AAC Accepted Manuscript Posted Online 1 June 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00708-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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TXA709: A FtsZ-Targeting Benzamide Prodrug with Improved Pharmacokinetics

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and Enhanced In Vivo Efficacy against Methicillin-Resistant Staphylococcus

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aureus

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Malvika Kaul,a Lilly Mark,b,c Yongzheng Zhang,b Ajit K. Parhi,b,c Yi Lisa Lyu,a Joan

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Pawlak,d Stephanie Saravolatz,d Louis D. Saravolatz,d,e Melvin P. Weinstein,f,g Edmond J.

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LaVoie,c and Daniel S. Pilcha#

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Department of Pharmacology, Rutgers Robert Wood Johnson Medical School, Piscataway, New

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Jersey, USAa; TAXIS Pharmaceuticals, Inc., North Brunswick, New Jersey, USAb; Department

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of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers-The State University of

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New Jersey, Piscataway, New Jersey, USAc; St. John Hospital and Medical Center, Detroit,

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Michigan, USAd; Wayne State University School of Medicine, Detroit, Michigan, USAe;

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Departments of Medicine (Infectious Disease)f and Pathology and Laboratory Medicineg,

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Rutgers Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA

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RUNNING TITLE: ADME/PK Properties and In Vivo Efficacy of TXA709

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#Address correspondence to Daniel S. Pilch, [email protected].

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ABSTRACT

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The clinical development of FtsZ-targeting benzamide compounds like PC190723 has been

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limited by poor drug-like and pharmacokinetic properties. First-generation prodrugs of

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PC190723 (e.g., TXY541) resulted in enhanced pharmaceutical properties, which, in turn,

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led to improved intravenous efficacy as well as the first demonstration of oral efficacy in

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vivo versus both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-

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resistant S. aureus (MRSA). Despite being efficacious in vivo, TXY541 still suffered from

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sub-optimal pharmacokinetics and the requirement of high efficacious doses. We describe

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herein the design of a second-generation prodrug (TXA709) in which the Cl group on the

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pyridyl ring has been replaced with a CF3 functionality that is resistant to metabolic attack.

31

As a result of this enhanced metabolic stability, the product of the TXA709 prodrug

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(TXA707) is associated with improved pharmacokinetic properties (a 6.5-fold longer half-

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life and a 3-fold greater oral bioavailability) and superior in vivo antistaphylococcal

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efficacy relative to PC190723. We validate FtsZ as the antibacterial target of TXA707, and

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demonstrate that the compound retains potent bactericidal activity against S. aureus

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strains resistant to the current standard-of-care drugs vancomycin, daptomycin, and

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linezolid.

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mammalian cells, establish the prodrug TXA709 as an antistaphylococcal agent worthy of

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clinical development.

These collective properties, coupled with minimal observed toxicity to

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INTRODUCTION

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Bacterial resistance has emerged as a global problem. The Centers for Disease Control and

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Prevention (CDC) have identified methicillin-resistant Staphylococcus aureus (MRSA) and

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vancomycin-resistant S. aureus (VRSA) as being two major antibiotic resistance threats (1).

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Typically, MRSA strains are resistant not only to the penicillins, but also to other classes of

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antibiotics, including the tetracyclines, the macrolides, the aminoglycosides, and clindamycin (2-

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4). Current standard-of-care (SOC) drugs for the treatment of MRSA infections are therefore

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limited to a few drugs, which include vancomycin, daptomycin, and linezolid (3). However,

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resistance to these SOC drugs is already on the rise, and the clinical utility of these drugs is

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likely to diminish in the future (3, 5-9).

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The bacterial protein FtsZ has been identified as an appealing new target for the

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development of antibiotics that can be used to treat infections caused by multidrug-resistant

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(MDR) bacterial pathogens (10-14). The appeal of FtsZ as an antibiotic target lies in the

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essential role the protein plays in bacterial cell division (cytokinesis). Furthermore, FtsZ is

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prokaryotic-specific with no known eukaryotic homolog.

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dependent manner to form a ring-like structure (the Z-ring) at midcell that serves as a scaffold

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for the recruitment and organization of other critical components for proteoglycan synthesis,

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septum formation, and cell division (15-20).

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FtsZ self-polymerizes in a GTP-

The substituted benzamide derivative PC190723 has been shown to inhibit bacterial cell

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division through disruption of FtsZ function (21-24).

PC190723 is associated with potent

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bactericidal activity against Staphylococcus spp., including MRSA (23, 24). However, the

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clinical development of this compound has been hindered by poor pharmaceutical and

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pharmacokinetic properties. We have previously reported the design and characterization of

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first-generation prodrugs of PC190723 (TXY436 and TXY541) with physico-chemical

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properties that significantly enhance the ease of formulation in vehicles suitable for in vivo

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administration (25, 26).

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efficacious in vivo against MRSA, the doses required for efficacy were high, and their

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pharmacokinetic properties were sub-optimal (25, 26). Here we describe a second-generation

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prodrug (TXA709) of an FtsZ-targeting benzamide compound (TXA707) with enhanced

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metabolic stability, improved pharmacokinetic properties, and superior in vivo efficacy versus

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MRSA. We provide results that highlight TXA709 as an attractive lead agent for clinical

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development.

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MATERIALS AND METHODS

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Bacterial strains. Methicillin-resistant S. aureus (MRSA) clinical strains (n = 30) were isolated

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from patients admitted to St. John Hospital and Medical Center (SJHMC) in Detroit, MI (n = 20)

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or to Robert Wood Johnson University Hospital (RWJUH) in New Brunswick, NJ (n = 10). The

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MRSA isolates were cultured from blood (n = 26), wound tissue (n = 2), pleural fluid (n = 1),

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and arm drainage (n = 1). Daptomycin-nonsusceptible S. aureus (DNSSA) isolates (n = 7) were

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cultured from blood samples collected from patients at SJHMC. Vancomycin-intermediate S.

