents 2.High-throughput screening: 3.Antimicrobial-susceptibility testing 4.Nebraska transposon mutant library screen(NTML) 5.Selection and characterization of spontaneous resistant mutants 6.General molecular techniques 7.Bioluminescence assays: 8.Protein overexpression and purification: 9.Isothermal titration calorimetry binding assays 10.Circular dichroism spectroscopy: 11.Biofilm formation assays 12.In vivo infection of Galleria mellonella larvae: 13.Infection of RAW 264.7 macrophages 14.Synthesis of MAC-545496 analogs and their characterization 15.Statistical Analysis
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Summary and conclusion References To conclude, this work is a proof of concept that drugs with double antivirulence effect and antibiotic adjuvant properties can be a powerful tool in our fight against nulti-drug resistant bugs. MAC-545496 on nanomolar doses poses both β-lactams adjuvance, and attenuate the virulence of MRSA USA300. The drug did not affect the growth of commensal nor gram-negative bacteria. Although the drug has low water solubility, it poses a lead compound from which other GraR inhibitors can be developed.
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Proposed Modification References We propose the addition of a phosphate group salt with Sodium instead of the Cl to increase water solubility. This would be a prodrug, once inside the system, phosphatases of the body may remove the phosphate group leaving a hydroxyl group which is a classical chemical isomer of the Cl.
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References References References: 1. Prioritization of Pathogens to Guide Discovery, Research and Development of New Antibiotics for Drug-Resistant Bacterial Infections Including Tuberculosis (World Health Organization, 2017). 2. Boucher, H. W. et al. Bad bugs, no drugs: no ESKAPE! an update from the Infectious Diseases Society of America. Clin. Infect. Dis. 48, 1–12 (2009). 3. Nannini, E., Murray, B. E. & Arias, C. A. Resistance or decreased susceptibility to glycopeptides, daptomycin, and linezolid in methicillinresistant Staphylococcus aureus. Curr. Opin. Pharmacol. 10, 516–521 (2010). 4. Lowy, F. D. Staphylococcus aureus infections. New Engl. J. Med. 339, 520–532 (1998). 5. Wright, G. D. & Sutherland, A. D. New strategies for combating multidrugresistant bacteria. Trends Mol. Med. 13, 260–267 (2007). 6. Maura, D., Ballok, A. E. & Rahme, L. G. Considerations and caveats in anti-virulence drug development. Curr. Opin. Microbiol. 33, 41–46 (2016). 7. Lee, K., Campbell, J., Swoboda, J. G., Cuny, G. D. & Walker, S. Development of improved inhibitors of wall teichoic acid biosynthesis with potent activity against Staphylococcus aureus. Bioorg. Med. Chem. Lett. 20, 1767–1770 (2010). 8. Sewell, E. W. & Brown, E. D. Taking aim at wall teichoic acid synthesis: new biology and new leads for antibiotics. J. Antibiot. 67, 43–51 (2014). 9. D’Elia, M. A., Henderson, J. A., Beveridge, T. J., Heinrichs, D. E. & Brown, E. D. Te N-acetylmannosamine transferase catalyzes the frst committed step of teichoic acid assembly in Bacillus subtilis and Staphylococcus aureus. J. Bacteriol. 191, 4030–4034 (2009).
