Blockade of Anthrax Cytotoxicity Using Furin Inhibitors

描述(申请人提供)：炭疽杆菌产生一种三组分的外毒素，其生物活性的一个基本元素是被称为保护性抗原或PA的蛋白质。PA与细胞表面受体结合，并被切割产生63 kDa的蛋白质，另一种炭疽毒素LF和EF可以与之结合。PA的蛋白分解和结合允许PA-LF复合体内化到胞浆中，在那里它能够攻击细胞机械，导致细胞死亡。由于PA炭疽毒素的蛋白水解性裂解是表现毒性活性所必需的，这一裂解步骤代表了药物干预的自然靶点。以前的研究表明，这种切割是由一种被称为Furin的细胞表面酶执行的，Furin是真核枯草杆菌蛋白家族的成员。这一应用旨在通过抑制激活的裂解事件来减弱炭疽毒素的细胞毒性作用，从而减少毒性和细胞保护。 在过去的十年里，几个研究小组已经证明，使用工程蛋白呋喃的抑制剂来阻止细菌毒素的分解是可能的。我们最近利用组合化学技术鉴定了一种稳定的六肽，D6R(D-六精氨酸)，它代表了一种有效的、稳定的呋喃类小分子抑制剂。我们的初步数据表明，D6R可以有效地抑制Furin介导的对来自假单胞菌的细菌毒素的切割，从而阻断对这两种细胞系和活动物的致死作用。我们建议系统地将我们的呋喃抑制研究应用于阻断炭疽毒素的激活。具体地说，我们将研究D6R本身在减轻炭疽毒素细胞毒性方面的潜在治疗作用；研究D6R相关分子抑制Furin介导的细胞毒性的结构要求；以及在炭疽毒性的动物模型中测试D6R和/或其他稳定的Furin抑制剂。我们的初步数据显示D6R对PA细胞毒性的有效抑制支持这样的想法，即小分子呋喃抑制剂将是生物减弱炭疽毒素细胞毒性的有效试剂，此类抗毒素药物的开发将显著增强我们保护生物免受这种病原体侵袭的能力。

DESCRIPTION (provided by applicant): The anthrax bacillus produces a three-component exotoxin of which an essential element for bioactivity is the protein known as protective antigen, or PA. PA binds to a cell surface receptor and is cleaved to generate a 63 kDa protein to which the one of the other anthrax toxins, LF and EF, can bind. Proteolysis and binding of PA permits internalization of a PA-LF complex into the cytosol, where it is able to attack cellular machinery, resulting in cell death. Since proteolytic cleavage of the PA anthrax toxin is obligatory for the manifestation of toxic activity, this cleavage step represents a natural target for pharmacologic intervention. Previous research has shown that this cleavage is performed by a cellular surface enzyme known as furin, a member of the family of eukaryotic subtilisins. This application is directed toward the idea that the cytotoxic action of anthrax toxin can be attenuated through inhibition of the activating cleavage event, resulting in lessened toxicity and cellular protection. In the last decade, several groups have shown that it is possible to block cleavage of bacterial toxins using engineered protein inhibitors of furin. We have recently used combinatorial chemistry techniques to identify a stable hexapeptide, D6R {D-hexa-arginine) which represents a potent, stable small molecule inhibitor of furin. Our preliminary data indicate that D6R can effectively inhibit furin-mediated cleavage of a bacterial toxin derived from Pseudomonas, blocking lethal effects in both cell lines as well as live animals. We here propose to systematically apply our studies of furin inhibition to the blockade of anthrax toxin activation. Specifically, we will investigate the use of D6R itself as a potential therapeutic in the attenuation of anthrax toxin cytotoxicity; examine the structural requirements for inhibition of furin-mediated cytotoxicity by D6R related molecules; and test D6R and/or other stable furin inhibitors identified in this work in animal models of anthrax toxicity. Our preliminary data showing potent inhibition of PA cytotoxicity by D6R support the idea that small molecule furin inhibitors will represent effective agents for the biologic attenuation of anthrax toxin cytotoxicity, the development of such antitoxin agents will add significantly to our ability to protect organisms against this pathogen.