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aureus (VISA) isolates (n = 20) were obtained from 20 patients. The VISA isolates were

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cultured from blood (n = 12), wound tissue (n = 1), bile (n = 1), cerebral spinal fluid (n = 1), and

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an unknown source (n = 5). Vancomycin-resistant S. aureus (VRSA) isolates (n = 11) were

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obtained from 11 patients, with these isolates being cultured from wound tissue (n = 8),

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prosthetic knee drainage (n = 1), urine (n = 1), and a catheter exterior (n = 1). Linezolid-

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nonsusceptible S. aureus (LNSSA) isolates (n = 6) were cultured from blood (n = 2), sputum (n =

While TXY436 and TXY541 were both orally and intravenously

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1), and an unknown source (n = 3). The VRSA, 17 of the VISA, and 4 of the LNSSA isolates

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were provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA)

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for distribution by BEI Resources, NIAID, NIH. The NARSA website (http://www.narsa.net)

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describes the location of origin of each isolate.

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collected at SJHMC, and the remaining two LNSSA isolates were collected at Robinson

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Memorial Hospital in Ravenna, OH. Methicillin-sensitive S. aureus (MSSA) isolates (n = 10)

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were cultured from the blood of 10 patients at RWJUH. MRSA Mu3 was a gift from Dr. George

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M. Eliopoulos (Beth Israel Deaconess Medical Center, Boston, MA) and MSSA 8325-4 was a

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gift from Dr. Glenn W. Kaatz (John D. Dingell VA Medical Center, Detroit, MI). All other

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MSSA and MRSA strains were obtained from the American Type Culture Collection (ATCC).

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Bacillus subtilis FG347 was a gift from Dr. Richard Losick (Harvard University, Boston, MA).

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Compound synthesis.

The remaining three VISA isolates were

TXY541 and PC190723 were synthesized as described

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previously (25, 27). TXA707 and TXA709 were synthesized as detailed in the Supplementary

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Material.

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Compound stability studies. The conversion of TXA709 to TXA707 in either cation-

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adjusted Mueller-Hinton (CAMH) broth (Becton, Dickinson and Co.; Franklin Lakes, NJ) or

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100% filtered mouse serum (Lampire Biological Laboratories, Inc.; Ottsville, PA) at 37 °C was

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monitored as described previously for the conversion of TXY541 to PC190723 (25). The mouse

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serum was filtered through a 0.2 µm filter before use.

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In Vitro susceptibility assays.

In vitro susceptibility assays were conducted with

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TXA707, TXA709, and PC190723 using vancomycin-HCL, daptomycin, and linezolid as

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comparator control antibiotics (all obtained from Sigma). Determinations of minimal inhibitory

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concentrations (MICs) and minimal bactericidal concentrations (MBCs) were conducted in

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duplicate according to Clinical and Laboratory Standards Institute (CLSI) guidelines (28).

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Microdilution assays with CAMH broth were used to determine the MICs of all agents. For the

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daptomycin assays, calcium was added to a final concentration of 50 mg/l. MIC is defined as the

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lowest compound or drug concentration with no visible growth after 16-24 hours of incubation.

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MBC is defined as the compound or drug concentration that reduced the number of viable cells

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by ≥99.9%, as determined by colony counts relative to antibiotic-free controls.

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Assay for metabolism of TXY541 and TXA709 in the presence of mouse and human

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hepatocytes. Metabolism experiments were conducted by SAI Life Sciences Ltd. (Pune, India).

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Mouse hepatocytes, human hepatocytes, and cryopreserved hepatocyte recovery medium

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(CHRM) were obtained from Invitrogen BioServices India Pvt. Ltd. Cryopreserved hepatocytes

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were revived in CHRM medium supplemented with Krebs Henseleit Buffer (KHB), pH 7.4

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(Sigma). A 500 µL cell suspension containing 2 x 106 cells/mL was added to individual wells of

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24 well plates and incubated at 37 °C for 15 minutes. Duplicate reactions were initiated by

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adding 500 µL of test compound diluted in KHB (prewarmed at 37 °C), and further incubated at

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37 °C for 0 and 60 minutes with gentle shaking. The final cell density was 1 x 106 cells/mL, and

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the final concentration of test compound was 10 µM. Reactions were terminated by adding 1 mL

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of ice-cold acetonitrile to a final volume of 2 mL. The terminated reaction mixes from each well

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were transferred to individual tubes and sonicated for 5 minutes prior to centrifugation at 2147 ×

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g for 15 minutes at 4 °C. A 1.2 mL aliquot of each supernatant was then transferred to a clean

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tube and the metabolites identified by LC-MS/MS analysis. Testosterone (Sigma) and 7-OH-

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coumarin (Apin Chemicals Ltd.) were used as positive controls.

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Pharmacokinetic studies. Pharmacokinetic experiments in male BALB/c mice were

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conducted by SAI Life Sciences Ltd. (Pune, India) as described previously (25). When used, 1-

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aminobenzotriazole (ABT) was administered orally at a standard dose 50 mg/kg (29) one hour

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prior to administration of test prodrug (TXY541 or TXA709) formulated in 10 mM citrate, pH

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2.6. Plasma concentrations of the prodrug products PC190723 and TXA707 were quantified by

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LC-MS/MS, with the lower limit of quantitation (LLOQ) being 10.02 and 4.93 ng/mL,

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respectively.

