Combretastatin A-4 (CA-4) in phosphate and serine pro-drug forms is under phase II clinical trials. With our interest of discovering CA-4 inspired new chemical entities, a novel series of 4,5-diaryl-2-aminoimidazole analogues of the compound was designed and synthesized by an efficient and diversity feasible route involving atom economical arene C-H bond arylation. Interestingly, four compounds showed potent cell-based antiproliferative activities in nanomolar concentrations. Among the compounds, compound 12 inhibited the proliferation of several types of cancer cells much more efficiently than CA-4. It depolymerized microtubules, induced spindle defects, and stalled mitosis in cells. Compound 12 bound to tubulin and inhibited the polymerization of tubulin in vitro. In addition, podophyllotoxin and CA-4 inhibited the binding of compound 12 to tubulin. The distinctive pharmacophoric features of the bridging motif as well as quinoline nucleus were explored. We noted also a valuable quality of compound 12 as a potential probe in characterizing new CA-4 analogues.

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.

A cathelin-related antimicrobial peptide (CRAMP) of 37 amino acid residues is thought to regulate innate immunity and provide a host defense mechanism in mammals. Here, a part of the CRAMP peptide, CRAMP (16-33) (GEKLKKIGQKIKNFFQKL), was found to bind to FtsZ and to inhibit the assembly and GTPase activity of FtsZ in vitro. A computational analysis indicated that CRAMP (16-33) binds in the cavity of the T7 loop of FtsZ. Both hydrophobic and ionic interactions were involved in the binding interactions. Further, CRAMP (16-33) inhibited the formation of the FtsZ ring in bacteria, indicating that it inhibited bacterial cell division by inhibiting FtsZ assembly.

FtsZ monomers assemble to form a dynamic Z-ring at the midcell position in bacteria that coordinates bacterial cell division. Antibacterial agents plumbagin and SB-RA-2001 were found to bind to FtsZ and to inhibit Z-ring formation in bacteria. Docking analysis indicated similar binding regions for these two inhibitors on FtsZ, and residue R191 was involved in the binding interaction with both compounds. In this work, the importance of R191 in FtsZ assembly and in bacterial cell division was analyzed. R191A-FtsZ exhibited significantly poorer polymerization ability. Further, the mutant FtsZ could poison the assembly of wild-type FtsZ (WT-FtsZ). The expression of R191A-FtsZ in Bacillus subtilis strain PL2084 perturbed Z-ring formation and produced filamentous cells, indicating that the mutation hindered the division of these cells. The results suggested that the R191A mutation is a dominant negative mutation of FtsZ. Molecular dynamics simulations of R191A-FtsZ and WT-FtsZ revealed a kink in helices H5 and H7 in the active site of R191A-FtsZ compared to that of WT-FtsZ, which is required for FtsZ assembly. The findings suggested that R191 is an important residue for FtsZ assembly, which can be targeted for the design of FtsZ inhibitors.

The assembly and disassembly of the FtsZ ring drives the division of bacteria cells, including Streptococcus pneumoniae, which causes pneumonia and meningitis. In contrast to FtsZ from other bacterial species, Streptococcus pneumoniae (Spn) FtsZ contains two tryptophan residues. Here, we demonstrate that the assembly and disassembly of Streptococcus pneumoniae FtsZ (SpnFtsZ) monomers can be monitored by the intrinsic tryptophan fluorescence of FtsZ. We found that the assembly of SpnFtsZ is closely associated with its GTPase activity. Guanosine 5′‐[β,γ‐imido]triphosphate, a nonhydrolyzable analog of GTP, stabilized the FtsZ filaments without inducing their bundling. Using intrinsic tryptophan fluorescence, light scattering, and electron microscopy, we could differentiate the effects of divalent calcium and magnesium on the assembly of FtsZ. Though Mg2+ increased the stability of the FtsZ filaments, it could not prevent the disassembly of the filaments under conditions where GTP was limiting. Thus, our results indicate that Mg2+ primarily enhances the longitudinal assembly of FtsZ. Low concentrations of Ca2+ strongly promoted the bundling of FtsZ filaments and inhibited the disassembly of the filaments, suggesting that low concentrations of Ca2+ enhance the lateral interactions between the FtsZ filaments. Interestingly, Ca2+ delayed the nucleation process of FtsZ assembly, indicating that Ca2+ exerts paradoxical effects on the assembly of FtsZ. However, higher concentrations of Ca2+ did not enhance the bundling of FtsZ filaments. In addition, Ca2+ altered the secondary structure of FtsZ and increased the fluorescence of the FtsZ‐1‐anilinonaphthalene‐8‐sulfonic acid complex, indicating that Ca2+ induces conformational changes in FtsZ. The study provides an interesting insight into the assembly of SpnFtsZ and its regulation by divalent cations.