Blockade of Anthrax Cytotoxicity Using Furin Inhibitors

描述（由申请人提供）：炭疽杆菌产生一种三组分外毒素，其生物活性的基本要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，其他炭疽毒素LF和EF之一可以与之结合。PA的蛋白水解和结合允许PA-LF复合物内化到胞质溶胶中，在胞质溶胶中它能够攻击细胞机器，导致细胞死亡。由于PA炭疽毒素的蛋白水解裂解对于毒性活性的表现是必须的，因此该裂解步骤代表了药理学干预的天然靶标。先前的研究表明，这种切割是由一种称为弗林蛋白酶的细胞表面酶进行的，弗林蛋白酶是真核枯草杆菌蛋白酶家族的一员。本申请涉及这样的想法，即炭疽毒素的细胞毒性作用可以通过抑制活化裂解事件而减弱，从而导致毒性和细胞保护的降低。 在过去的十年中，几个小组已经表明，有可能使用弗林蛋白酶的工程蛋白抑制剂来阻断细菌毒素的裂解。我们最近已经使用组合化学技术来鉴定稳定的六肽D 6 R（D-六-精氨酸），其代表弗林蛋白酶的有效、稳定的小分子抑制剂。我们的初步数据表明，D 6 R可以有效地抑制弗林蛋白酶介导的裂解的细菌毒素来源于假单胞菌，阻断在两个细胞系以及活的动物的致死作用。在这里，我们建议系统地应用我们的研究弗林蛋白酶抑制炭疽毒素激活的封锁。具体而言，我们将研究使用D 6 R本身作为一种潜在的治疗在炭疽毒素细胞毒性的衰减;检查的结构要求，抑制弗林蛋白酶介导的细胞毒性D 6 R相关分子;和测试D 6 R和/或其他稳定的弗林蛋白酶抑制剂确定在这项工作中的炭疽毒性的动物模型。我们的初步数据显示D 6 R对PA细胞毒性的有效抑制支持小分子弗林蛋白酶抑制剂将代表炭疽毒素细胞毒性生物衰减的有效试剂的想法，这种抗毒素试剂的开发将显著增加我们保护生物体对抗这种病原体的能力。

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.