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Plasma protein binding studies. Plasma protein binding studies of TXA707 were

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conducted by SAI Life Sciences Ltd. using human, dog, rat, and mouse plasma. Protein binding

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was measured via rapid equilibrium dialysis (RED) using an RED device (Thermo Scientific)

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containing dialysis membrane with a molecular weight cut-off of 8,000 Da. Each plasma type

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was spiked in triplicate with 5 µM TXA709. Dialysis was then performed with shaking (at 100

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rpm) for four hours at 37 °C, as per the manufacturer’s recommendation. Following dialysis, the

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concentration of TXA707 in each well (both plasma and buffer side) was quantified by LC-

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MS/MS, and the resulting peak area ratios were used to determine the percentages of compound

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unbound and bound to plasma proteins.

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S. aureus FtsZ polymerization assay. S. aureus FtsZ was expressed and purified as

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described previously (30).

Polymerization of S. aureus FtsZ was monitored as described

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previously (26), with the exception that FtsZ was used at a concentration of 15 µM.

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Fluorescence microscopy. Fluorescence microscopy studies with B. subtilis FG347

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were conducted as described previously (26), with the exception that the bacteria were cultured

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for two hours in the presence of DMSO vehicle or 4 µg/mL TXA707 (8x MIC).

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Transmission electron microscopy (TEM).

Log-phase MSSA 8325-4 cells were

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cultured in CAMH broth at 37 °C for 0, 1, 4, or 9 hours in the presence of DMSO (solvent

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vehicle) or TXA707 at a concentration of 4 µg/mL (8x MIC). At each time point, a 1-mL

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sample was withdrawn from the culture and centrifuged at 16,000 × g for 3 minutes at room

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temperature. The supernatant was removed, and the bacterial pellet was washed with 1 mL of

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phosphate-buffered saline (PBS). The final bacterial pellet was fixed by resuspension in 500 µL

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of 0.1 M cacodylate buffer (pH 7.2) containing 2.5% gluteraldehyde and 4% paraformaldehyde.

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The fixed bacterial cells were then post-fixed in buffered osmium tetroxide (1%), and

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subsequently dehydrated in a graded series of ethanol and embedded in epon resin. Thin sections

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(90 nm) were cut on a Leica EM UC6 ultramicrotome. Sectioned grids were then stained with a

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saturated solution of uranyl acetate and lead citrate. Images were captured with an AMT XR111

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digital camera at 80 Kv on a Philips CM12 transmission electron microscope.

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Assay for the frequency of resistance (FOR) and identification of resistant ftsZ

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mutations. FOR studies were conducted as described elsewhere (25), and the ftsZ genes in

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resistant mutants were sequenced (30).

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In vivo efficacy assays. Antistaphylococcal efficacy in vivo was assessed in a mouse

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peritonitis model of systemic infection, as well as in a mouse tissue (thigh) model of infection.

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The experimental details associated with the in vivo efficacy studies are described in the

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Supplementary Material.

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MTT cytotoxicity assay. The cytotoxicity of TXA709 was assessed in human cervical

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cancer (HeLa) and Madin-Darby canine kidney (MDCK) epithelial cells using a four-day

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continuous 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as

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described previously (31), with vehicle and the anticancer drug camptothecin serving as negative

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and positive controls, respectively. TXA707 was not included in these characterizations, due to

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its limited solubility at the higher concentrations necessitated by the assay.

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RESULTS AND DISCUSSION

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Metabolic attack of the Cl atom on the pyridyl ring of the first-generation prodrug

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TXY541 results in sub-optimal pharmacokinetics of the active product PC190723. In our

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previous studies, we demonstrated that the oral and intravenous doses of the prodrug TXY541

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required for efficacy in mouse models of MRSA and MSSA infection are large (96 to 192

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mg/kg) due to the rapid elimination and modest oral bioavailability of the active product

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PC190723 (25). As a first step toward understanding the basis for the rapid elimination of

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PC190723 upon TXY541 administration, we conducted a metabolic study of TXY541 in the

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presence of either mouse or human hepatocytes. Several metabolites were observed in the

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presence of both mouse and human hepatocytes (Fig. 1A). The active product PC190723 was

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one of these metabolites (M2), detected by mass spectrometry peak analysis at an abundance of

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≈20% with mouse and ≈30% with human hepatocytes. An even more abundant metabolite (M1)

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(detected at an abundance of ≈70% with mouse and ≈50% with human hepatocytes) was one

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resulting from the dechlorination and subsequent monooxygenation of TXY541. Thus, the Cl

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atom on the pyridyl ring of TXY541 appears to be susceptible to metabolic dehalogenation.

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Dehalogenation and oxygenation reactions are typically catalyzed by phase I cytochrome

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P450 (CYP) enzymes. We therefore hypothesized that pretreatment with a CYP inhibitor would

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retard the elimination of PC190723. To this end, we assessed the impact of pretreatment with

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CYP inhibitor 1-aminobenzotriazole (ABT) on the pharmacokinetics of PC190723 in mice

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following administration of TXY541. As shown in Table 1, pretreatment with ABT resulted in

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an elimination half-life (t½) of 1.95 hours, a value approximately 3.5-times greater than that

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observed in the absence of ABT (0.56 hours). This increased t½ results from a correspondingly

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reduced clearance (CL) (13.2 versus 55.1 mL/min/kg). Thus, inhibition of CYP enzymatic

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activity with ABT retards the elimination of the PC190723 upon administration of the TXY541

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prodrug.

