The mechanism of action of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), which is involved in fatty acid biosynthesis, was confirmed by using biochemistry, macromolecular synthesis, genetics, and cocrystallization of an AFN-1252-FabI complex. AFN-1252 demonstrated a low propensity for spontaneous resistance development and a time-dependent reduction of the viability of both methicillin-susceptible and methicillin-resistant S. aureus, achieving a >2-log 10 reduction in S. aureus counts over 24 h, and was extremely potent against clinical isolates of S. aureus (MIC 90 , 0.015 g/ml) and coagulase-negative staphylococci (MIC 90 , 0.12 g/ml), regardless of their drug resistance, hospital-or community-associated origin, or other clinical subgroup. AFN-1252 was orally available in mouse pharmacokinetic studies, and a single oral dose of 1 mg/kg AFN-1252 was efficacious in a mouse model of septicemia, providing 100% protection from an otherwise lethal peritoneal infection of S. aureus Smith. A median effective dose of 0.15 mg/kg indicated that AFN-1252 was 12 to 24 times more potent than linezolid in the model. These studies, demonstrating a selective mode of action, potent in vitro activity, and in vivo efficacy, support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.

AFN-1252, a potent inhibitor of enoyl-acyl carrier protein reductase (FabI), inhibited all clinical isolates of Staphylococcus aureus (n ‫؍‬ 502) and Staphylococcus epidermidis (n ‫؍‬ 51) tested, including methicillin (meticillin)-resistant isolates, at concentrations of <0.12 g/ml. In contrast, AFN-1252 was inactive (MIC 90 , >4 g/ml) against clinical isolates of Streptococcus pneumoniae, beta-hemolytic streptococci, Enterococcus spp., Enterobacteriaceae, nonfermentative gram-negative bacilli, and Moraxella catarrhalis. These data support the continued development of AFN-1252 for the treatment of patients with resistant staphylococcal infections.

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bThis open-label noncontrolled, phase II multicenter trial was designed to evaluate the safety, tolerability, and efficacy of 200 mg of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), given by mouth twice daily in the treatment of acute bacterial skin and skin structure infections (ABSSSI) due to staphylococci. Important aspects of the current study included a comparison of early response efficacy endpoints with end-of-treatment and follow-up endpoints. Many patients in the intent-to-treat population (n ‫؍‬ 103) had significant comorbidities. The overall early response rate at day 3 was 97.3% (wound, 100%; abscess, 96.6%; cellulitis, 94.4%) in the microbiologically evaluable (ME) population. Within the ME population, 82.9% of patients had a >20% decrease in the area of erythema, and 77.9% of patients had a >20% decrease in the area of induration, on day 3. S. aureus was detected in 97.7% of patients (n ‫؍‬ 37 patients with methicillin-resistant S. aureus W idespread overuse of broad-spectrum antibiotics over the past 20 years has been associated with significant rates of antibiotic resistance, with multidrug resistance prevalent among a broad range of bacterial pathogens (1), including Staphylococcus aureus. Many experts have advocated the development of speciestargeted agents which reduce off-target selection pressures on the human microbiome and could potentially decrease such conditions as antibiotic-induced colitis and candidiasis (2). Antiinfectives that specifically target Staphylococcus spp. may be less likely to lead to the development of resistant enterococcal, pneumococcal or other common bacterial pathogens.FabI catalyzes the last step in the essential bacterial fatty acid biosynthetic pathway and is the sole form of enoyl-acyl carrier protein (ACP) reductase present in S. aureus, Staphylococcus epidermidis, and other staphylococci (3, 4, 5, 6). Other enoyl-ACP reductase enzymes are present in many bacterial species, but these are sufficiently different to enable species-specific selectivity (7). AFN-1252, a FabI inhibitor, is the result of a program tasked with finding a new antimicrobial agent specifically active against Staphylococcus spp., including methicillin-resistant S. aureus (MRSA) (8).The objective of the current proof-of-concept study was to investigate the efficacy and safety of orally administered AFN-1252 in patients with acute bacterial skin and skin structure infection (ABSSSI) due to staphylococci. The study design followed recent FDA guidance (9) on the development of drugs for the treatment of ABSSSI. The inclusion criteria were designed to identify patients with a high likelihood of having a proven staphylococcal infection to ensure a high number of microbiologically evaluable patients. MATERIALS AND METHODSAntibacterial agent. AFN-1252 (free base, AFN-12520000) was formulated as immediate-release (IR) tablets.Study design. This noncontrolled, open-label, phase II trial was designed as a proof-of-concept study to evaluate the s...

AFN-1252, a new antimicrobial agent, specifically and potently inhibits fatty acid synthesis in Staphylococcus aureus. We characterized in vivo pharmacokinetic and pharmacodynamic profiles of AFN-1252 administered orally to neutropenic mice inoculated in thighs (∼106 CFU) with methicillin-susceptible S. aureus (MSSA) ATCC 29213. Efficacy was also assessed in mice inoculated with MSSA, hospital-acquired Methicillin-resistant Staphylococcus aureus (HA-MRSA) or community-acquired (CA)-MRSA, and administered AFN-1252 or linezolid orally. Bacterial density was determined after 24 hours and efficacy defined as the change in CFU/thigh versus untreated controls at time 0. With MSSA, antibacterial reductions of ≧1 log were observed at ≧20 mg/kg doses, with ƒAUC/minimum inhibitory concentration (MIC) best describing the pharmacodynamic profile of AFN-1252. The 80, 50 and 5% maximum effects were observed with ƒAUC/MIC values of 22·3, 17·0, and 9·6, respectively. Similar values were obtained for CA-MRSA and HA-MRSA. AFN-1252 was 4–40 fold more effective than linezolid against CA-MRSA and HA-MRSA. These data demonstrate the excellent in vivo potency of AFN-1252 against phenotypically diverse S. aureus.

The heavily encapsulated Acinetobacter calcoaceticus BD4 and the "miniencapsulated" single-step mutant A. calcoaceticus BD413 produced extracellular polysaccharides in addition to the capsular material. The molar ratio of rhamnose to glucose (3:1) in the extracellular BD413 polysaccharide fraction was similar to the composition of the capsular material. In both strains, the increase in capsular polysaccharide was parallel to cell growth and remained constant in stationary phase. The extracellular polysaccharides were detected starting from mid-logarithmic phase and continued to accumulate in the growth medium for 5 to 8 h after the onset of stationary phase. Strain BD413 produced one-fourth the total rhamnose exopolysaccharide per cell that strain BD4 did. Depending on the growth medium, 32 to 63% of the rhamnose polysaccharide produced by strain BD413 was extracellular, whereas in strain BD4 only 7 to 14% was extracellular. In all cases, strain BD413 produced more extracellular rhamnose polysaccharide than strain BD4 did. In glucose medium, strain BD413 also produced approximately 10 times more extracellular emulsifying activity than strain BD4 did. The isolated capsular polysaccharide obtained after shearing of BD4 cells showed no emulsifying activity. Thus, strain BD413 either produces a modified extracellular polysaccharide or excretes an additional substance(s) that is responsible for the emulsifying activity. Emulsions induced by the ammonium sulfate-precipitated BD413 extracellular emulsifier require the presence of magnesium ion and a mixture of an aliphatic and an aromatic hydrocarbon.

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