Alkoxy- and amino-benzamides as inhibitors of the bacterial cell division protein FtsZ and antibacterial agents

Abstract

Since their discovery in the 20th century, antibiotics have been prescribed for patients with bacterial infections. The first commercially available antibiotic was penicillin, which was discovered in 1928 by Alexander Fleming in St. Mary’s Hospital, UK. Penicillin was effective to inhibit the growth of disease-causing microorganisms. However, in 1947, four years after the mass-production of penicillin, the first penicillin resistance case was identified. Since then, scientists have been looking for new targets to inhibit the bacterial growth. Among them, the bacterial cell division protein, filament temperature-sensitive Z (FtsZ), is a promising target for the development of new antibiotics. FtsZ protein is an essential protein in bacterial cytoplasmic division. A GTPase active site is formed when two FtsZ monomers are joined together in head-to-tail manner. The presence of GTP induces the polymerization of FtsZ in the middle of the cell. FtsZ polymers act as a platform to recruit other cell division proteins in subsequent cytoplasmic division. The FtsZ homologue in eukaryotic cell is tubulin, which has similar three-dimensional structure and functions in prokaryotic cells. However, tubulin only shares a limited sequence identity with FtsZ. Therefore, scientists believe FtsZ inhibitor can perturb the normal functions of FtsZ without affecting tubulin in human. 3-Methoxybenzamide (3-MBA) has been shown to inhibit cell division in Bacillus subtilis 168. PC190723, a derivative of 3-MBA, inhibits the growth of Bacillus subtilis 168 at 0.5 μM. Although PC190723 is by far the most effective FtsZ inhibitor, the oral bioavailability of PC190723 in mice is only 57%. In this project, derivatives of alkoxybenzamide and aminobenzamide were synthesized. Several of them can effectively inhibit the growth of Bacillus subtilis 168. Among them, 4-F332 inhibits the growth of Bacillus subtilis 168 and Staphylococcus aureus 29213 with MICs at 3.13 μM and 12.5 μM whereas 4-F361 does the same at 1.57 μM and 3.13 μM respectively. Both 4-F332 and 4-F361 inhibit the polymerization of Staphylococcus aureus FtsZ protein as demonstrated in light scattering assay. In GTPase activity assay, however, no significant inhibition was observed. Both 4-F332 and 4-F361 induce filamentation in Bacillus subtilis 168. The morphology of FtsZ polymers was observed by transmission electron microscope. Both 4-F332 and 4-F361 can reduce the length and thickness of Staphylococcus aureus FtsZ polymers. Cytotoxicity assay showed that 4-F361 has a selectivity ratio of 19, which indicates it has the potential to be developed into an antibiotic for clinical use.