Interfering with Protein-Protein Interaction for the Treatment of Leishmaniasis

描述(申请人提供)：利什曼病是一种地方性疾病，分布在80多个国家的热带和亚热带地区，每年有200多万新病例发生http://www.who.int/tdr/diseases/leish/diseaseinfo.htm.活化C激酶受体1(RACK1)是一种普遍存在的高度保守的支架蛋白，它与多种参与细胞存活、生长和分化的信号转导过程中的蛋白结合。我们已经成功地产生了短肽调节剂，可以破坏或增强RACK1与这些信号蛋白的相互作用，从而调节它们的生物学效应。关于RACK1的利什曼原虫同源物、活化C激酶受体的利什曼原虫同源物LASK的细胞功能知之甚少。我们认为，由于其高度保守的性质，LACK也是参与利什曼原虫基本信号传递过程的关键支架蛋白。我们提出了一种新的方法，利用我们成熟的多肽设计方法来识别和表征LACK结合伙伴，并针对LACK和这些利什曼病信号转导酶之间的蛋白质-蛋白质相互作用。这些多肽可能成为开发治疗利什曼病新疗法的先导。具体地说，在这个早期的可行性提案中，我们计划：1.鉴定和鉴定作为缺失结合伙伴的利什曼原虫信号蛋白(LACK-BP)。2.合理设计选择性抑制支架LINK和LACK-BP相互作用的多肽，并检测其对利什曼原虫的活性。增殖和体外感染。为了鉴定和鉴定LACK-BP，我们将利用几种方法，包括蛋白质覆盖(Far Western)、体外下拉试验和表达相互作用克隆。为了设计蛋白质-蛋白质相互作用的多肽调节剂，我们将使用Rack-BP中与Rack结合所需的序列，并在LACK-BP中鉴定同源序列。此外，我们将在Rack-BP的同源物中鉴定进化保守的序列，并使用结构建模来寻找多肽设计的同源性区域。最后，我们将识别来自LACK的短肽，这些短肽可能会破坏LACK-BPS的结合，从而破坏LACK-BPS的信号转导。这些肽调节剂将被拴在细胞渗透载体上，并在培养的利什曼原虫前鞭毛体和感染利什曼原虫的小鼠巨噬细胞上测试它们对增殖的生物活性。作为拟议项目的一部分开发的多肽可能会产生显著的生物效应。重要的是，识别干扰其生物活性的缺乏结合的蛋白质和多肽有助于指导利什曼原虫感染患者的新疗法的开发。 项目简介：每年报告200万新的利什曼原虫感染病例，其结果死亡率为3%。目前治疗利什曼病的方法是化疗，毒性很大，效果不佳。我们提议的研究将使用一种新的方法来确定参与利什曼原虫生存能力的关键因素，使我们能够确定并创造高度特异性的疗法来扰乱这些细胞过程。这种治疗方法可能提供一种毒性较小、高度特异和有效的治疗利什曼病的方法。

DESCRIPTION (provided by applicant): Leishmaniasis is an endemic disease in tropical and subtropical regions of over 80 countries with more than 2 million new cases occurring annually http://www.who.int/tdr/diseases/leish/diseaseinfo.htm. Receptor for activated C kinase 1 (RACK1), a ubiquitous and highly conserved scaffold protein, binds to proteins involved in many signaling processes leading to cell survival, growth and differentiation. We have successfully generated short peptide regulators that either disrupt or enhance the interactions of RACK1 with these signaling proteins and thus regulate their biological effects. Little is known about the cellular function of the Leishmania homolog of RACK1, Leishmania homolog of receptors for activated C kinase, LACK. We believe that LACK, due to its highly conserved nature, is also a key scaffolding protein involved in essential signaling processes of the Leishmania parasite. We propose a novel approach, utilizing our well established methods of peptide design, to identify and characterize LACK- binding partners and to target protein-protein interactions between LACK and these Leishmaniasis- signaling enzymes. These peptides may serve as leads for the development of new therapeutics for the treatment of Leishmaniasis. Specifically, in this early feasibility proposal, we plan to: 1. Identify and characterize Leishmania signaling proteins that are LACK-binding partners (LACK-BP). 2. Rationally design peptides that selectively inhibit the interactions between the scaffold LACK and LACK-BP and test their activity towards Leishmania sp. proliferation and in vitro infection. To identify and characterize LACK-BP, we will utilize several methodologies including protein overlays (Far Western), in vitro pull-down assays, and expression interaction cloning. To design peptide modulators of protein-protein interactions, we will use sequences in RACK-BP that are required for RACK binding and identify homologous sequences in LACK-BP. Additionally, we will identify evolutionary conserved sequences in homologs of RACK-BP and use structural modeling to look for regions of homology for peptide design. Finally, we will identify short peptides derived from LACK that are likely to disrupt the binding and hence the signaling of LACK-BPs. The peptide modulators will be tethering to cell-permeable vectors and tested for their biological activity on proliferation of Leishmania promastigotes in culture and on Leishmania-infected murine macrophages. The peptides developed as part of the proposed project are likely to exert significant biological effects. Importantly, identification of LACK-binding proteins and peptides that interfere with their biological activities can help guide the development of new therapeutics for patients suffering from Leishmania infection. PROJECT NARRATIVE: Two million new cases of Leishmania infection are reported annually, with a resultant death rate of 3%. The current treatment for Leishmaniasis, chemotherapy, is highly toxic and ineffective. Our proposed studies will use a new approach to identify key players involved in Leishmania viability, allowing us to identify and create highly specific therapeutics that disrupt these cellular processes. This therapeutic approach is likely to provide a less toxic, highly specific and effective way of treating Leishmaniasis.