Discovery of some FtsZ and FtsA inhibitors by virtual screening and studies on their inhibitory effects

Abstract

Antibiotic resistance is a major threat to human health. Finding new antibiotic drug targets is a way to solve this serious problem. Filamenting temperature-sensitive mutant A (FtsA) and filamenting temperature-sensitive mutant Z (FtsZ) are two conserved proteins among different bacteria strains, which play crucial roles in bacterial cell division. FtsZ is the first protein that localizes at the future division site and polymerizes into a cytokinetic ring (Z-ring) which is responsible for the recruitment of downstream cell division proteins. The bacterial cell can normally divide only when localization, polymerization, and constriction of the Z-ring occur correctly. FtsA is known to anchor the Z-ring to the inner cell membrane. Considering the essential roles of FtsA and FtsZ in cell division, both FtsA and FtsZ are considered to be promising drug targets in antibiotic development. PC190723 is one of the most potent inhibitors of FtsZ and its co-crystal structure with FtsZ is known (PDB: 4DXD). In this work, inhibitors of FtsZ targeting the PC190723 binding site were screened from compound libraries using high-throughput virtual screening and compound S2303 and S2727 were identified. Both compounds show antimicrobial activities against the Gram-positive methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Neither S2303 nor S2727 can inhibit the growth of the Gram-negative E. coli. When S2303 or S2727 was used in combination with Polymyxin B nonapeptide (PMBN), which is known to increase the permeability of the outer membrane of Gram-negative bacteria, both compounds demonstrated their antimicrobial activity against E. coli. Following antimicrobial tests, biological assays were used to establish the target of S2303 and S2727. Elongation of B. subtilis and delocalization of FtsZ were observed when cells were treated with S2303 or S2727. Additionally, delocalization of other cell division proteins, including EzrA, FtsA, and PBP2B, were also detected in cells treated by S2303. However, S2303 was also found to induce changes in membrane permeability. Both S2303 and S2727 inhibit the GTPase activity of FtsZ. The results of ITC assay demonstrate that S2303 and S2727 can bind to FtsZ. Therefore, in vivo and in vitro assays are consistent with that FtsZ is a target of S2303 and S2727. Inhibitors of FtsA targeting at its ATP binding site and site 1 were virtually screened based on the X-ray crystal structure of S. aureus FtsA. Compounds S7460 and S1324 were identified as inhibitors of FtsA. Compound S7460 was predicted to bind to the ATP binding site in silico whereas S1324 was identified as potential to bind to the site 1. Both compounds show antimicrobial activities against MRSA and MSSA. With PMBN to increase outer membrane permeability, these inhibitors of FtsA also inhibit the growth of E. coli. In vivo biological assays show that B. subtilis 168 cells are elongated and the localizations of cell division proteins are disrupted by S7460 and S1324. In vitro assay shows that S7460 enhances the ATPase activity while S1324 decreases the ATPase activity of S. aureus FtsA. However, the enhancement of the ATPase activity by S7460 is contrary to the in silico prediction that S7460 binds to the ATP binding site. It is because that S7460 is able to bind to another site of FtsA as well. The spontaneous bindings of these compounds to FtsA were confirmed using ITC assay. The mutual enhancement by FtsZ and FtsA in the GTPase activity of FtsZ and the ATPase activity of FtsA, respectively, were also studied. Considering that both the ATPase activity and the GTPase activity are essential in regulating the assembly and dynamics of divisome in cell division, interactions between FtsA and FtsZ are believed to play an essential role in regulating the enzyme activities and the cell division process. The fractional inhibition concentration indices of inhibitors of FtsA and FtsZ are in the range of 0.5 to 4, demonstrating that the two groups of inhibitors have no interactions. The interaction between FtsA and FtsZ can be affected by inhibitors of FtsZ or FtsA, as demonstrated in the ITC assay.