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We next sought to determine whether the retarded elimination of PC190723 induced by

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ABT would also reduce the dose of TXY541 required for antistaphylococcal efficacy in vivo. In

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the absence of ABT, oral administration of TXY541 required a dose of 128 mg/kg for efficacy

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(83% survival; 5/6) in mice systemically infected with MSSA ATCC 19636 (Fig. 2A) and a dose

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of 192 mg/kg for efficacy (100% survival; 6/6) in mice systemically infected with MRSA ATCC

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43300 (Fig. 2B). By contrast, with ABT pretreatment, the dose of TXY541 required for efficacy

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(100% survival; 6/6) in both MSSA- and MRSA-infected mice was 64 mg/kg. Pretreatment with

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the CYP inhibitor therefore reduces the dose of TXY541 required for efficacy by at least 2- and

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3.5-fold in the MSSA-infected and MRSA-infected mice, respectively.

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Design of a second-generation prodrug (TXA709) that can circumvent the CYP-

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mediated dechlorination/oxygenation reactions associated with TXY541. As indicated by

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our studies described above, the Cl atom on TXY541 is vulnerable to CYP-mediated metabolic

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attack. Our goal in designing a second-generation prodrug was to substitute the Cl functionality

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on the pyridyl ring with an electron-withdrawing group that would not undergo dehalogenation.

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In addition to being resistant to metabolism, the introduced group needed to be hydrophobic in

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nature, as polar functionalities at this position of the pyridyl ring tend to reduce

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antistaphylococcal potency (32). One of the most promising second-generation prodrugs to

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emerge from our design efforts is TXA709 (shown in Fig. 1B) in which the Cl atom present in

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TXY541 has been replaced with a CF3 functionality. Just as hydrolysis of TXY541 yields the

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active product PC190723, corresponding hydrolysis of TXA709 yields an active product we

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denote as TXA707 (also shown in Fig. 1B). In fact, both TXY541 and TXA709 have similar

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prodrug-to-product conversion kinetics. Fig. 3 shows HPLC chromatograms demonstrating the

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time-dependent conversion of TXA709 to TXA707 at 37 °C in two different media at pH 7.4,

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CAMH broth (the media used for culturing S. aureus) and mouse serum. Analysis of the

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TXA709 peak areas yielded conversion half-lives (7.7 ± 0.1 hours in CAMH broth and 3.0 ± 0.2

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minutes in mouse serum) of similar magnitude to those previously reported for the conversion of

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TXY541 to PC190723 (8.2 ± 0.4 hours in CAMH broth and 3.4 ± 0.2 minutes in mouse serum)

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(25). In other words, substitution of CF3 for Cl on the pyridyl ring does not significantly alter the

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prodrug-to-product conversion kinetics.

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The CF3 functionality of TXA709 is resistant to metabolic attack in the presence of

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mouse and human hepatocytes. To determine if the introduced CF3 functionality of TXA709

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imparts resistance to metabolism, we examined the metabolism of TXA709 in the presence of

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mouse or human hepatocytes. With both hepatocyte types, only two metabolites were observed

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(Fig. 1B), with the major metabolite (M1) being the active product TXA707. Significantly, no

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metabolic reactions targeting the CF3 group were observed, confirming the metabolic stability of

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this functionality.

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TXA709 is associated with improved pharmacokinetic properties relative to

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TXY541. We next sought to determine whether the metabolic stability of TXA709 would

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translate to improved pharmacokinetic properties in mice. To this end, we evaluated the plasma

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pharmacokinetics of the TXA709 prodrug and its TXA707 product following both intravenous

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(i.v.) and oral (p.o.) administration of TXA709 to mice. TXA709 was not detectable in the

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plasma at any time point (from 0.08 to 24 hours) following administration by either route. By

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contrast, the plasma concentration of TXA707 was detectable and quantifiable up to 24 hours

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following both i.v. and p.o. administration of TXA709. This observation is consistent with the

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rapid conversion of TXA709 to TXA707 that we observed in the presence of mouse serum at 37

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°C (Fig. 3).

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The time-dependent plasma concentrations of TXA707 after both p.o. and i.v.

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administration of TXA709 are graphically depicted in Fig. S1 of the Supplementary Material.

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Pharmacokinetic analysis of these data yielded the pharmacokinetic parameters summarized in

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Table 1. A comparison of these parameters with the corresponding pharmacokinetic parameters

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for PC190723 following TXY541 administration reveals that TXA707 is associated with an

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elimination t½ following i.v. administration of TXA709 that is approximately 6.5-times longer

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than the corresponding t½ of PC109723 following i.v. administration of TXY541 (t½ = 3.65 hours

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for TXA707 versus 0.56 hours for PC190723). The longer t½ of TXA707 relative to PC190723

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is due to a correspondingly reduced CL (9.40 mL/min/kg for TXA707 versus 55.11 mL/min/kg

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for PC190723). In addition, the bioavailability (%F) of TXA707 following p.o. administration

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of TXA709 was found to be 95%, approximately 3.2-times the corresponding value (%F = 29.6)

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we have previously shown for PC190723 following p.o. administration of TXY541 (25).

