Inhibition of Anthrax Lethal Factor by alpha-defensins

描述（申请人提供）：炭疽毒素由炭疽芽孢杆菌分泌的三种蛋白组成——保护性抗原（PA）、水肿因子（EF）和致死因子（LF）。LF是一种依赖Zn2+的金属蛋白酶，可切割丝裂原活化蛋白激酶（MAPKK）家族成员，阻断专业吞噬细胞中的MARK信号通路。单独的PA和LF二元复合物，称为致死毒素或LeTx，足以诱导靶细胞死亡并杀死实验动物，表现出与炭疽有关的症状。LF作为炭疽发病的主要毒力因子之一，已成为药物开发的重要治疗靶点。许多基于小分子的LF抑制剂已被证明能够在细胞培养中中和LeTx的细胞毒性和在动物试验中中和LeTx的致死性，在治疗炭疽感染方面具有潜在的治疗价值。人类中性粒细胞a-防御素（HNPs）是一个不断发展的小阳离子抗菌肽家族的成员，它能杀死广泛的微生物，如细菌、真菌和病毒，在对抗感染性病原体的先天免疫防御中起着重要作用。Kaufmann及其同事最近发现HNP1非竞争性地抑制LF，在letx处理的巨噬细胞中阻止MAPKK的切割并恢复受损的MAPK信号传导（Kim et al., 2005）。此外，HNP1可以使小鼠巨噬细胞免受炭疽杆菌诱导的细胞毒性，并且在体内用HNP1-3治疗可以保护小鼠免受LeTx的致命后果（Kim et al . 2005）。在随后的报道中，Mayer-Scholl等人（2005）证明了被人中性粒细胞吞噬并在细胞内萌发的炭疽芽孢杆菌孢子可以被HNPs有效杀死。这些发现提出了一种可能性，即a-防御素可能被开发成一种新型的抗毒素来对抗炭疽感染。尽管基于a-防御素的抗毒素在炭疽治疗中具有治疗潜力，但a-防御素抑制LF的分子基础仍然知之甚少。在本研究中，我们试图破译a-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.