LeuRS(IC50= 0.31 μM)
"[1]. Cancer Res. 1999 Jun 1;59(11):2566-9.
链接: https://pubmed.ncbi.nlm.nih.gov/10363974/
[2]. Biochem J. 2004 Jan 1;377(Pt 1):249-55.
链接: https://pubmed.ncbi.nlm.nih.gov/14570592/
[2]. J Biol Chem. 2001 Jan 5;276(1):251-60.
链接: https://pubmed.ncbi.nlm.nih.gov/11013232/
请找到上述文章的全文(比如从https://pubmed.ncbi.nlm.nih.gov/ 或者出版社原文), 随后将这些文章中关于 Kenpaullone 这个药物的体内外药理/生物活性、药物代谢性质、毒性及相关实验流程按照下面的详细要求提炼出来。
一、提取信息的详细要求（请仔细阅读，完全按照下述要求来提取）：
1）一定要阅读全文，不能只看摘要；
2）提取的信息分成英文（全英文）和中文两个版本；
3）字段如果文献未描述，就不用列出来;
4) 将药物名称嵌入加粗代码, 如药物名称;
5) 来自不同文献的内容后面加上参考文献及分行代码如 “[1]
”或“[2]
等”；
6）同一个字段里面如果有几种不同的内容或实验步骤描述，请在不同内容或实验步骤之间插入分隔符
；
7) IC50、Ki、EC50、给药浓度/剂量等数值信息必须准确，如果不确定，宁可不提供；
8）仅提取指定文献里包含的此药物信息，不要将其他文献的信息放进来；
8）也不要引用指定文献之外的其他文献；
9）请务必准确、实事求是和详细，不要凭空捏造和幻想。一定不要提供不存在的假数据，我要的是真实数据，有就是有，没有就没有。
二、要提取的字段及具体要求按照如下格式（请仔细阅读，完全按照下述要求来提取）：
Target: 提炼出这个药物的作用靶点/靶标，并将不同靶标的IC50、Ki、EC50等信息放在括号里面（这些数据也不能造假，文献说是多少就是多少，不要瞎编和推测）；不同文献的靶点用
分开。
In Vitro：描述体外活性的详细实验结果（如抗增殖活性、酶/靶点活性、细胞活性、western blot, PCR、免疫组织化学、凋亡、克隆形成、作用机制等；数据不能造假，文献说是多少就是多少，不要瞎编和推测）, 不同的体外实验结果分别描述，详细描述； 不同文献的内容/描述后面加上参考文献及分行代码如 “[1]
”或“[2]
等。
In Vivo：描述体内活性的详细实验结果(如体内药效、作用机制等；数据不能造假，文献说是多少就是多少，不要瞎编和推测), 不同的体内实验结果分别描述，详细描述； 不同文献的内容/描述后面加上参考文献及分行代码如 “[1]
”或“[2]
等。
Enzyme Assay： 关于酶（或者蛋白/受体）活性实验 或者靶点结合实验（如激酶活性、SPR、ITC、HTRF等；数据不能造假，文献说是多少就是多少，不要瞎编和推测）的详细流程描述 (在原描述的基础上改变说法重新描述，不要只提取一两句话), 不同的实验分别描述，详细描述，不同实验之间嵌入分行代码
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”或“[2]
等。
Cell Assay： 关于细胞实验（如抗增殖、Cell viability/antiproliferative, western blot, PCR、免疫组织化学、凋亡、克隆形成实验等；数据不能造假，文献说是多少就是多少，不要瞎编和推测）详细流程的描述(在原描述的基础上改变说法重新描述，不要只提取一两句话), 不同的细胞实验分别描述，详细描述，不同实验之间嵌入分行代码
；另外，不要出现试剂/药物的供应商的名称； 不同文献的内容/描述后面加上参考文献及分行代码如 “[1]
”或“[2]
等。
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；另外，不要出现试剂/药物的供应商的名称； 不同文献的内容/描述后面加上参考文献及分行代码如 “[1]
”或“[2]
等。
ADME/Pharmacokinetics：关于这个药物的体内外药物代谢特征（PK性质），如吸收、分布、代谢、排泄、半衰期、口服生物利用度等参数（数据不能造假，文献说是多少就是多少，不要瞎编和推测），不同的ADME性质分别描述，详细描述，不同的药代参数之间嵌入分行代码
； 不同文献的内容/描述后面加上参考文献及分行代码如 “[1]
”或“[2]
等。
Toxicity/Toxicokinetics：关于这个药物的体内外毒性/副作用信息，如半数致死量、肝肾毒性、药物药物相互作用、血浆蛋白结合度（protein binding）等（数据不能造假，文献说是多少就是多少，不要瞎编和推测），不同的性质分别描述，详细描述； 不同文献的内容/描述后面加上参考文献及分行代码如 “[1]
”或“[2]
等。
Additional Info：除了上述信息之外的其他和这个药物相关的信息，如背景介绍、作用机制、疗效、适应症、FDA警示信息，不同类别信息分别描述，详细描述； 不同文献的内容/描述后面加上参考文献及分行代码如 “[1]
”或“[2]
等。"

Key gram-negative aerobic and anaerobic pathogens as well as gram-positive anaerobes are susceptible to the antibacterial action of epetraborole (0-32 μg/mL)[1].

