Inhibition of Anthrax Lethal Factor by alpha-defensins

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水解酶，它能切割 丝裂原活化蛋白激酶(MAPKK)家族，阻断MARK信号通路 专业吞噬细胞。PA和LF的二元复合体，称为致死毒素或LeTx，足以 导致靶细胞死亡并杀死实验动物，表现出与 炭疽热。作为炭疽致病的主要毒力因子之一，Lf已成为重要的致病因子。 药物开发的治疗目标。一些基于小分子的LF抑制剂已经被发现 证明能够中和细胞培养中LeTx的细胞毒性和动物试验中LeTx的致命性， 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)是小分子阳离子抗菌剂家族中的一员。 杀死多种微生物的多肽，如细菌、真菌和病毒，在 对传染性病原体的先天免疫防御。考夫曼和他的同事最近发现 HNP1非竞争性抑制LF，阻止MAPKK的切割并恢复受损的MAPK LeTx处理的巨噬细胞中的信号传递(Kim等人，2005年)。此外，HNP1将小鼠巨噬细胞从 B.A/tfftrac/S诱导的细胞毒性，并在WVO治疗中用HNP1-3保护小鼠免受致死性 LeTx的后果(Kim等人，2005年)。在随后的一份报告中，Mayer-Scholl等人。(2005)演示 炭疽杆菌孢子被人中性粒细胞吞噬并在细胞内萌发能有效地 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 用来对抗炭疽感染的抗毒素。 尽管基于a-防御素的抗毒素治疗炭疽病具有治疗潜力，但其分子基础 对于α-防御素对LF的抑制作用仍知之甚少。在这项提案中，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 第二代防御素分子具有显著提高的抑制活性。

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 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 nduce 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 cytotoxicityof 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. a/tfftrac/s-induced cytotoxicity, and in wVo 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.