Inhibition of Anthrax Lethal Factor by alpha-defensins

描述(申请人提供)：炭疽毒素由炭疽杆菌分泌的三种蛋白质组成--保护性抗原(PA)、水肿因子(EF)和致死因子(LF)。Lf是一种依赖于锌离子的金属蛋白酶，它能切割丝裂原活化蛋白激酶(MAPKK)家族的成员，阻断专业吞噬细胞中的Mark信号通路。PA和LF的二元复合体，称为致死毒素或LeTx，足以诱导目标细胞死亡和杀死实验动物，表现出与炭疽病相关的症状。作为炭疽病发病机制中的主要毒力因子之一，Lf已成为药物开发的重要治疗靶点。一些基于Lf的小分子抑制剂在细胞培养中能够中和LeTx的细胞毒性，在动物试验中能够中和LeTx的致死性，在治疗炭疽感染方面具有潜在的治疗价值。人中性粒细胞α-防御素(HNPs)是一个不断壮大的小阳离子抗菌肽家族的成员，它可以杀死细菌、真菌和病毒等多种微生物，在对抗感染性病原体的先天免疫防御中发挥着重要作用。Kaufmann和他的同事最近发现，HNP1非竞争性地抑制LF，防止MAPKK的切割，并恢复LeTx处理的巨噬细胞中受损的MAPK信号(Kim等人，2005年)。此外，HNP1从炭疽杆菌引起的细胞毒性中拯救了小鼠巨噬细胞，并在体内用HNP1-3治疗保护小鼠免受LeTx的致命后果(Kim等人，2005年)。在随后的一份报告中，Mayer-Scholl等人。(2005)证明了被人类中性粒细胞吞噬并在细胞内萌发的炭疽芽胞可以有效地被HNPs杀死。这些发现增加了a-防御素可能被开发为一类新的抗毒素以对抗炭疽感染的可能性。尽管基于α-防御素的抗毒素具有治疗炭疽的潜力，但α-防御素抑制LF的分子基础仍然知之甚少。在这个提议中，我们试图破译决定α-防御素抑制LF的序列规则和结构决定因素，并设计具有显著提高抑制活性的第二代防御素分子。

DESCRIPTION (provided by applicant): Anthrax toxin consists of three proteins secreted by Bacillus anthracis - protective antigen (PA), edema factor (EF) and lethal factor (LF). LF is a Zn2+dependent metalloprotease that cleaves members of the mitogen-activated protein kinase (MAPKK) family, blocking the MARK signaling pathway in professional phagocytes. The binary complex of PA and LF alone, termed lethal toxin or LeTx, is sufficient to induce death of target cells and kill experimental animals, manifesting the symptoms associated with anthrax. As one of the primary virulence factors in the pathogenesis of anthrax, LF has become an important therapeutic target for drug development. A number of small-molecule-based inhibitors of LF have been shown to be able to neutralize cytotoxicity of LeTx in cell cultures and lethality of LeTx in animal trials, promising potential therapeutic value in the treatment of anthrax infection. Human neutrophil a-defensins (HNPs) are members of a growing family of small cationic antimicrobial peptides that kill a broad range of microbes such as bacteria, fungi and viruses, playing an important role in the innate immune defense against infectious pathogens. Kaufmann and colleagues recently discovered that HNP1 non-competitively inhibits LF, preventing cleavage of a MAPKK and restoring impaired MAPK signaling in LeTx-treated macrophages (Kim et al., 2005). Further, HNP1 rescued murine macrophages from B. anthracis-induced cytotoxicity, and in vivo treatment with HNP1-3 protected mice against the fatal consequences of LeTx (Kim et al 2005). In a subsequent report, Mayer-Scholl et al. (2005) demonstrated that B. anthracis spores engulfed by human neutrophils and germinated intracellularly can be effectively killed by HNPs. These findings raise the possibility that a-defensins may be developed as a novel class of antitoxins to combat anthrax infection. Despite the therapeutic potential of a-defensin-based antitoxins for anthrax treatment, the molecular basis for the inhibition of LF by a-defensins remains poorly understood. In this proposal, we seek to decipher the sequence rules and structural determinants that dictate the inhibition of LF by a-defensins, and to design second-generation defensin molecules with significantly improved inhibitory activity.