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To determine whether the observed pharmacokinetic differences between TXA707 and

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PC190723 might be due to differences in plasma protein binding, we assessed the protein

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binding properties of TXA707 in mouse, human, rat, and dog plasma. The results of these

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studies (summarized in Table 2) revealed that TXA707 is ≈86% bound to mouse plasma proteins

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and ≈91% bound to human, rat, and dog plasma proteins. Significantly, the percentage bound to

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mouse plasma proteins is nearly identical to that (85.4%) previously reported for PC190723 (32),

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indicating that the observed pharmacokinetic differences between the two compounds do not

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reflect differences in plasma protein binding properties. Our collective results therefore indicate

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that the metabolic stability of the CF3 moiety present in TXA709 confers the second-generation

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prodrug with markedly improved pharmacokinetic properties relative to TXY541, properties that

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include an active product with (i) reduced CL, (ii) longer elimination t½, and (iii) enhanced oral

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bioavailability.

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TXA709 and TXA707 exhibit potent bactericidal activity versus clinical isolates of

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MRSA, VISA, VRSA, DNSSA, LNSSA, and MSSA. We evaluated the minimal inhibitory

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concentrations (MICs) and minimal bactericidal concentrations (MBCs) of TXA709 and

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TXA707 against a panel of 84 clinical S. aureus isolates, including 30 MRSA, 20 VISA, 11

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VRSA, 7 DNSSA, 6 LNSSA, and 10 MSSA isolates. The results of these determinations are

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summarized in Table 3. The modal MIC for TXA707 was 1 µg/mL against all six types of

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clinical isolates tested (MRSA, VISA, VRSA, DNSSA, LNSSA, and MSSA). We observe an

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identical modal MIC for PC190723 against the six types of clinical isolates. Thus, the Cl-to-CF3

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substitution on the pyridyl ring does not diminish antibacterial potency. Note that the modal

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MIC of TXA707 against the MRSA isolates is similar, if not identical, to the corresponding

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modal MICs of vancomycin (1 µg/mL), daptomycin (1 µg/mL), and linezolid (2 µg/mL), which

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were included as comparator control antibiotics representative of current standard-of-care (SOC)

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drugs for the treatment of MRSA infections. Significantly, TXA707 maintains a modal MIC of

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1 µg/mL against S. aureus strains that are associated with varying degrees of resistance to

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vancomycin (VISA and VRSA), daptomycin (DNSSA), or linezolid (LNSSA). Thus, TXA707

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maintains its antistaphylococcal potency even against strains where current SOC drugs do not.

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While the prodrug TXA709 also exhibits activity against all 84 S. aureus isolates tested, its

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modal MICs are 2- to 4-fold higher than those of TXA707. This discrepancy likely reflects the

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lack of total conversion of TXA709 to TXA707, due to the slow conversion kinetics in CAMH

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medium (t½ = 7.7 ± 0.1 hours). For all the S. aureus isolates examined, the modal MBCs for

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TXA707 and TXA709 were similar, if not identical, to the corresponding modal MICs, with the

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difference between the modal MBCs and MICs never being >2-fold. These results indicate that

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the antibacterial activity of both compounds against the S. aureus isolates tested is bactericidal in

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nature.

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For comparative purposes, we also tested TXA707 and TXA709 for activity versus

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quality control and laboratory strains of both MSSA and MRSA (two quality control strains and

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one laboratory strain each), with the results being summarized in Table 4. As expected, the

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antibacterial potencies of both compounds versus these MSSA and MRSA strains were similar in

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magnitude (MIC = 0.5 µg/mL for TXA707 and 2-4 µg/mL for TXA709) to those observed

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versus the clinical isolates described above. These quality control and laboratory strains of S.

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aureus were used in the in vivo efficacy experiments described below.

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Validation of the cell division protein FtsZ as the bactericidal target of TXA707. We

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next sought to establish that the antibacterial activity of TXA707, the active product of the

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TXA709 prodrug, reflects its ability to target FtsZ. To this end, we conducted the following four

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series of studies:

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(i) Impact on FtsZ polymerization. We and others have shown that the antibacterial

307

activities of FtsZ-targeting benzamides like PC190723 result from over-stimulation of FtsZ

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polymerization dynamics and stabilization of nonfunctional FtsZ polymeric structures (21-24,

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26, 33, 34). Thus, as a first step in validating FtsZ as the antibacterial target of TXA707, we

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assessed the impact of TXA707 on the polymerization dynamics of purified S. aureus FtsZ using

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a microtiter plate-based spectrophotometric assay in which changes in FtsZ polymerization are

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reflected by corresponding changes in absorbance at 340 nm (A340). Fig. 4A shows the time-

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dependent A340 profiles of S. aureus FtsZ in the absence and presence of TXA707 at

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concentrations ranging from 0.5 to 5 µg/mL. Note that TXA707 stimulates FtsZ polymerization

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in concentration-dependent manner, a similar behavior to that previously demonstrated for

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PC190723 (21-24, 26, 33, 34). As expected, vancomycin, included as a non-FtsZ-targeting

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control antibiotic in this assay, did not impact FtsZ polymerization.

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(ii) Impact on FtsZ Z-ring formation. We also evaluated the impact of TXA707 on FtsZ

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Z-ring formation in bacteria using fluorescence microscopy. To this end, we used a strain of B.

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subtilis (FG347) that expresses a GFP-tagged Z-ring marker protein (ZapA) (35). We have

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previously shown that PC190723 induces a filamentous phenotype in these bacteria, as well as

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mislocalization of FtsZ from the septal Z-ring at midcell to multiple punctate sites throughout the

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cell (26, 34). As shown in Fig. 4B, our studies with TXA707 have demonstrated an identical

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pattern of behavior, consistent with inhibition of cell division by the compound through

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disruption of FtsZ function.

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(iii) Impact on septum formation and cell division. As a third approach to establishing

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TXA707 as an inhibitor of cell division, we also used transmission electron microscopy (TEM)

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to examine the impact of TXA707 on cell morphology and septum formation in MSSA 8325-4.

