FabI/enoyl-acyl carrier protein reductase
AFN-1252 targets FabI, the enoyl-acyl carrier protein reductase enzyme in Staphylococcus spp. FabI is a key enzyme in the type II fatty acid synthesis (FASII) pathway, which is essential for bacterial membrane synthesis. By inhibiting FabI with high affinity (Ki = 12.8 nM), AFN-1252 blocks the final step in each round of fatty acid elongation, depleting the bacterial cell of essential membrane components. This action is specific to the bacterial FASII pathway, which is structurally and functionally distinct from the fatty acid synthesis machinery in human cells, thereby providing a basis for the compound's selective antibacterial activity.

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 4 microg/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[1].
In vitro, AFN-1252 demonstrates extremely potent activity against clinical isolates of Staphylococcus aureus, with a minimum inhibitory concentration (MIC90) of 0.015 µg/mL. It also shows potent activity against coagulase-negative staphylococci, with an MIC90 of 0.12 µg/mL. The compound inhibits all clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis at concentrations of ≤0.12 µg/mL. AFN-1252 is inactive (MIC90 >4 µg/mL) against clinical isolates of Streptococcus pneumoniae, beta-hemolytic streptococci, Enterococcus spp., Enterobacteriaceae, and nonfermentative gram-negative bacilli, confirming its narrow, Staphylococcus-specific spectrum of activity.

Melioidosis is a tropical bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei; Bpm), a Gram-negative bacterium. Current therapeutic options are largely limited to trimethoprim-sulfamethoxazole and β-lactam drugs, and the treatment duration is about 4 months. Moreover, resistance has been reported to these drugs. Hence, there is a pressing need to develop new antibiotics for Melioidosis. Inhibition of enoyl-ACP reducatase (FabI), a key enzyme in the fatty acid biosynthesis pathway has shown significant promise for antibacterial drug development. FabI has been identified as the major enoyl-ACP reductase present in B. pseudomallei. In this study, we evaluated AFN-1252, a Staphylococcus aureus FabI inhibitor currently in clinical development, for its potential to bind to BpmFabI enzyme and inhibit B. pseudomallei bacterial growth. AFN-1252 stabilized BpmFabI and inhibited the enzyme activity with an IC50 of 9.6 nM. It showed good antibacterial activity against B. pseudomallei R15 strain, isolated from a melioidosis patient (MIC of 2.35 mg/L). X-ray structure of BpmFabI with AFN-1252 was determined at a resolution of 2.3 Å. Complex of BpmFabI with AFN-1252 formed a symmetrical tetrameric structure with one molecule of AFN-1252 bound to each monomeric subunit. The kinetic and thermal melting studies supported the finding that AFN-1252 can bind to BpmFabI independent of cofactor. The structural and mechanistic insights from these studies might help the rational design and development of new FabI inhibitors[2].
In vivo, AFN-1252 is efficacious in a mouse model of septicemia. In a study using female CD-1 mice infected with a lethal peritoneal inoculum of S. aureus Smith, a single oral dose of AFN-1252 at 1 mg/kg provided 100% protection. The compound was administered via oral gavage at doses ranging from 0.03 to 3 mg/kg, and the calculated ED50 (95% confidence limits) was 0.15 (0.11 to 0.21) mg/kg. These results demonstrate the potent in vivo efficacy of AFN-1252 against systemic staphylococcal infections.

BpmFabI enzyme inhibition assay for AFN-1252[2]
AFN-1252 was synthesized in-house using a published synthetic scheme.38 The potency of AFN-1252 to inhibit BpmFabI was evaluated in a spectrophotometric assay by monitoring the oxidation of the cofactor NADH.33 Buffer used for the assay was 30 mM PIPES, pH 6.8, containing 150 mM NaCl, and 1 mM EDTA. 175 nM BpmFabI enzyme was used in the assay. Michaelis–Menton constant (Km) and Kcat were determined from the enzyme activity at increasing concentrations of crotonyl-CoA. The values of Km (257 µM) and Kcat (307 min−1) were slightly higher than the reported values (188 µM and 215 min−1, respectively). To determine the IC50, AFN-1252 was preincubated with BpmFabI for 30 min and the reaction was started by adding substrate mix containing crotonyl-CoA (300 µM) and NADH (375 µM). The oxidation of NADH was monitored by following the decrease of absorbance at 340 nm. IC50 value was determined by fitting the dose-response data to sigmoidal dose response (variable slope) curve using Graphpad Prism software V4. To determine the mechanism of binding, kinetic studies were carried out at different concentrations of inhibitor and varying the concentration of NADH at a fixed concentration of crotonoyl-CoA (300 µM) and also by varying the concentrations of crotonoyl-CoA keeping NADH concentration fixed at 375 µM. Lineweaver–Burk plots were subsequently generated to determine the mechanism of binding of AFN-1252 to BpmFabI.

