FabI/enoyl-acyl carrier protein reductase

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].

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].

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.

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].

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].

[1]. Karlowsky\nJA, et al. AFN-1252, a FabI inhibitor, demonstrates a\nStaphylococcus-specific spectrum of activity. Antimicrob Agents\nChemother. 2009 Aug;53(8):3544-8.

[2]. Narasimha\nRao K, et al. AFN-1252 is a potent inhibitor of enoyl-ACP reductase\nfrom Burkholderia pseudomallei-Crystal structure, mode of action, and\nbiological activity. Protein Sci. 2015 May;24(5):832-40.

[3]. Yao\nJ, et al. Resistance to AFN-1252 arises from missense mutations in\nStaphylococcus aureus enoyl-acyl carrier protein reductase (FabI). J\nBiol Chem. 2013 Dec 20;288(51):36261-71.

[4]. Parsons\nJB, et al. Perturbation of Staphylococcus aureus gene expression by the\nenoyl-acyl carrier protein reductase inhibitor AFN-1252. Antimicrob\nAgents Chemother. 2013 May;57(5):2182-90.

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

AFN-1252 is a potent antibiotic against Staphylococcus aureus, targeting enoyl-acyl carrier protein reductase (FabI). To identify the acquired resistance mechanism, we comprehensively screened AFN-1252-resistant strains. We isolated 49 fabI missense mutants predicted to encode FabI (M99T) and one strain predicted to encode FabI (Y147H). AFN-1252 binds only to the NADPH form of FabI, and the tight interaction between the drug and Met-99 and Tyr-147 explains how these mutations lead to the development of the resistance enzyme. Clones expressing FabI (Y147H) exhibited significant growth defects, but could be rescued by exogenous fatty acid supplementation, and the enzymatic activity of purified FabI (Y147H) protein was less than 5% of that of FabI. FabI(Y147F) also exhibits catalytic defects but remains sensitive to AFN-1252, indicating that the conserved Tyr-147 hydroxyl group is crucial for FabI function. Strains expressing FabI(M99T) grew normally, and the biochemical properties of the purified protein were identical to those of FabI. The Ki(app) value for AFN-1252 increased from 4 nM for FabI to 69 nM for FabI(M99T), explaining the enhanced resistance of the corresponding mutant. FabI(Y147H) showed lower activity, making accurate Ki value determination impossible. Strains expressing FabI(Y147H) also exhibited resistance to triclosan; however, strains expressing FabI(M99T) were more susceptible. No strains with higher AFN-1252 resistance were obtained. AFN-1252-resistant strains remained sensitive to submicromolar concentrations of AFN-1252, which blocked their growth by inhibiting fatty acid biosynthesis in the Fabi step. [3]

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This study investigated the changes in gene expression in Staphylococcus aureus after treatment with the type II fatty acid synthesis inhibitor AFN-1252. Affymetrix microarray analysis showed that AFN-1252 rapidly increased the expression of fatty acid synthesis genes and inhibited the expression of virulence genes controlled by the SaeRS two-component regulatory factor in cells in the exponential growth phase. In saeR-deficient strains or Newman strains expressing constitutively active SaeS kinase, AFN-1252 did not alter the virulence mRNA level. AFN-1252 led to a significant increase in fabH mRNA levels in cells entering the stationary phase, while the inhibitory effect on virulence factor transcription was weakened. This study used a mouse subcutaneous granuloma infection model to determine the effect of AFN-1252 on gene expression in vivo. The results showed that AFN-1252 had a therapeutic effect, and the exposure level in the bag solution (area under the concentration-time curve [AUC(0-48)]) was comparable to that of the plasma concentration in the orally administered animals. AFN-1252 inhibited the biosynthesis of fatty acids in the infected bag, as evidenced by a significant and sustained increase in the level of fabH mRNA in the related bacteria in the bag; while the decrease in the level of virulence factor mRNA in the bacteria after AFN-1252 treatment was not as obvious as that in cells in the exponential growth phase in vitro. The trend of fabH and virulence factor gene expression in animals was similar to that of bacteria with slower growth in vitro. These data suggest that the effect of AFN-1252 on virulence factor gene expression depends on the physiological state of the bacteria. [4] The enoyl-acyl carrier protein reductase catalyzes the last step of each extended cycle of fatty acid synthesis in type II bacteria and is a key regulatory protein in bacterial fatty acid synthesis. The facultative intracellular pathogen Listeria monocytogenes encodes two functional enoyl-acyl carrier protein isoforms that complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but as expected, the FabK isoform was unaffected. AFN-1252 inhibition of FabI reduced endogenous fatty acid synthesis by 80% and decreased the growth rate of Listeria monocytogenes in laboratory media. Effective exogenous fatty acid incorporation was not detected in Listeria monocytogenes unless this pathway was partially inhibited by treatment with AFN-1252. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented intracellular growth of Listeria monocytogenes, indicating that neither FabK nor host cell fatty acid incorporation is sufficient to support intracellular growth of Listeria monocytogenes. Our results indicate that FabI is the major enoyl-acyl reductase for fatty acid synthesis in type II bacteria and is crucial for the intracellular growth of Listeria monocytogenes. [5]

CC29H29N3O6S

547.622066259384

547.178

C, 63.61; H, 5.34; N, 7.67; O, 17.53; S, 5.85

1047981-31-6

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

24880177

Typically exists as solid at room temperature

6.1

2

7

5

39

829

0

S(C1C=CC(C)=CC=1)(=O)(=O)O.O1C2C=CC=CC=2C(C)=C1CN(C)C(/C=C/C1C=NC2=C(C=1)CCC(N2)=O)=O

NHTYVWVPSCBFQW-HCUGZAAXSA-N

InChI=1S/C22H21N3O3.C7H8O3S/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)23-12-151-6-2-4-7(5-3-6)11(8,9)10/h3-7,10-12H,8-9,13H2,1-2H3,(H,23,24,26)2-5H,1H3,(H,8,9,10)/b10-7+

2-Propenamide, N-methyl-N-((3-methyl-2-benzofuranyl)methyl)-3-(5,6,7,8-tetrahydro-7-oxo-1,8-naphthyridin-3-yl)-, (2E)-, 4-methylbenzenesulfonate (1

AFN-1252 tosylate; API-1252 tosylate; AFN-1252; Debio 1452; AFN 1252; AFN1252; AFN-12520000; API-1252; Debio-1452; AFN12520000; API1252; Debio1452; AFN-1252 tosylate; 1047981-31-6; API-1252 tosylate; UNII-VDH5PP94F0; VDH5PP94F0; 2-Propenamide, N-methyl-N-((3-methyl-2-benzofuranyl)methyl)-3-(5,6,7,8-tetrahydro-7-oxo-1,8-naphthyridin-3-yl)-, (2E)-, 4-methylbenzenesulfonate (1:1); 4-methylbenzenesulfonic acid;(E)-N-methyl-N-[(3-methyl-1-benzofuran-2-yl)methyl]-3-(7-oxo-6,8-dihydro-5H-1,8-naphthyridin-3-yl)prop-2-enamide; SCHEMBL725467;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)