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The ever increasing problem of antibiotic resistance necessitates a search for new drug molecules that would target novel proteins in the prokaryotic system. FtsZ is one such target protein involved in the bacterial cell division machinery. In this study, we have shown that berberine, a natural plant alkaloid, targets Escherichia coli FtsZ, inhibits the assembly kinetics of the Z-ring, and perturbs cytokinesis. It also destabilizes FtsZ protofilaments and inhibits the FtsZ GTPase activity. Saturation transfer difference NMR spectroscopy of the FtsZ-berberine complex revealed that the dimethoxy groups, isoquinoline nucleus, and benzodioxolo ring of berberine are intimately involved in the interaction with FtsZ. Berberine perturbs the Z-ring morphology by disturbing its typical midcell localization and reduces the frequency of Z-rings per unit cell length to half. Berberine binds FtsZ with high affinity ( K D approximately 0.023 microM) and displaces bis-ANS, suggesting that it may bind FtsZ in a hydrophobic pocket. Isothermal titration calorimetry suggests that the FtsZ-berberine interaction occurs spontaneously and is enthalpy/entropy-driven. In silico molecular modeling suggests that the rearrangement of the side chains of the hydrophobic residues in the GTP binding pocket may facilitate the binding of the berberine to FtsZ and lead to inhibition of the association between FtsZ monomers. Together, these results clearly indicate the inhibitory role of berberine on the assembly function of FtsZ, establishing it as a novel FtsZ inhibitor that halts the first stage in bacterial cell division.

A photoaffinity analogue of taxol, N-([3,5-3H]-4-azidobenzoyl)-N-debenzoyltaxol (7), was synthesized and used to photolabel microtubules. Approximately 20% of the noncovalently bound analogue becomes covalently bound upon irradiation at 300 nm. Incorporated label was stable to a 50% ethanol solution and sodium dodecyl sulfate. About 80% of the incorporated label was found in the beta-subunit and 20% in the alpha-subunit. Incorporation did not occur into unpolymerized tubulin, consistent with the fact that taxol binds only to polymerized tubulin, and was decreased by the presence of taxol. Little or no nonspecific labeling occurs. This analogue is currently being used to identify taxol binding site(s) on tubulin.

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