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The prevalence of multidrug resistance among clinically significant bacteria calls for the urgent development of new antibiotics with novel mechanisms of action. In this study, a new small molecule exhibiting excellent inhibition of bacterial cell division with potent antibacterial activity was discovered through cell-based screening. The compound exhibits a broad spectrum of bactericidal activity, including the methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and NDM-1 Escherichia coli. The in vitro and in vivo results suggested that this compound disrupts the dynamic assembly of FtsZ protein and Z-ring formation through stimulating FtsZ polymerization. Moreover, this compound exhibits no activity on mammalian tubulin polymerization and shows low cytotoxicity on mammalian cells. Taken together, these findings could provide a new chemotype for development of antibacterials with FtsZ as the target.

Inhibition of the functional activity of Filamenting temperature-sensitive mutant Z (FtsZ) protein, an essential and highly conserved bacterial cytokinesis protein, is a promising approach for the development of a new class of antibacterial agents. Berberine, a benzylisoquinoline alkaloid widely used in traditional Chinese and native American medicines for its antimicrobial properties, has been recently reported to inhibit FtsZ. Using a combination of in silico structure-based design and in vitro biological assays, 9-phenoxyalkyl berberine derivatives were identified as potent FtsZ inhibitors. Compared to the parent compound berberine, the derivatives showed a significant enhancement of antibacterial activity against clinically relevant bacteria, and an improved potency against the GTPase activity and polymerization of FtsZ. The most potent compound 2 strongly inhibited the proliferation of Gram-positive bacteria, including methicillin-resistant S. aureus and vancomycin-resistant E. faecium, with MIC values between 2 and 4 µg/mL, and was active against the Gram-negative E. coli and K. pneumoniae, with MIC values of 32 and 64 µg/mL respectively. The compound perturbed the formation of cytokinetic Z-ring in E. coli. Also, the compound interfered with in vitro polymerization of S. aureus FtsZ. Taken together, the chemical modification of berberine with 9-phenoxyalkyl substituent groups greatly improved the antibacterial activity via targeting FtsZ.

The increasing emergence of new bacterial [beta]-lactamases that can efficiently hydrolyze [beta]-lactam antibiotics to clinically inactive carboxylic acids has created an intractable problem in the treatment of bacterial infections, and it is highly desirable to develop a useful tool that can rapidly screen bacteria for [beta]-lactamases against a variety of antibiotic candidates in a high-throughput manner. This paper describes the use of a fluorescein-labeled [beta]-lactamase mutant (E166Cf) as a convenient fluorescent tool to screen [beta]-lactamases, including the Bacillus cereus [beta]-lactamase I (PenPC), B. cereus [beta]-lactamase II, Bacillus licheniformis PenP, Escherichia coli TEM-1, and Enterobacter cloacae P99 against various [beta]-lactam antibiotics (penicillin G, penicillin V, ampicillin, cefuroxime, cefoxitin, moxalactam, cephaloridine), using a 96-well microplate reader. The E166Cf mutant was constructed by replacing Glu166 on the flexible [OMEGA]-loop, which is close to the enzyme's active site, with a cysteine residue on a class A [beta]-lactamase (B. cereus PenPC) and subsequently labeling the mutant with thiol-reactive fluorescein-5-maleimide. Such modifications significantly impaired the hydrolytic activity of the E166Cf mutant compared to that of the wild-type enzyme. The fluorescence intensity of the E166Cf mutant increases in the presence of [beta]-lactam antibiotics. For antibiotics that are resistant to hydrolysis by the E166Cf mutant (cefuroxime, cefoxitin, moxalactam), the fluorescence signal slowly increases until it reaches a plateau. For antibiotics that can be slowly hydrolyzed by the E166Cf mutant (penicillin G, penicillin V, ampicillin), the fluorescence signal rapidly increases to the plateau and then declines after a prolonged incubation. The E166Cf mutant retains its characteristic pattern of fluorescence signals in the presence of both bacterial [beta]-lactamases and [beta]-lactamase-resistant antibiotics. In contrast, in the presence of both bacterial [beta]-lactamases and [beta]-lactamase-sensitive antibiotics, the fluorescence signals of the E166Cf mutant were decreased. The fluorescence signals from the E166Cf mutant allow an unambiguous differentiation of [beta]-lactamase-resistant antibiotics from [beta]-lactamase-sensitive ones in the screening of bacterial [beta]-lactamases against a panel of antibiotic candidates. This simple method may provide an alternative tool in choosing potent [beta]-lactam antibiotics for treatment of bacterial infections.