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Restoring Tarnished Gold

Assay could identify compounds effective against antibiotic-resistant bacteria

“The so-called golden age of antibiotics is drawing to a close,” remarks Christopher Davies, PhD, Professor in the Department of Biochemistry and Molecular Biology at MUSC. The advent of penicillin and other β-lactam antibiotics in the early 20th century seemed to spell victory in the war against pathogens. It turns out we underestimated our enemy. In recent decades, bacteria have fought back by developing resistance to our most powerful antibiotics.
It’s an arms race in a way,” says Davies. “The more we throw at bacteria, the more they evolve resistance. The writing is on the wall for current antibiotics.”

β-lactam antibiotics inhibit penicillin-binding proteins (PBPs), transpeptidases that synthesize the bacterial cell wall. Structurally similar to the peptide substrates of PBPs, β-lactam antibiotics are able to bind – and block – the active site of the PBPs. As a result, holes appear in the bacterial cell wall, leading the cell to rupture and die.

Bacteria resist β-lactam antibiotics by attacking them with an enzyme, by blocking their entry via reduction in the size of channels in the outer membrane, by expelling them with pumps, or by altering the active site of the PBP to make it a less attractive target.

The story of our efforts to control Neisseria gonorrhoeae illustrates just how effective such bacterial resistance can be. After seemingly being dealt a death blow by the introduction of β-lactam antibiotics in the 1940s, N. gonorrhoeae began to strike back, developing resistance first to penicillin and then to a long line of other antibiotics, including narrow-spectrum cephalosporins, tetracyclines, macrolides, and fluoroquinolones. In recent years, only two extended-spectrum cephalosporins – ceftriaxone and cefixime – have remained effective. In 2011, a cephalosporin-resistant strain of N. gonorrhoeae was reported in Japan. Subsequently, the Centers for Disease Control and Prevention withdrew its recommendation of cefixime as a first-line treatment for gonorrhea in the U.S. due to this growing resistance. This has left treatment options very limited and may portend a new era of untreatable gonorrhea.

In an effort to identify critically needed compounds with efficacy against resistant strains of N. gonorrhoeae, Davies and his team developed a high-throughput assay with which they screened a 50,000-compound library in MUSC’s Drug Discovery Center.1 To develop the assay, they focused on a tried-and-true target – a PBP, specifically PBP2. At the heart of their assay is Bocillin-FL, a fluorescent penicillin that competes with the tested compound to bind to PBP2 and emits different intensities of fluorescence in its bound and free forms. If the tested compound is a potent PBP2 inhibitor, it will successfully bind with PBP2, and Bocillin will remain in solution in its free form. In contrast, if the compound is a poor inhibitor, Bocillin binds to PBP2. Using this assay, the team identified several compounds with antimicrobial activity against N. gonorrhoeae – some effective even against resistant strains.

Because PBPs occur in most bacteria, the assay can be used to identify novel compounds against virtually any bacterial pathogen. The golden age of β-lactam antibiotics may be almost over, but Davies is restoring some of their luster by using PBPs, their primary target, to identify compounds that could one day protect us against increasingly resistant pathogens.

Reference

1 Fedarovich A, Djordjevic KA, Swanson SM, Peterson YK, Nicholas RA, Davies C. High-throughput screening for novel inhibitors of Neisseria gonorrhoeae penicillin-binding protein 2. PLoS One. 2012;7(9):e44918.