Chrysophaentins A-H, antibacterial bisdiarylbutene macrocycles that inhibit the bacterial cell division protein FtsZ

Abstract

Eight new antimicrobial natural products named chrysophaentins A-H belonging to a new structural class have been isolated from the marine chrysophyte alga Chrysophaeum taylori. Their structures were determined by extensive 2D NMR and MS techniques and are characterized by the presence of two polyhalogenated, polyoxygenated omega,omega'-diarylbutene units connected by two ether bonds to form the suite of macrocyclic natural products. Chrysophaentin A, the most potent of these antibiotics, inhibited the growth of clinically relevant Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MIC(50) 1.5 +/- 0.7 microg/mL), multidrug-resistant S. aureus (1.3 +/- 0.4 microg/mL), and vancomycin-resistant Enterococcus faecium (MIC(50) 2.9 +/- 0.8 microg/mL). In vitro enzyme assays and transmission electron microscopy showed chrysophaentin A to inhibit the GTPase activity of the bacterial cytoskeletal protein FtsZ with an IC(50) value of 6.7 +/- 1.7 microg/mL, as well as GTP-induced formation of FtsZ protofilaments. Saturation Transfer Difference (STD) NMR experiments further confirmed chrysophaentin A binds to FtsZ, and NMR competition experiments with GTPgammaS showed chrysophaentin A and GTP to bind competitively to FtsZ. Last, molecular docking simulations provided a low energy model in which chrysophaentin A binds in and occludes a large portion of the GTP binding site of FtsZ in a manner that is consistent with the binding epitope determined by STD NMR.

Figure 1

Key HMBC correlations used to establish partial structures corresponding to fragments I and II in compound 1. ¹H–¹³C and OH–¹³C correlations are shown by black and red arrows, respectively.

Figure 2

Global minimum energy conformer of 1 obtained by molecular dynamics and PRCG minimization. Selected strong ROE’s are indicated with double-sided black arrows together with corresponding interproton distances observed in the model. Blue arrows show key ROEs used to connect fragments I and II.

Figure 3

Transmission electron micrographs of FtsZ in the presence of (A) GTP, and (B) GTP and chrysophaentin A. (A) Polymerization of recombinant E. coli FtsZ was induced with addition of GTP for 5 min at rt before transferring to a carbon grid, staining with 3% uranyl acetate, and visualizing by TEM, 44 000 x. (B) Incubation of FtsZ with 1 (50 μM) prior to addition of GTP inhibits polymerization and protofilament formation. No filaments were observed over the entire grid shown in panel B.

Figure 4

Reference (black) and STD NMR difference (red) spectra of chrysophaentin A in complex with FtsZ. Spectral expansion shows non-overlapped aromatic and olefinic signals of 1 displaying the strongest enhancements upon binding to FtsZ. Samples comprising 100:1 1:FtsZ were prepared in 20 mM NaPO₄, 50 mM NaCl, 5 mM MgCl₂, pH 6.8, and spectra recorded at 298 K. Overlaid spectra were normalized to the signal for H-3 (δ 6.85), which gave the strongest enhancement.

Figure 5

Competition of chrysophaentin A and GTPγS binding to FtsZ by STD NMR. (a) Expanded ¹H STD NMR spectrum of chrysophaentin A (1.25 mM) in the presence of FtsZ (12.5 μM). (b-e) STD NMR spectra recorded on the same sample after addition of (b) 0.5, (c) 1.0, (d) 2.0 and (e) 3.0 eq GTPγS. Final concentrations of GTPγS were 625 μM, 1.25 mM, 2.50 mM and 3.75 mM, respectively.

Figure 6

Molecular docking of 1 to FtsZ with chrysophaentin A bound in the GTP binding site. (A) Docked model of 1 bound to an E. coli FtsZ homology model; FtsZ is displayed as a white surface where atoms within 5 Å of docked 1 are colored yellow; chrysophaentin A is shown in ball and stick representation with chlorine and oxygen atoms colored green and red, respectively. Protons displaying the strongest enhancements are labeled in italics. (B) Superposition of the docked model shown in panel A with GDP bound FtsZ (pdb accession number 1ofu.pdb17). GDP (blue) is shown in a ball and stick representation, and surfaces of residues comprising the GTP binding site of FtsZ are colored blue. Docking was performed using the program Autodock Vina 1.0.3.