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Thermofluor assay[2]
Melting temperature of BpmFabI protein in presence and absence of inhibitors were determined with ABI Prism 7500 instrument (Applied Biosystems, Carlsbad) in the presence of 5× SYPRO Orange dye. 2 µM BpmFabI in 20 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole with/without 50 µM NADH was incubated with 20 µM AFN-1252/Triclosan for 1 h followed by the addition of SYPRO Orange dye. Melting temperatures were determined with Boltzmann equation using Protein thermal shift™ software version 1.1.
In vitro enzyme or receptor binding assays for AFN-1252 involve measuring its direct inhibition of FabI enzymatic activity. The assay typically uses a purified recombinant FabI enzyme from Staphylococcus aureus and a substrate, such as trans-2-dodecenoyl-CoA, in the presence of NADH. The enzymatic activity is monitored spectrophotometrically by following the decrease in absorbance at 340 nm, which corresponds to the oxidation of NADH. The compound is incubated with the enzyme and substrate at varying concentrations, and the inhibition constant (Ki) is calculated from the resulting kinetic data. For AFN-1252, a Ki value of 12.8 nM has been determined using this method.

Minimum inhibitory concentration (MIC) determination was carried out using the microdilution technique described by Wiegand et al. with some modifications.39 A twofold serial dilution of the compound was prepared in Brain Heart infusion broth (BHIB) and dispensed into 96-well plate. An inoculum of 1 × 108 cfu/mL of BpR15 was added into each well and incubated at 37°C for 24 h. Growth control (bacterial inoculum only), sterility control (broth only) and positive control (bacterial inoculum with Triclosan) wells were prepared and incubated simultaneously. The MIC, defined as the lowest concentration of AFN-1252 that inhibited visible growth of BpR15, was recorded. The MIC results are average of n = 2[2].
In vitro cell-based assays for AFN-1252 are performed using standard broth microdilution methods to determine the minimum inhibitory concentration (MIC). Clinical isolates of Staphylococcus spp. are cultured in cation-adjusted Mueller-Hinton broth and incubated with serial two-fold dilutions of the compound. The MIC is defined as the lowest concentration of the compound that inhibits visible growth of the organism after overnight incubation at 35°C. For AFN-1252, this assay has demonstrated MIC90 values of 0.015 µg/mL against S. aureus and 0.12 µg/mL against coagulase-negative staphylococci. Time-kill studies can also be performed to assess the bactericidal activity of the compound.

Burkholderia pseudomallei strain R15 (herein referred to as BpR15) was isolated from an individual who succumbed to melioidosis at the Kuala Lumpur Hospital in Malaysia.40 BpR15 was routinely cultured on Ashdown agar at 37°C and overnight bacterial cultures were prepared in BHIB. AFN-1252 was dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C until use[2].
In vivo animal experiments for AFN-1252 are conducted using a murine septicemia model. Female CD-1 mice (5-6 weeks old, 18-22 g) are infected intraperitoneally with a lethal inoculum of S. aureus Smith. The compound is administered orally as a single dose at various concentrations (e.g., 0.03, 0.1, 0.3, 1, 3 mg/kg) one hour post-infection. Survival is monitored over a period of 7 days. The protective efficacy is expressed as the percentage of surviving animals and as the ED50, which is calculated using probit analysis. This model is standard for evaluating the in vivo efficacy of novel antibacterial agents against systemic staphylococcal infections.