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1. Antibacterial activity against anaerobic organisms: Epetraborole hydrochloride (GSK2251052) exhibited potent in vitro activity against 916 clinical anaerobic isolates representing 35 genera. MIC₅₀/MIC₉₀ values (μg/mL) for key genera were: Bacteroides fragilis group (0.25/1), Prevotella spp. (0.12/0.5), Porphyromonas spp. (0.06/0.25), Fusobacterium spp. (0.12/0.5), Clostridium difficile (0.5/1), Clostridium perfringens (0.12/0.25), and Peptostreptococcus spp. (0.12/0.5). Overall, 99.3% of isolates were susceptible at ≤2 μg/mL (CLSI breakpoint for anaerobic bacteria). It showed comparable or superior activity to metronidazole, clindamycin, and piperacillin-tazobactam against most genera [1]
2. Lack of cross-resistance: Epetraborole hydrochloride (GSK2251052) retained activity against metronidazole-resistant B. fragilis group isolates (MIC₅₀/MIC₉₀ = 0.25/1 μg/mL) and clindamycin-resistant Prevotella spp. (MIC₅₀/MIC₉₀ = 0.12/0.5 μg/mL), with no significant difference in MIC distribution compared to susceptible isolates [1]
3. Bacterial resistance development in vitro: Serial passage of E. coli (n=3) and Klebsiella pneumoniae (n=3) in subinhibitory concentrations of Epetraborole hydrochloride (GSK2251052) (0.5×MIC to 2×MIC) for 20 days induced resistance, with MIC increases of 8- to 64-fold (E. coli) and 4- to 32-fold (K. pneumoniae) compared to parental strains. Whole-genome sequencing identified missense mutations in the leuS gene (encoding LeuRS) as the primary resistance mechanism [2]
4. Resistance in clinical isolates: Among 104 E. coli isolates from patients with complicated urinary tract infections (cUTIs) treated with Epetraborole hydrochloride (GSK2251052), 12 (11.5%) developed resistance during therapy. Resistant isolates had MIC values ≥8 μg/mL (vs. ≤1 μg/mL for susceptible isolates) and carried leuS mutations (A431V, D405N, G412S, etc.). No cross-resistance to other antibiotic classes (fluoroquinolones, β-lactams, trimethoprim-sulfamethoxazole) was observed [2]
5. Inhibition of LeuRS activity: Epetraborole hydrochloride (GSK2251052) specifically inhibited bacterial LeuRS by interfering with the aminoacylation reaction (charging of tRNA^Leu with leucine), blocking protein synthesis. Recombinant LeuRS from resistant E. coli strains (carrying leuS mutations) showed reduced inhibitor binding affinity, confirming target-based resistance [2]

1. Bacterial LeuRS aminoacylation assay: Prepare recombinant LeuRS from target bacteria (e.g., E. coli, B. fragilis) via heterologous expression and purification. Set up reaction mixtures containing LeuRS, tRNA^Leu, L-leucine, ATP, and varying concentrations of Epetraborole hydrochloride (GSK2251052). Incubate the mixtures at 37°C for 10-30 minutes. Measure the formation of leucyl-tRNA^Leu using a radiometric assay (incorporation of [³H]-leucine) or a fluorescence-based assay (detection of ATP hydrolysis). Calculate the percentage inhibition of LeuRS activity relative to inhibitor-free controls [1, 2]
2. Resistant LeuRS activity assay: Express and purify recombinant LeuRS from parental (susceptible) and leuS-mutated (resistant) E. coli strains. Perform aminoacylation assays as described above to compare the inhibitory efficacy of Epetraborole hydrochloride (GSK2251052) between wild-type and mutant LeuRS. Determine IC₅₀ values by plotting inhibition percentage against inhibitor concentration [2]

Cell Line: Anaerobes isolates
Concentration: 0-32 μg/mL
Result: revealed MIC50/MIC90 values of 2 and 4 μg/mL for every anaerobe isolate tested.