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The precise localization of FtsZ and the penicillin binding proteins (PBPs) to the septum is

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critical for the proper coordination of septal proteoglycan biosynthesis and cell division in S.

331

aureus (18-20). The FtsZ-targeting activity of PC190723 in S. aureus has been shown to cause

332

the disruption of cell division through mislocalization of both FtsZ and PBP2 (and thus septal

333

biosynthesis) from midcell to the cell periphery (18, 20). Our TEM studies demonstrate that

334

within 1 hour of treatment with TXA707, no septa are observed in the S. aureus bacteria (Fig. 5).

335

Instead, peptidoglycan synthesis appears to have shifted to discrete locations at the cell

336

periphery. In addition, the cells become enlarged by approximately 3-fold over time, eventually

337

leading to cell lysis.

338

(iv) Isolation of resistance mutations that map to the FtsZ coding sequence. Perhaps the

339

most definitive approach for validating a particular bacterial protein as the specific target of an

340

antibacterial agent is through genetic studies demonstrating that mutations in the target protein

341

can result in resistance. In this connection, we used a large inoculum approach in an effort to

342

raise spontaneous mutants of MRSA that are resistant to TXA707. This approach yielded

343

resistant clones at a frequency of ≈3 x 10-8 in MRSA ATCC 43300 and ≈1 x 10-8 in a MRSA

344

clinical isolate from a patient at RWJUH. These FOR values are similar in magnitude to those

345

previously reported for PC190723 in various MSSA and MRSA strains (20, 23, 25). Twenty of

346

the TXA707-resistant clones (10 from each MRSA strain) were isolated, and the ftsZ gene in

347

each clone was sequenced. All 20 resistant clones carried a mutation that mapped to the ftsZ

348

gene. Of the 20 clones, 11 (55%) carried the G196S mutation, 3 (15%) carried the N263K

349

mutation, 3 (15%) carried the G193D nutation, 2 (10%) carried the G196C mutation, and 1 (5%)

350

carried the G196A mutation (Fig. 4C). All of these FtsZ mutations have been previously shown

351

to cause S. aureus resistance to PC190723 at similar relative frequencies (20, 23).

352

mutational analysis is therefore fully consistent with FtsZ being the antibacterial target of

353

TXA707.

Our

354

TXA709 is associated with enhanced oral and intravenous efficacy relative to

355

TXY541 in a mouse systemic (peritonitis) model of MSSA and MRSA infection. Armed

356

with the in vitro susceptibility, FtsZ-targeting, and pharmacokinetic results described above, we

357

next evaluated the in vivo antistaphylococcal efficacy of TXA709 using a mouse peritonitis

358

model of systemic infection with S. aureus.

359

intraperitoneally with a lethal inoculum of MSSA ATCC 19636, against which TXA707 exhibits

360

an MIC of 0.5 µg/mL (Table 4). None of the mice treated p.o. (0/6) or i.v. (0/5) with vehicle

361

alone survived beyond one day post infection (Figs. 6A and 6D). By contrast 50% (3/6) of the

362

mice receiving a p.o. dose of 32 mg/kg TXA709 and 100% (6/6) of the mice receiving a p.o.

363

dose of 64 mg/kg TXA709 survived the MSSA infection. Furthermore, 100% (5/5) of the mice

364

receiving an i.v. dose of 36 or 72 mg/kg TXA709 also survived the MSSA infection. Thus, an

365

i.v. dose of 36 mg/kg TXA709 and a p.o. dose of 64 mg/kg were sufficient for 100% survival of

366

the MSSA infection. Significantly, these efficacious i.v. and p.o. doses of TXA709 are 2- to 2.7-

367

times lower than the corresponding efficacious doses of TXY541 (Fig. 2).

In our initial studies, mice were inoculated

368

In our next set of studies, mice were administered a lethal inoculum of MRSA ATCC

369

43300. None of the infected mice treated p.o. (0/6) or i.v. (0/4) with vehicle alone survived

370

beyond two days post infection (Figs. 6B and 6E). In striking contrast, an i.v. dose of 36 mg/kg

371

TXA709 and a p.o. dose of 48 mg/kg TXA709 resulted in 100% survival (6/6 and 4/4 of the

372

mice treated i.v. and p.o., respectively). Recall that the p.o. dose of TXY541 required for 100%

373

survival in the systemic MRSA 43300 infection model was 192 mg/kg (Fig. 2), 4-times higher

374

than the corresponding efficacious p.o. dose of TXA709 (48 mg/kg). Additional studies with

375

mice infected with MRSA Mu3 revealed efficacious TXA709 doses of 72 mg/kg i.v. and 96

376

mg/kg p.o. (Figs. 6C and 6F). The larger doses of TXA709 required for efficacy against Mu3

377

relative to ATCC 43300 do not reflect a difference in the antibacterial potency of TXA707

378

against the two bacterial strains, as the MIC of TXA707 is 0.5 µg/mL against either strain (Table

379

4). Instead, the reduced efficacy of TXA709 against Mu3 compared to ATCC 43300 may reflect

380

an enhanced virulence on the part of the Mu3 strain, the lethal inoculum for which is 2.5-times

381

lower than that for ATCC 43300 (see Supplementary Material).

382

In the aggregate, our in vivo antistaphylococcal studies demonstrate that TXA709 is

383

associated with enhanced in vivo efficacy relative to TXY541 against both MSSA and MRSA

384

infections.

385

TXA709 is also orally efficacious in a mouse tissue (thigh) model of MRSA infection.

