Chrysophaentins are competitive inhibitors of FtsZ and inhibit Z-ring formation in live bacteria

Abstract

The bacterial cell division protein FtsZ polymerizes in a GTP-dependent manner to form a Z-ring that marks the plane of division. As a validated antimicrobial target, considerable efforts have been devoted to identify small molecule FtsZ inhibitors. We recently discovered the chrysophaentins, a novel suite of marine natural products that inhibit FtsZ activity in vitro. These natural products along with a synthetic hemi-chrysophaentin exhibit strong antimicrobial activity toward a broad spectrum of Gram-positive pathogens. To define their mechanisms of FtsZ inhibition and determine their in vivo effects in live bacteria, we used GTPase assays and fluorescence anisotropy to show that hemi-chrysophaentin competitively inhibits FtsZ activity. Furthermore, we developed a model system using a permeable E. coli strain, envA1, together with an inducible FtsZ-yellow fluorescent protein construct to show by fluorescence microscopy that both chrysophaentin A and hemi-chrysophaentin disrupt Z-rings in live bacteria. We tested the E. coli system further by reproducing phenotypes observed for zantrins Z1 and Z3, and demonstrate that the alkaloid berberine, a reported FtsZ inhibitor, exhibits auto-fluorescence, making it incompatible with systems that employ GFP or YFP tagged FtsZ. These studies describe unique examples of non-nucleotide, competitive FtsZ inhibitors that disrupt FtsZ in vivo, together with a model system that should be useful for in vivo testing of FtsZ inhibitor leads that have been identified through in vitro screens but are unable to penetrate the Gram-negative outer membrane.

Recent advances in the discovery and development of antibacterial agents targeting the cell-division protein FtsZ

“…52 Keffer et al synthesized hemi-chrysophaentin and found that its mechanism of action was similar to that of chrysophaentin A. 53 Both chrysophaentin A and hemi-chrysophaetin did not exhibit antimicrobial activity against gram-negative bacteria, but inhibited Ec -FtsZ in vitro , which could be ascribed to these compounds’ inability to pass through the outer membrane of gram-negative bacteria. Thus, in order to test this hypothesis, the activities of chrysophaentin A and hemi-chrysophaetin were determined against E. coli envA1 , a strain with increased compound permeability, and found the IC 50 values to be 27 μM and 84 μM, respectively, which proved the hypothesis.…”

Understanding Nucleotide-Regulated FtsZ Filament Dynamics and the Monomer Assembly Switch with Large-Scale Atomistic Simulations

“…FtsZ has been validated as the target of the in vivo effective antibacterial compound PC190723 (7), which modulates FtsZ assembly by stabilizing its polymers (8,9) and binds into the cleft between the C-terminal domain and the core helix H7 of this protein (9)(10)(11). Moreover, several nucleotide analogs (12,13) and nonnucleotide compounds that selectively target the nucleotide site of FtsZ have also been identified (14)(15)(16).…”

Identification of Agents Targeting Ftsz Assembly

“…NMR experiments further demonstrated that chrysophaentin A 18 occludes a large portion of the GTP-binding site of FtsZ, which may explain its mechanism of action [72]. Hemichrysophaentin 19 also inhibits the GTPase activity of both organisms (E. coli IC 50 37 ± 7 μM; S. aureus 38 ± 9 μM) [102]. Hemichrysophaentin 19 competes with GTP for binding to the FtsZ nucleotide pocket and, like chrysophaentin A, decreases the polymerization of FtsZ, resulting in the disruption of Z-ring localization and integrity.…”