SB-RA-2001 Inhibits Bacterial Proliferation by Targeting FtsZ Assembly

Abstract

FtsZ has been recognized as a promising antimicrobial drug target because of its vital role in bacterial cell division. In this work, we found that a taxane SB-RA-2001 inhibited the proliferation of Bacillus subtilis 168 and Mycobacterium smegmatis cells with minimal inhibitory concentrations of 38 and 60 μM, respectively. Cell lengths of these microorganisms increased remarkably in the presence of SB-RA-2001, indicating that it inhibits bacterial cytokinesis. SB-RA-2001 perturbed the formation of the FtsZ ring in B. subtilis 168 cells and also affected the localization of the late cell division protein, DivIVA, at the midcell position. Flow cytometric analysis of the SB-RA-2001-treated cells indicated that the compound did not affect the duplication of DNA in B. subtilis 168 cells. Further, SB-RA-2001 treatment did not affect the localization of the chromosomal partitioning protein, Spo0J, along the two ends of the nucleoids and also had no discernible effect on the nucleoid segregation in B. subtilis 168 cells. The agent also did not appear to perturb the membrane potential of B. subtilis 168 cells. In vitro, SB-RA-2001 bound to FtsZ with modest affinity, promoted the assembly and bundling of FtsZ protofilaments, and reduced the GTPase activity of FtsZ. GTP did not inhibit the binding of SB-RA-2001 to FtsZ, suggesting that it does not bind to the GTP binding site on FtsZ. A computational analysis indicated that SB-RA-2001 binds to FtsZ in the cleft region between the C-terminal domain and helix H7, and the binding site of SB-RA-2001 on FtsZ resembled that of PC190723, a well-characterized inhibitor of FtsZ. The findings collectively suggested that SB-RA-2001 inhibits bacterial proliferation by targeting the assembly dynamics of FtsZ, and this can be exploited further to develop potent FtsZ-targeted antimicrobials.

Lessons from bacterial homolog of tubulin, FtsZ for microtubule dynamics

“…A taxane derivative, SB-RA-2001, was recently identified to have minimum host cytotoxicity and MIC 99 value (Huang et al 2006). The compound promotes the assembly of purified FtsZ and stabilizes FtsZ filaments and unlike paclitaxel, SB-RA-2001 did not promote and stabilize the assembly of tubulin (Singh et al 2014). Without affecting karyokinesis, SB-RA-2001 seems to inhibit the localization of FtsZ to form the Z-ring (Fig.…”

“…It inhibited the proliferation of Bacillus subtilis and Mycobacterium smegmatis cells and induced filamentation in these cells. It is proposed that the mechanism of action of SB-RA-2001 on FtsZ is similar to that of paclitaxel on microtubules (Singh et al 2014).…”

“…This lead to reporting PC as a new class of FtsZ polymer-stabilizing agents (Andreu et al 2010). 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).…”

Small molecules targeting at the bacterial cell division protein FtsZ as potential antimicrobial agents

“…The main scaffold of taxane polycyclic core was kept unchanged in this study. SB-RA-2001 (40 in Figure 7) shows differences from paclitaxel (41 in Figure 7) mainly in two parts: (i) the alcohol at C-10 without the acetyl group; (ii) an unsaturated ester replaces the α-hydroxy-β-amido ester at C-13 [114]. These structural modifications resulted in better inhibition of B. subtilis FtsZ activity and showed antibacterial activity against both B. subtilis and M. smegmatis.…”

Identification of Agents Targeting Ftsz Assembly

“…Examples of commonly employed assays are a cell-based assay for inhibitors of the discovery of cell division inhibitors [63], malachite green determination of phosphate release during the turnover of GTP to GDP for the inhibition of GTPase activity [64], monitoring FtsZ assembly using light scattering [65,66], examination of cell morphology by difference interference contrast microscopy to determine the effect of agents on bacterial cell filamentation [64,67], bacterial growth inhibition and in vivo efficacy in a murine septicaemia model [64], (Table 1). For example, GTPase assays in the presence and absence of the neutral detergent Triton X-100 have been proposed in order to distinguish between specific and nonspecific (aggregation-based) ligand effects [35,68], but other research has shown that this detergent inhibits the assembly of both B. subtilis and E. coli FtsZ, casting doubt on its use in this regard [69].…”