386

Among the pharmacokinetic properties of TXA707 upon i.v. administration of TXA709 was a

387

volume of distribution at steady state (Vd-ss) of 2.02 L/kg (see Table 1). This Vd-ss value is

388

approximately 3-times greater than the total water volume in the mouse (0.7 L/kg), indicating

389

that TXA707 distributes beyond the vascular space and into the tissue. This property is desirable

390

for antistaphylococcal agents, since staphylococcal infections frequently occur in soft tissue. In

391

this connection, we characterized the oral efficacy of TXA709 in a mouse tissue (thigh) model of

392

infection with MRSA ATCC 33591. An oral dose of either 120 or 160 mg/kg TXA709 resulted

393

in a ≈2-log reduction in bacterial CFUs relative to vehicle-treated mice (p ≤ 0.003) at 24 hours

394

post-infection (Fig. 7). Previous studies with PC190723 in a mouse thigh model of MRSA

395

infection yielded only a ≈0.5-log reduction in bacterial CFUs when the compound was

396

administered orally at a dose of 200 mg/kg every 6 hours over a 24-hour period (20). Viewed as

397

a whole, these results indicate that TXA709 is orally efficacious against MRSA not only in a

398

mouse systemic model of infection, but also in a mouse tissue model of infection. Furthermore,

399

the efficacy of orally administered TXA709 against MRSA in the mouse tissue model of

400

infection is significantly greater than that of orally administered PC190723.

401

TXA709 exhibits minimal toxicity to mammalian cells. To probe for any potential

402

mammalian cytotoxicity, we used a four-day tetrazole (MTT)-based assay to assess the

403

cytotoxicity of TXA709 against human cervical cancer (HeLa) and Madin-Darby canine kidney

404

(MDCK) cells. TXA709 was found to be minimally toxic to both cell types, with IC50 values

405

>120 µg/mL. While these IC50 values reflect cell culturing conditions that include the presence

406

of 10% fetal bovine serum, our control studies reveal a modal antistaphylococcal MIC of 2

407

µg/mL for TXA709 in the presence of the same percentage of serum. Thus, the large difference

408

between the mammalian cell IC50s and the antistaphylococcal MICs is due to the targeting

409

specificity of the compound for staphylococci as opposed to serum protein binding effects.

410

In conclusion, TXA709 is a second-generation prodrug of the FtsZ-targeting benzamide

411

compound TXA707.

TXA709 is associated with enhanced metabolic stability, improved

412

pharmacokinetic properties, and superior in vivo antistaphylococcal efficacy (both oral and

413

intravenous) relative to first-generation prodrugs (TXY436 and TXY541) of PC190723. In

414

addition, the TXA707 hydrolysis product of TXA709 maintains potent activity against S. aureus

415

strains (e.g., MRSA, VRSA, DNSSA, and LNSSA) that are resistant to current SOC antibiotics.

416

These characteristics, coupled with minimal cytotoxicity versus mammalian cells, make TXA709

417

an attractive lead compound for development into a clinically useful agent for the treatment of

418

drug-resistant staphylococcal infections.

419

ACKNOWLEDGEMENTS

420

This study was supported by research agreements between TAXIS Pharmaceuticals, Inc. and

421

Rutgers RWJMS (D.S.P. and M.P.W.), Rutgers EMSP (E.J.L.), and SJHMC (L.D.S.). We are

422

indebted to Drs. Glenn W. Kaatz, Richard Losick, and George M. Eliopoulos for providing us

423

with S. aureus 8325-4, B. subtilis FG347, and S. aureus Mu3, respectively. We also wish to

424

thank Raj Patel (Rutgers RWJMS, Core Imaging Lab) for his assistance with the TEM

425

experiments, and Giovanni Divinagracia (RWJUH) for his assistance with the in vitro

426

susceptibility assays.

427

428

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544

545 TABLE 1 Pharmacokinetic parameters of PC190723 and TXA707 following administration of their respective prodrugs (TXY541 and TXA709) to male BALB/c micea Prodrug Dose Administered (mg/kg)

ABT Pretreatment b

Product Measured

tmax (h)

Cmax AUClast t½ (ng/mL) (h•ng/mL) (h)

CL (mL/min/kg)

Vd-ss (L/kg)

%F

TXY541

24 (i.v.)

Yes

PC190723

0.50

7491

28589

1.95

13.20

2.11

NA

TXY541

24 (i.v.)

No

PC190723

0.25

6646

7216

0.56

55.11

2.18

NA

TXA709

24 (i.v.)

No

TXA707

0.50

13794

42299

3.65

9.40

2.02

NA

TXA709

32 (p.o.)

No

TXA707

1.00

9850

53679

2.66

NA

NA

95

a

Parameters were calculated using the sparse sampling mode in the non-compartmental analysis (NCA) module of the Phoenix WinNonlin version 6.3 software package. ABT, 1-aminobenzotriazole; NA, not applicable; i.v., intravenous; p.o., peroral.

b

Mice were pretreated for one hour with 50 mg/kg ABT administered orally.

546 547 548 549 TABLE 2 Protein binding of TXA707 in mouse, human, dog, and rat plasma

550 551

Species

% Protein Bounda

Mouse Human Dog Rat a Values reflect three replicates.