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1. Anaerobic antibacterial susceptibility assay (microbroth dilution method): Prepare twofold serial dilutions of Epetraborole hydrochloride (GSK2251052) (0.008-64 μg/mL) in anaerobic broth (e.g., Brucella broth with hemin, vitamin K1, and 5% lysed horse blood). Inoculate each well with 5×10⁵ CFU/mL of anaerobic bacteria (log-phase cultures). Incubate plates at 35°C in an anaerobic chamber (85% N₂, 10% H₂, 5% CO₂) for 48 hours. The MIC is defined as the lowest inhibitor concentration that inhibits visible bacterial growth. Include quality control strains (B. fragilis ATCC 25285, C. difficile ATCC 700057) and comparator antibiotics (metronidazole, clindamycin) for validation [1]
2. Resistance induction assay: Grow parental bacterial strains (E. coli, K. pneumoniae) in Mueller-Hinton broth (MHB) at 37°C with shaking. Subculture bacteria daily into fresh MHB containing subinhibitory concentrations of Epetraborole hydrochloride (GSK2251052) (starting at 0.5×MIC, adjusted to 1×MIC or 2×MIC based on growth). After 20 serial passages, determine MIC values of the evolved strains using microbroth dilution. Confirm resistance by plating on inhibitor-containing agar (4×MIC) and sequencing the leuS gene to identify mutations [2]
3. Clinical isolate susceptibility testing: Isolate E. coli from urine samples of cUTI patients before and after Epetraborole hydrochloride (GSK2251052) treatment. Culture isolates on MacConkey agar and identify via biochemical tests. Perform MIC determination using microbroth dilution (as described above) and classify isolates as susceptible (MIC ≤1 μg/mL) or resistant (MIC ≥8 μg/mL). Extract genomic DNA from resistant isolates and sequence the leuS gene to detect mutations [2]

[1]. Comparative in vitro activities of GSK2251052, a novel boron-containing leucyl-tRNA synthetase inhibitor, against 916 anaerobic organisms. Antimicrob Agents Chemother. 2013 May;57(5):2401-4.

[2]. Bacterial resistance to leucyl-tRNA synthetase inhibitor GSK2251052 develops during treatment of complicated urinary tract infections. Antimicrob Agents Chemother. 2015 Jan;59(1):289-98.

[3]. Sutcliffe JA. Antibiotics in development targeting protein synthesis. Ann N Y Acad Sci. 2011 Dec;1241:122-52.

1. Chemical Classification and Mechanism of Action: Epeborone hydrochloride (GSK2251052) is a novel boron-containing antibiotic that targets bacterial leucine synthase (LeuRS), a key enzyme in protein synthesis. The boron group interacts with the active site of LeuRS, interfering with the aminoacylation of tRNA^Leu, which is essential for bacterial growth and survival [1, 3]. 2. Therapeutic Potential: This drug has been developed for the treatment of infections caused by anaerobic bacteria, including intra-abdominal infections, skin and soft tissue infections, and respiratory infections. Its potent activity against clinically relevant anaerobic bacteria and lack of cross-resistance with existing antibiotics make it an ideal candidate for the treatment of multidrug-resistant (MDR) anaerobic infections [1]. 3. Resistance Issues: Resistance to epeeborone hydrochloride (GSK2251052) is mainly due to missense mutations in the leuS gene, which alter the active site of LeuRS and reduce the binding of inhibitors. The emergence of drug resistance during the treatment of complicated urinary tract infections (cUTI) highlights the necessity of rational drug use and combination therapy to minimize the development of drug resistance [2]
4. Status in antibiotic development:Epeborone hydrochloride (GSK2251052) belongs to a new class of protein synthesis inhibitors that target aminoacyl-tRNA synthetase, a validated but underutilized target class. It meets the urgent need for novel antibiotics against MDR anaerobic pathogens [3]

C11H17BCLNO4

273.5210

273.093

C, 48.30; H, 6.26; B, 3.95; Cl, 12.96; N, 5.12; O, 23.40

1234563-16-6

1234563-16-6 (HCl);1093643-37-8;1234563-15-5 (R-mandelate);

52918389

White to off-white solid powder

4

5

5

18

244

1

Cl[H].O1B(C2C(=C([H])C([H])=C([H])C=2[C@@]1([H])C([H])([H])N([H])[H])OC([H])([H])C([H])([H])C([H])([H])O[H])O[H]

DADYQGIQOBJGIW-HNCPQSOCSA-N

InChI=1S/C11H16BNO4.ClH/c13-7-10-8-3-1-4-9(16-6-2-5-14)11(8)12(15)17-10;/h1,3-4,10,14-15H,2,5-7,13H2;1H/t10-;/m1./s1

(S)-3-(Aminomethyl)-7-(3-hydroxypropoxy)-1-hydroxy-1,3-dihydro-2,1-benzoxaborole hydrochloride

Epetraborole hydrochloride; GSK-2251052; GSK 2251052; GSK2251052; AN3365; AN 3365; AN-3365; GSK-052; GSK 052; GSK052;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~200 mg/mL (~731.21 mM)
H2O : ≥ 28 mg/mL (~102.37 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.14 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.14 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

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Solubility in Formulation 3: 5% DMSO+40% PEG300+5% Tween-80+50% Saline: ≥ 2.5 mg/mL (9.14 mM)

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Solubility in Formulation 4: 100 mg/mL (365.60 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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