抗炭疽致死毒素药物的发现研究

Anthrax attack is a reality bioterrorism threat.Currently there are only two counter measures available for anthrax: antibiotics and vaccine. After infection of bacteria bacillus anthracis,it is known that the bacteria secrete three proteins in vivo: edema factor (EF) and lethal factor (LF) each of which combine with protective antigen (PA) to form two binary toxins edema toxin and lethal toxin (ET and LT, respectively).Even with successful antibiotic treatment and vaccination, anthrax toxins can remain in the blood stream and cause lethality.Thus both of the two measures are drawbacks for treatment of anthrax infection.Antibiotics have no effect on the exotoxin itself and vaccination against anthrax has been confined to people at risk, such as members of the armed forces. Among the toxin components, LF is the dominant virulence factor in anthrax pathogenesis, suggesting a key role for the toxin. Since weaponized anthrax continues to pose a threat to society, there remains a critical need for small-molecule LF inhibitors that can be administered concurrent with antibiotics to increase the probability of host survival.So it is an urgent requirement of safety and stabilization of country to discover and develop LF inhibitors as a clinical antidote for anthrax toxin. Several classes of chemically distinct classes of LF inhibitors have been identified in worldwide.In the previous studies of this project,we designed and synthesized a series of LF inhibitors by analyzing the structural characteristic of LF active site and discloesd LF inhibitors.The evaluation methods of LF inhibitor,including binding assay,cell and animal model,were established.Some of those compounds showed activity against rathrax LF in low micromolar to nanomolar concentrations and one of them was identitied with significant activity in the rat anthrax LT challenge model.On the basis of previous work, this project will further focus on design,synthesis and structural optimization of the LF inhibitors. Combining the interaction model between LF and known inhibitors,and SAR of the inhibitors obtained in our previous work,a structural optimiaztion strategy was suggested in this project.Two series of new compounds were designed and feasibilty synthetic routes were proposed.The inhibitory activities against anthrax LF and other metalloprotease (MMPs and ACE, ECE, NEP, TACE) of these compounds will be evaluated by a binding assy.The antitoxin of compounds against anthrax letal toxin will be evaluated through RAW 264.7 cell lines and a mouse anthrax LT challenge model.Early ADME/TOX of active compounds will also be evaluated.We hope that new LF inhibitors with high activity,selectivity and acceptability ADME/TOX will be discovered to lay a foundation for developing a clinical antidote for anthrax lethal toxin.

炭疽攻击是一种现实的恐怖威胁，应用疫苗与抗生素治疗炭疽感染都有局限性。炭疽毒素是炭疽感染导致死亡的重要原因，目前还没有抗炭疽毒素中毒的药物。致死因子是炭疽毒素的主要毒力因子。因此，研究炭疽毒素致死因子抑制剂，进而开发抗炭疽致死毒素药物，是关乎国家安全稳定的迫切需要。 在前期研究中，我们根据炭疽毒素致死因子结构特征，结合国际在研活性化合物，进行了致死因子抑制剂的设计、合成和活性评价工作，建立了从分子、细胞到动物模型的抗炭疽致死毒素化合物的评价方法并获得了在动物体内具有显著抗炭疽致死毒素的化合物。在此基础上，本项目进一步开展炭疽毒素致死因子抑制剂的设计、合成和结构优化研究，通过分子水平抑制炭疽毒素致死因子的活性和选择性评价、细胞模型和动物体内抗炭疽致死毒素的药效评价、化合物早期毒理学和药代动力学的评价，得到具有良好活性、选择性、安全性和代谢性质的化合物，为抗炭疽致死毒素药物的开发奠定基础。

炭疽攻击是一种现实的恐怖威胁，应用疫苗与抗生素治疗炭疽感染都有局限性。炭疽毒素是炭疽感染导致死亡的重要原因，目前还没有抗炭疽毒素中毒的药物。致死因子（LF）是炭疽毒素的主要毒力因子。因此，研究炭疽毒素致死因子抑制剂，进而开发抗炭疽致死毒素药物，是关乎国家安全稳定的迫切需要。.本项目开展如下几个方面的研究内容：.（1）根据对已知的 LF 抑制剂分别结合 LF的结构特征分析，利用基于分子片段的药物先导物发现方法，进行了新结构化合物的设计。.（2）通过五条合成路线，合成包含有五种结构类型的新化合物47个，并对其结构进行了1H-NMR、LC-MS的确证。.（3）利用荧光多肽裂解试验，测定化合物抑制 LF蛋白酶催化水解其底物的活性，确定了新化合物抑制LF的活性及其构效关系。.（4）评价了13个对LF抑制活性较高的化合物对其他9种金属蛋白酶（MMP-1、MMP-2、MMP-3、MMP-14、MMP-9、TACE、NEP、ACE和ECE）的抑制活性，得出了对LF强抑制活性和高选择性的化合物。 .（5）建立了鼠类巨噬细胞样细胞系RAW 264.7模型，评价了12个化合物抵抗炭疽致死毒素（LF+PA）的活性，发现了在细胞水平上活性较好的化合物。.（6）建立了炭疽致死毒素所致小鼠毒血症模型，评价了化合物III-1静脉给药抵抗炭疽致死毒素（PA + LF）的药效，结果表明化合物III-1可以有效保护PA/LF引起的毒血症，且具有剂量相关性。.综上所述，通过本项目的研究，发现了新结构的LF抑制剂III-1，该化合物制备较为容易；其对LF具有强抑制活性（IC50 = 0.001 μmol•L-1）和高选择性（高出其对MMP-2、3、9 14抑制活性的10000倍，高出TACE抑制活性3700倍以上，在10 mol•L-1浓度下对ACE、ECE和MMP-1没有抑制活性，对NEP的抑制率为48.33%）；化合物III-1在鼠类巨噬细胞样细胞系RAW 264.7模型抵抗炭疽致死毒素（LF+PA）的EC50值为2.264 mol•L-1；化合物III-1可以剂量相关性的保护PA/LF引起的小鼠毒血症，在剂量30 mg/kg时，小鼠48 h存活率为70 %，且存活的小鼠行为正常。化合物III-1作为新结构的LF抑制剂，具有发展为抗炭疽毒素中毒的药物前景。