FabI/enoyl-acyl carrier protein reductase

AFN-1252是烯酰基载体蛋白还原酶（FabI）的强效抑制剂，在浓度<=0.12微克/毫升时，抑制了所有测试的金黄色葡萄球菌（n=502）和表皮葡萄球菌（t=51）的临床分离株，包括耐甲氧西林（甲氧西林）的分离株。相比之下，AFN-1251对肺炎链球菌、β-溶血性链球菌、肠球菌属、肠杆菌科、非发酵革兰氏阴性杆菌和卡他莫拉菌的临床分离物没有活性（MIC（90）>4微克/毫升）。这些数据支持AFN-1252继续开发用于治疗耐药葡萄球菌感染患者[1]。

类鼻疽是一种热带细菌感染，由革兰阴性菌假鼻疽伯克氏菌（B.pseudomallei；Bpm）引起。目前的治疗选择主要限于甲氧苄啶-磺胺甲恶唑和β-内酰胺类药物，治疗时间约为4个月。此外，有报道称对这些药物有耐药性。因此，迫切需要开发新的类鼻疽抗生素。抑制脂肪酸生物合成途径中的关键酶烯酰基ACP还原酶（FabI）在抗菌药物开发方面显示出巨大的前景。FabI已被确定为假鼻疽杆菌中存在的主要烯酰基ACP还原酶。在这项研究中，我们评估了目前正在临床开发的金黄色葡萄球菌FabI抑制剂AFN-1252与BpmFabI酶结合并抑制拟鼻疽杆菌生长的潜力。AFN-1252稳定了BpmFabI并抑制了酶活性，IC50为9.6 nM。它对从类鼻疽患者中分离出的拟鼻疽杆菌R15菌株显示出良好的抗菌活性（MIC为2.35mg/L）。以2.3Å的分辨率确定了含有AFN-1252的BpmFabI的X射线结构。BpmFabI与AFN-1252的复合物形成了一个对称的四聚体结构，每个单体亚基上都结合有一个AFN-125二分子。动力学和热熔融研究支持了AFN-1252可以独立于辅因子与BpmFabI结合的发现。这些研究的结构和机理见解可能有助于新FabI抑制剂的合理设计和开发[2]。

AFN-1252的BpmFabI酶抑制试验[2] AFN-1252是使用已发表的合成方案在内部合成的。38通过监测辅因子NADH.33的氧化，在分光光度测定中评估了AFN-1252中抑制BpmFabI的效力。用于测定的缓冲液为30 mM PIPES，pH 6.8，含有150 mM NaCl和1 mM EDTA。试验中使用了175 nM BpmFabI酶。米氏常数（Km）和Kcat是根据巴豆酰辅酶A浓度增加时的酶活性确定的。Km（257µM）和Kcat（307分钟-1）的值略高于报告的值（分别为188µM和215分钟-1）。为了测定IC50，将AFN-1252与BpmFabI预孵育30分钟，并通过加入含有巴豆酰辅酶A（300µM）和NADH（375µM）的底物混合物开始反应。通过跟踪340nm处吸光度的降低来监测NADH的氧化。IC50值是通过使用Graphpad Prism软件V4将剂量反应数据拟合到S形剂量反应（可变斜率）曲线来确定的。为了确定结合机制，在不同浓度的抑制剂下进行了动力学研究，并在固定浓度的巴豆酰辅酶a（300µM）下改变NADH的浓度，同时通过改变巴豆酰CoA的浓度将NADH浓度固定在375µM。随后生成Lineweaver–Burk图，以确定AFN-1252与BpmFabI结合的机制。

使用Wiegand等人描述的微量稀释技术进行最低抑菌浓度（MIC）测定，并进行了一些修改。39在脑心输注肉汤（BHIB）中制备化合物的两倍连续稀释液，并将其分配到96孔板中。将1×108cfu/mL的BpR15接种物加入每个孔中，在37°C下孵育24小时。同时制备生长对照（仅细菌接种物）、无菌对照（仅肉汤）和阳性对照（含三氯生的细菌接种）孔并孵育。记录了MIC，定义为抑制BpR15可见生长的AFN-1252的最低浓度。MIC结果为n=2[2]的平均值。

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 that targets the enoyl-acyl carrier protein reductase (FabI). A thorough screen for AFN-1252-resistant strains was undertaken to identify the spectrum of mechanisms for acquired resistance. A missense mutation in fabI predicted to encode FabI(M99T) was isolated 49 times, and a single isolate was predicted to encode FabI(Y147H). AFN-1252 only bound to the NADPH form of FabI, and the close interactions between the drug and Met-99 and Tyr-147 explained how the mutations would result in resistant enzymes. The clone expressing FabI(Y147H) had a pronounced growth defect that was rescued by exogenous fatty acid supplementation, and the purified protein had less than 5% of the enzymatic activity of FabI. FabI(Y147F) was also catalytically defective but retained its sensitivity to AFN-1252, illustrating the importance of the conserved Tyr-147 hydroxyl group in FabI function. The strains expressing FabI(M99T) exhibited normal growth, and the biochemical properties of the purified protein were indistinguishable from those of FabI. The AFN-1252 Ki(app) increased from 4 nm in FabI to 69 nm in FabI(M99T), accounting for the increased resistance of the corresponding mutant strain. The low activity of FabI(Y147H) precluded an accurate Ki measurement. The strain expressing FabI(Y147H) was also resistant to triclosan; however, the strain expressing FabI(M99T) was more susceptible. Strains with higher levels of AFN-1252 resistance were not obtained. The AFN-1252-resistant strains remained sensitive to submicromolar concentrations of AFN-1252, which blocked growth through inhibition of fatty acid biosynthesis at the FabI step.[3]

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This study examines the alteration in Staphylococcus aureus gene expression following treatment with the type 2 fatty acid synthesis inhibitor AFN-1252. An Affymetrix array study showed that AFN-1252 rapidly increased the expression of fatty acid synthetic genes and repressed the expression of virulence genes controlled by the SaeRS 2-component regulator in exponentially growing cells. AFN-1252 did not alter virulence mRNA levels in a saeR deletion strain or in strain Newman expressing a constitutively active SaeS kinase. AFN-1252 caused a more pronounced increase in fabH mRNA levels in cells entering stationary phase, whereas the depression of virulence factor transcription was attenuated. The effect of AFN-1252 on gene expression in vivo was determined using a mouse subcutaneous granuloma infection model. AFN-1252 was therapeutically effective, and the exposure (area under the concentration-time curve from 0 to 48 h [AUC(0-48)]) of AFN-1252 in the pouch fluid was comparable to the plasma levels in orally dosed animals. The inhibition of fatty acid biosynthesis by AFN-1252 in the infected pouches was signified by the substantial and sustained increase in fabH mRNA levels in pouch-associated bacteria, whereas depression of virulence factor mRNA levels in the AFN-1252-treated pouch bacteria was not as evident as it was in exponentially growing cells in vitro. The trends in fabH and virulence factor gene expression in the animal were similar to those in slower-growing bacteria in vitro. These data indicate that the effects of AFN-1252 on virulence factor gene expression depend on the physiological state of the bacteria. [4]

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Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to 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 the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. 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

141.68

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。