85.9 ± 0.4 90.9 ± 0.5 90.9 ± 0.3 90.8 ± 1.3 the mean ± SD of

TABLE 3 Activities against clinical isolates of MRSA, VISA, VRSA, DNSSA, LNSSA, and MSSAa Isolate and agent

MIC (µg/mL) Range

MBC (µg/mL) Modal

MRSA (n = 30) TXA707 0.25-1 1 TXA709 2-4 2 PC190723 0.5-1 1 VAN 0.5-1 1 DAP 0.5-2 1 LZD 2-4 2 VISA (n = 20) TXA707 0.5-2 1 TXA709 2-4 2 PC190723 0.5-1 1 VAN 4-8 4 DAP 1-8 1 LZD 0.5-2 2 VRSA (n = 11) TXA707 0.5-1 1 TXA709 2 2 PC190723 0.5-1 1 VAN 32->64 >64 DAP 0.25-1 1 LZD 0.5-4 2 DNSSA (n = 7) TXA707 0.5-1 1 TXA709 2-4 2 PC190723 0.5-1 1 VAN 1-2 2 DAP 4 4 LZD 1-2 2 LNSSA (n = 6) TXA707 1 1 TXA709 2 2 PC190723 0.5-1 1 VAN 1-2 1 DAP 0.5-1 0.5 LZD 16-64 16 MSSA (n = 10) TXA707 0.5-1 0.5/1b TXA709 2-4 4 PC190723 0.5-1 1 VAN 1-2 1 DAP 1 1 a VAN, vancomycin; DAP, daptomycin; LZD, linezolid; ND, not for MRSA were determined with 20 isolates. b Bimodal behavior.

Range

Modal

1-2 2-4 0.5-1 0.5-1 0.5-1 8->8

1 4 1 1 1 >8

0.5-2 2-4 0.5-2 4-8 1-16 2->8

1 2 1 4 2 >8

1 2 0.5-1 64->64 0.25-1 8->8

1 2 1 >64 1 >8

1-2 2-4 0.5-1 2 4-8 2->8

1 4 1 2 8 >8

1-2 2-4 1 1-2 0.5-1 32->64

1 2 1 1 0.5/1b >64

1-2 1/2b 4-8 8 1-2 2 1-4 2 ND ND determined. MBC values

552 553 TABLE 4 Activities against reference quality control and laboratory strains of MRSA and MSSA MIC (µg/mL)

Strain

TXA707

TXA709

PC190723

VAN

0.5

4

0.5

2

1

4

1

2

0.5

2

0.5

2

ATCC 19636a

0.5

2

0.5

1

ATCC 29213

0.5

2

1

1

0.5

2

0.5

0.5

MRSA ATCC 43300a b

ATCC 33591 Mu3

a

MSSA

8325-4 a

c

Strains used in the in vivo mouse systemic (peritonitis) infection studies. b Strain used in the in vivo mouse tissue (thigh) infection studies. c Strain used in the TEM studies. 554 555

FIGURE 1

556

557 558 559 560

A

B

FIG 1 Metabolites of the prodrugs TXY541 (A) and TXA709 (B) that are observed upon exposure to either human or mouse hepatocytes for 60 minutes at 37 °C.

FIGURE 2

561

MSSA - ATCC 19636 100

% Survival

80

60

Oral (p.o.) 40

TXY541 (128 mg/kg) TXY541 (64 mg/kg) ABT + Vehicle

20

0

ABT + TXY541 (64 mg/kg)

A 0

1

2

3 Days

4

5

MRSA - ATCC 43300 100

% Survival

80

60

Oral (p.o.) TXY541 (192 mg/kg)

40

TXY541 (64 mg/kg) ABT + Vehicle

20

0 0

562 563 564 565 566

ABT + TXY541 (64 mg/kg)

B 1

2

3 Days

4

5

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 hour prior to infection.

FIGURE 3

567

CAMH @ 37 °C (t½ = 7.7 ± 0.1 h) 20

0

0 20

TXA709 0.5 min

10

10

0

0

Absorbance @ 296 nm (mAU)

10

10

0

0

0

0

TXA709 7h

20

0

0

20

TXA709 23 h

20 10

0

0

10

10

0

0

10

TXA709 30 min

20

TXA707 23 h

9

TXA709 10 min

10

10

20

TXA709 5 min

10

20 10

TXA709 2 min

20

TXA709 4h

10

TXA709 0.5 min

20

TXA709 2h

20

Serum Alone

10

20

20

569 570 571 572 573 574 575 576 577

20

CAMH Alone

10

568

Mouse Serum @ 37 °C (t½ = 3.0 ± 0.2 min)

11

12

13

Retention Time (min)

14

TXA707 30 min

9

10

11

12

13

14

Retention Time (min)

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 hours in CAMH (bottom left) or 30 minutes 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 (t½) in each medium is indicated.

578

FIGURE 4

579 580 581 582

583 584 585 586 587 588 589 590 591 592 593 594 595

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 (nonFtsZ-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 two hours in the presence of DMSO vehicle (left) or 4 µg/mL TXA707 (8x MIC) (right). The arrows in in the left panel highlight FtsZ Z-rings at midcell. (C) Relative frequency of FtsZ amino acid substitutions conferring resistance to TXA707 among 20 independently isolated TXA707-resistant clones.

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FIG 5 Transmission electron micrographs of MSSA 8325-4 bacteria treated with DMSO vehicle or 4 µg/mL TXA707 (8x MIC) for the indicated periods of time.

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FIG 6 Oral (A-C) and intravenous (D-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.

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FIG 7 Oral efficacy of TXA709 in a mouse tissue (thigh) model of infection with MRSA ATCC 33591. The number of CFUs recovered from the infected thighs after 24 hours are shown. Mean CFU reductions resulting from TXA709 treatment were significant (p ≤ 0.003).

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

The clinical development of FtsZ-targeting benzamide compounds like PC190723 has been limited by poor drug-like and pharmacokinetic properties. Develo...
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