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References References 10. D’Elia, M. A., Millar, K. E., Beveridge, T. J. & Brown, E. D. Wall teichoic acid polymers are dispensable for cell viability in Bacillus subtilis. J. Bacteriol. 188, 8313–8316 (2006). 11. D’Elia, M. A. et al. Lesions in teichoic acid biosynthesis in Staphylococcus aureus lead to a lethal gain of function in the otherwise dispensable pathway. J. Bacteriol. 188, 4183–4189 (2006). 12. Lee, S. H. et al. TarO-specifc inhibitors of wall teichoic acid biosynthesis restore β-lactam efcacy against methicillin-resistant staphylococci. Sci. Transl. Med. 8, 329ra332 (2016). 13. Czarny, T. L. & Brown, E. D. A small-molecule screening platform for the discovery of inhibitors of undecaprenyl diphosphate synthase. ACS Infect. Dis. 2, 489–499 (2016). 14. Farha, M. A. et al. Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase. Proc. Natl Acad. Sci. USA 112, 11048–11053 (2015). 15. Santa Maria, J. P. Jr. et al. Compound-gene interaction mapping reveals distinct roles for Staphylococcus aureus teichoic acids. Proc. Natl Acad. Sci. USA 111, 12510–12515 (2014). 16. Falord, M., Karimova, G., Hiron, A. & Msadek, T. GraXSR proteins interact with the VraFG ABC transporter to form a fve-component system required for cationic antimicrobial peptide sensing and resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 56, 1047–1058 (2012). 17. Falord, M., Mader, U., Hiron, A., Debarbouille, M. & Msadek, T. Investigation of the Staphylococcus aureus GraSR regulon reveals novel links to virulence, stress response and cell wall signal transduction pathways. PLoS One 6, e21323 (2011). 18. Yang, S. J. et al. Te Staphylococcus aureus two-component regulatory system, GraRS, senses and confers resistance to selected cationic antimicrobial peptides. Infect. Immun. 80, 74–81 (2012).
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References References 19. Kraus, D. et al. Te GraRS regulatory system controls Staphylococcus aureus susceptibility to antimicrobial host defenses. BMC Microbiol. 8, 85 (2008). 20. Li, M. et al. Te antimicrobial peptide-sensing system aps of Staphylococcus aureus. Mol. Microbiol. 66, 1136–1147 (2007). 21. Flannagan, R. S., Kuiack, R. C., McGavin, M. J. & Heinrichs, D. E. Staphylococcus aureus uses the GraXRS regulatory system to sense and adapt to the acidifed phagolysosome in macrophages. MBio 9, e01143-18 (2018). 22. Mannala, G. K. et al. Whole-genome comparison of high and low virulent Staphylococcus aureus isolates inducing implant-associated bone infections. Int J. Med. Microbiol. 308, 505–513 (2018). 23. Shanks, R. M. et al. Genetic evidence for an alternative citrate-dependent bioflm formation pathway in Staphylococcus aureus that is dependent on fbronectin binding proteins and the GraRS two-component regulatory system. Infect. Immun. 76, 2469–2477 (2008). 24. Campbell, J. et al. An antibiotic that inhibits a late step in wall teichoic acid biosynthesis induces the cell wall stress stimulon in Staphylococcus aureus. Antimicrob. Agents Chemother. 56, 1810–1820 (2012). 25. Strauss, L. et al. Origin, evolution, and global transmission of communityacquired Staphylococcus aureus ST8. Proc. Natl Acad. Sci. USA 114, E10596–E10604 (2017). 26. Performance Standards for Antimicrobial Susceptibility Testing 27th Edition, CLSI Supplement M100 (Clinical and Laboratory Standards Institute, 2017). 27. Fey, P. D. et al. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. MBio 4, e00537-12 (2013). 28. Fridman, M. et al. Two unique phosphorylation-driven signaling pathways crosstalk in Staphylococcus aureus to modulate the cell-wall charge: Stk1/Stp1 meets GraSR. Biochemistry 52, 7975–7986 (2013).
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References References 29. Vestergaard, M. et al. Inhibition of the ATP synthase eliminates the intrinsic resistance of Staphylococcus aureus towards polymyxins. MBio 8, e01114-17 (2017). 30. Rajagopal, M et al. Multidrug intrinsic resistance factors in Staphylococcus aureus identifed by profling ftness within high-diversity transposon libraries. MBio 7, e00950-16 (2016). 31. Vestergaard, M. et al. Genome-wide identifcation of antimicrobial intrinsicresistance determinants in Staphylococcus aureus. Front. Microbiol. 7, 2018 (2016). 32. Martinez-Hackert, E. & Stock, A. M. Structural relationships in the OmpR family of winged-helix transcription factors. J. Mol. Biol. 269, 301–312 (1997). 33. Kaiser, J. C. et al. Repression of branched-chain amino acid synthesis in Staphylococcus aureus is mediated by isoleucine via CodY, and by a leucine-rich attenuator peptide. PLoS Genet. 14, e1007159 (2018). The rest is in the report.
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