TXA709, an FtsZ-Targeting Benzamide Prodrug with Improved Pharmacokinetics and Enhanced In Vivo Efficacy against Methicillin-Resistant Staphylococcus aureus

Abstract

The clinical development of FtsZ-targeting benzamide compounds like PC190723 has been limited by poor drug-like and pharmacokinetic properties. Development of prodrugs of PC190723 (e.g., TXY541) resulted in enhanced pharmaceutical properties, which, in turn, led to improved intravenous efficacy as well as the first demonstration of oral efficacy in vivo against both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Despite being efficacious in vivo, TXY541 still suffered from suboptimal pharmacokinetics and the requirement of high efficacious doses. We describe here the design of a new prodrug (TXA709) in which the Cl group on the pyridyl ring has been replaced with a CF3 functionality that is resistant to metabolic attack. As a result of this enhanced metabolic stability, the product of the TXA709 prodrug (TXA707) is associated with improved pharmacokinetic properties (a 6.5-fold-longer half-life and a 3-fold-greater oral bioavailability) and superior in vivo antistaphylococcal efficacy relative to PC190723. We validate FtsZ as the antibacterial target of TXA707 and demonstrate that the compound retains potent bactericidal activity against S. aureus strains resistant to the current standard-of-care drugs vancomycin, daptomycin, and linezolid. These collective properties, coupled with minimal observed toxicity to mammalian cells, establish the prodrug TXA709 as an antistaphylococcal agent worthy of clinical development.

FIG 1

Metabolites of the prodrugs TXY541 (A) and TXA709 (B) that are observed upon exposure to either human or mouse hepatocytes for 60 min at 37°C.

FIG 2

Impact of pretreatment with 1-aminobenzotriazole (ABT) on the oral efficacy of TXY541 in a mouse peritonitis model of systemic infection with MSSA ATCC 19636 (A) or MRSA ATCC 43300 (B). ABT was administered orally at a dose of 50 mg/kg 1 h prior to infection.

FIG 3

Reverse-phase HPLC chromatograms of 20 μM TXA709 after the indicated times of incubation at 37°C in CAMH broth (left) or mouse serum (right). For comparative purposes, the corresponding chromatograms of 20 μM TXA707 after incubation for 23 h in CAMH (bottom left) or 30 min in mouse serum (bottom right) are also presented. The baseline chromatograms of CAMH broth and mouse serum alone are shown at the tops of the left and right panels, respectively. The solid arrows indicate the peaks corresponding to TXA709, while the dashed arrows indicate the peaks corresponding to TXA707. The TXA709-to-TXA707 conversion half-life (t_1/2) in each medium is indicated.

FIG 4

(A) Concentration dependence of the impact of TXA707 on the polymerization of S. aureus FtsZ, as determined by monitoring time-dependent changes in absorbance at 340 nm (A₃₄₀) at 25°C. Polymerization profiles were acquired in the presence of DMSO vehicle or the indicated concentrations of TXA707. Vancomycin (VAN) was included as a negative (non-FtsZ-targeting) control. Polymerization reactions were initiated by addition of GTP at the time indicated by the arrow. (B) Fluorescence micrographs of B. subtilis FG347 bacteria that express a GFP-tagged, Z-ring marker protein (ZapA). The bacteria were cultured for 2 h in the presence of DMSO vehicle (left) or 4 μg/ml TXA707 (8× MIC) (right). The arrows in the left panel highlight septal FtsZ Z-rings at midcell. (C) Relative frequency of FtsZ amino acid substitutions conferring resistance to TXA707 among 20 independently isolated TXA707-resistant clone of MRSA.

FIG 5

Transmission electron micrographs of MSSA 8325-4 bacteria treated with DMSO vehicle or 4 μg/ml TXA707 (8× MIC) for the indicated periods of time.

FIG 6

Oral (A to C) and intravenous (D to F) efficacy of TXA709 in a mouse peritonitis model of systemic infection with MSSA ATCC 19636 (A, D), MRSA ATCC 43300 (B, E), or MRSA Mu3 (C, F). The vehicle was 10 mM citrate (pH 2.6) in all experiments.

FIG 7

Oral efficacy of TXA709 in a mouse tissue (thigh) model of infection with MRSA ATCC 33591. The numbers of CFU recovered from the infected thighs after 24 h are shown. Mean CFU reductions resulting from TXA709 treatment were significant (P ≤ 0.003).