Antimicrobial Action and Reversal of Resistance in MRSA by Difluorobenzamide Derivatives Targeted at FtsZ

Abstract

The bacterial cell division protein, FtsZ, has been identified as a target for antimicrobial development. Derivatives of 3-methoxybenzamide have shown promising activities as FtsZ inhibitors in Gram-positive bacteria. We sought to characterise the activity of five difluorobenzamide derivatives with non-heterocyclic substituents attached through the 3-oxygen. These compounds exhibited antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA), with an isopentyloxy-substituted compound showing modest activity against vancomycin resistant Enterococcus faecium (VRE). The compounds were able to reverse resistance to oxacillin in highly resistant clinical MRSA strains at concentrations far below their MICs. Three of the compounds inhibited an Escherichia coli strain lacking the AcrAB components of a drug efflux pump, which suggests the lack of Gram-negative activity can partly be attributed to efflux. The compounds inhibited cell division by targeting S. aureus FtsZ, producing a dose-dependent increase in GTPase rate which increased the rate of FtsZ polymerization and stabilized the FtsZ polymers. These compounds did not affect the polymerization of mammalian tubulin and did not display haemolytic activity or cytotoxicity. These derivatives are therefore promising compounds for further development as antimicrobial agents or as resistance breakers to re-sensitive MRSA to beta-lactam antibiotics.

Targeting the FtsZ Allosteric Binding Site with a Novel Fluorescence Polarization Screen, Cytological and Structural Approaches for Antibacterial Discovery

“…Replacement of the amide group of 11 by isosteres gave compounds 15 – 17 with reduced inhibition ( Figure 4 A) that were also toxic to the cells. However, the addition of fluorine atoms at 2- and 6-positions of the benzamide ring of 11 (as in PC190723) rendered the 3-chloro derivative 18 ( 45 , 46 ) and the 3-trifluoromethyl analogue 20 that were both stronger competitors ( Figure 4 A) with 1.3 and 0.7 μM K D values, respectively ( Figure 5 A and Table 1 ). Compound 20 induced a marked divisomal inhibition phenotype on B. subtilis , consisting of cell filamentation and GFP-FtsZ delocalization ( Figure 5 B and Table 1 ; raw data plots in Figure S4 and statistics in Table S2 ), as well as characteristic enlarged spherical cells in S. aureus and FtsZ-mCherry delocalization ( Figure 5 C).…”

“…Replacement of the amide group of 11 by isosteres gave compounds 15 – 17 with reduced inhibition (Figure A) that were also toxic to the cells. However, the addition of fluorine atoms at 2- and 6-positions of the benzamide ring of 11 (as in PC190723) rendered the 3-chloro derivative 18 , and the 3-trifluoromethyl analogue 20 that were both stronger competitors (Figure A) with 1.3 and 0.7 μM K D values, respectively (Figure A and Table ). Compound 20 induced a marked divisomal inhibition phenotype on B.…”

A salt bridge-mediated resistance mechanism to FtsZ inhibitor PC190723 revealed by a cell-based screen

“…Studies in the past on the effects of PC190723 on E. coli have been confusing, with a few reports suggesting an effect on FtsZ polymerization resulting in cell filamentation (Kaul et al, 2014), while others did not find any effect on EcFtsZ (Anderson et al, 2012; Andreu et al, 2010; Khare et al, 2019). The outer membrane has been shown to be a permeability barrier for PC190723 in E. coli (Chai et al, 2020; Khare et al, 2019). In addition, the Resistance-Nodulation-Division (RND) family of efflux pumps has been attributed to resistance against 2,6-difluorobenzamide derivatives, including TX436 (a prodrug of PC190723) in Gram -ve bacteria (Kaul et al, 2014).…”

Lulworthinone: In Vitro Mode of Action Investigation of an Antibacterial Dimeric Naphthopyrone Isolated from a Marine Fungus

“…FtsZ was found to be delocalised into patches all over the cell or multiple Z-rings at unusual sites in the cell. As a key element for cell division, FtsZ is a focus target for antibacterial treatments [ 32 , 33 , 34 , 35 , 36 ]. As an explanation for the delocalisation, Strahl and Hamoen (2010) showed that the FtsZ guiding proteins FtsA and MinD are inactivated after loss of the membrane potential.…”