Pharmacokinetic (PK) properties of AFN-1252 indicate that it is orally active and bioavailable. The compound has a molecular weight of 375.42 and a molecular formula of C22H21N3O3. It is soluble in DMSO at a concentration of 5.8 mg/mL (15.45 mM) with the aid of sonication. For in vivo studies, the compound is typically formulated for oral administration. The powder is stable when stored at -20°C for up to 3 years and at 4°C for up to 2 years. In solution, it can be stored at -80°C for 2 years or at -20°C for 1 year. The compound exhibits typical pharmacokinetic parameters in preclinical species, supporting its once-daily oral dosing regimen.

Toxicology (toxicology) data for AFN-1252 are characteristic of a selective antibacterial agent. Its narrow spectrum of activity against Staphylococcus spp. is a key feature of its safety profile, as it minimizes disruption to the normal human microbiome. The compound's selective inhibition of the bacterial FASII pathway, which is absent in human cells, further contributes to its favorable toxicological profile. In preclinical studies, the compound is generally well-tolerated at therapeutic doses. Standard toxicology studies, including acute and repeat-dose toxicity assessments in rodent and non-rodent species, are conducted to support its development as a novel antibiotic.

[1]. AFN-1252, a FabI inhibitor, demonstrates a Staphylococcus-specific spectrum of activity. Antimicrob Agents Chemother. 2009 Aug;53(8):3544-8.

[2]. AFN-1252 is a potent inhibitor of enoyl-ACP reductase from Burkholderia pseudomallei-Crystal structure, mode of action, and biological activity. Protein Sci. 2015 May;24(5):832-40.

[3]. Resistance to AFN-1252 arises from missense mutations in Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI). J Biol Chem. 2013 Dec 20;288(51):36261-71.

[4].Perturbation of Staphylococcus aureus gene expression by the enoyl-acyl carrier protein reductase inhibitor AFN-1252. Antimicrob Agents Chemother. 2013 May;57(5):2182-90.

[5]. Yao J, Ericson ME, Frank MW, Rock CO. Enoyl-Acyl Carrier Protein Reductase I (FabI) is Essential for the Intracellular Growth of Listeria monocytogenes. Infect Immun. 2016 Oct 10. pii: IAI.00647-16. PubMed PMID: 27736774.

AFN-1252 has been used in research trials for the treatment of cellulitis, burn infections, wound infections, skin abscesses, and bacterial infections of the skin and subcutaneous tissue.
Other information: AFN-1252 is an investigational antibiotic being developed for the treatment of serious staphylococcal infections, including those caused by MRSA. It is a first-in-class FabI inhibitor with a unique mechanism of action. The compound has been the subject of extensive preclinical research, including studies on its in vitro activity, in vivo efficacy, and pharmacokinetic properties. AFN-1252 is available as a research compound for non-clinical studies and is not approved for human use.

C22H21N3O3

375.4204

375.158

C, 70.38; H, 5.64; N, 11.19; O, 12.78

620175-39-5

1047981-31-6;620175-39-5;1047981-30-5 (tosylate hydrate);

10407120

White to off-white solid powder.

3.777

1

4

4

28

622

0

O1C2=C([H])C([H])=C([H])C([H])=C2C(C([H])([H])[H])=C1C([H])([H])N(C([H])([H])[H])C(/C(/[H])=C(\[H])/C1C([H])=NC2=C(C=1[H])C([H])([H])C([H])([H])C(N2[H])=O)=O

QXTWSUQCXCWEHF-JXMROGBWSA-N

InChI=1S/C22H21N3O3/c1-14-17-5-3-4-6-18(17)28-19(14)13-25(2)21(27)10-7-15-11-16-8-9-20(26)24-22(16)23-12-15/h3-7,10-12H,8-9,13H2,1-2H3,(H,23,24,26)/b10-7+

(E)-N-methyl-N-((3-methylbenzofuran-2-yl)methyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide

AFN-1252; AFN 1252; AFN1252; API-1252; API1252; API 1252; AFN12520000; AFN 12520000; AFN12520000; Debio1452; Debio 1452; Debio1452; afabicin desphosphono; API 1252; DEBIO1452; DEBIO-1452; Debio 1452;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :5.8~9 mg/mL ( 15.45~23.97 mM）

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)