BT‐benzo‐29 inhibits bacterial cell proliferation by perturbing FtsZ assembly

Abstract

We have identified a potent antibacterial agent N‐(4‐sec‐butylphenyl)‐2‐(thiophen‐2‐yl)‐1H‐benzo[d]imidazole‐4‐carboxamide (BT‐benzo‐29) from a library of benzimidazole derivatives that stalled bacterial division by inhibiting FtsZ assembly. A short (5 min) exposure of BT‐benzo‐29 disassembled the cytokinetic Z‐ring in Bacillus subtilis cells without affecting the cell length and nucleoids. BT‐benzo‐29 also perturbed the localization of early and late division proteins such as FtsA, ZapA and SepF at the mid‐cell. Further, BT‐benzo‐29 bound to FtsZ with a dissociation constant of 24 ± 3 μm and inhibited the assembly and GTPase activity of purified FtsZ. A docking analysis suggested that BT‐benzo‐29 may bind to FtsZ at the C‐terminal domain near the T7 loop. BT‐benzo‐29 displayed significantly weaker inhibitory effects on the assembly and GTPase activity of two mutants (L272A and V275A) of FtsZ supporting the prediction of the docking analysis. Further, BT‐benzo‐29 did not appear to inhibit DNA duplication and nucleoid segregation and it did not perturb the membrane potential of B. subtilis cells. The results suggested that BT‐benzo‐29 exerts its potent antibacterial activity by inhibiting FtsZ assembly. Interestingly, BT‐benzo‐29 did not affect the membrane integrity of mammalian red blood cells. BT‐benzo‐29 bound to tubulin with a much weaker affinity than FtsZ and exerted significantly weaker effects on mammalian cells than on the bacterial cells indicating that the compound may have a strong antibacterial potential.

Lessons from bacterial homolog of tubulin, FtsZ for microtubule dynamics

“…One of the benzimidazoles, BT-benzo-29, was found to inhibit the formation of Z-ring and to perturb the localization of FtsZ-interacting proteins in bacteria as seen in Fig. 6 (Ray et al 2015). The localization of GFP-tagged proteins -FtsZ, FtsA, SepF and ZapA in Bacillus subtilis cells are disturbed in the presence of the drug (Ray et al 2015).…”

“…6 (Ray et al 2015). The localization of GFP-tagged proteins -FtsZ, FtsA, SepF and ZapA in Bacillus subtilis cells are disturbed in the presence of the drug (Ray et al 2015). It had a minimal effect on the assembly of tubulin.…”

“…Docking of SB-RA-2001 showed the compound to bind at the site of PC190723 binding, near the cleft at the C-terminal (Singh et al 2014). The putative binding site for BT-benzo-29 was identified using site-directed mutagenesis and molecular docking studies (Ray et al 2015). The compound binds to the C-terminal region of the protein involving hydrophobic residues leucine, glycine, proline and valine (Ray et al 2015).…”

“…The putative binding site for BT-benzo-29 was identified using site-directed mutagenesis and molecular docking studies (Ray et al 2015). The compound binds to the C-terminal region of the protein involving hydrophobic residues leucine, glycine, proline and valine (Ray et al 2015). STD NMR and docking studies suggested the binding site for cinnamaldehyde, an antibacterial agent that targets FtsZ, to be around the T7 loop (Domadia et al 2007) similar to SulA, a known cell division inhibitor that interacts with FtsZ (Cordell et al 2003).…”

“…However, agents targeting tubulin such as colchicine, paclitaxel, vinblastine and benomyl have no discernable effect on FtsZ assembly (Yu & Margolin 1998, White et al 2000, Beuria et al 2005. Most of the FtsZ inhibitors currently under study such as PC197023, BT-benzo-29 and OTBA also show minimal effects on tubulin polymerization (Haydon et al 2008, Beuria et al 2009, Ray et al 2015. The differential effect of these compounds raises the possibilities of finding inhibitors that specifically target FtsZ without harming the eukaryotic host.…”