Computationally guided design of helical peptide interaction reagents

描述（由申请人提供）：蛋白质-蛋白质相互作用调节所有细胞过程，是治疗抑制的有吸引力的靶点。这项工作的长期目标是加速发现可在研究、诊断和治疗中用作蛋白质-蛋白质相互作用抑制剂的修饰螺旋肽。短期目标是开发新的综合计算和实验方法，以提供有效和选择性的Bcl-2蛋白抑制剂。抗凋亡Bcl-2蛋白在许多癌症中都很重要，它们的过表达抵消了细胞死亡信号。Bcl-2蛋白提供对化疗的抗性，使其成为优先考虑的肿瘤靶点。许多Bcl-2蛋白的相互作用涉及一个保守的结合槽，它与伴侣蛋白中约20个残基的短α螺旋（称为BH3螺旋）结合。模拟BH3螺旋的合成肽可抑制抗凋亡功能并导致细胞死亡。然而，Bcl-2家族有多个成员，并不是所有的BH3肽都能同样有效地抑制所有Bcl-2蛋白。一个重要的目标是发现每个家族成员的高亲和力和选择性抑制剂。另一个挑战是，工程肽对蛋白酶非常敏感，很难穿过细胞膜，这限制了它们作为试剂的效用。最近的研究表明，稳定螺旋的化学修饰可以改善它们的性能。这项提议的具体目标是围绕紧密耦合的计算和实验技术组织起来的，这将加深我们对什么是好的螺旋肽抑制剂的理解，并帮助我们更有效地发现有用的分子。第一步将是使用基于计算结构的方法来设计预测与Bcl-2家族成员Bfl-1和BHRF1紧密和选择性结合的肽。这些信息将用于设计约107个多肽的组合文库，重点关注高优先级候选。在酵母表面显示程序中将筛选具有所需特性的分子，这将提供关于计算库设计方法质量的反馈。酵母展示的最佳多肽将通过生物物理测量和x射线晶体学进一步表征。计算模型的建立和分析将有助于确定结合亲和力和特异性的决定因素。最后，从这些过程中得到的最佳肽将使用化学技术进一步优化，引入稳定的交联成螺旋。目前关于交联改性的好坏的见解是有限的。在这项工作中，将对修饰和未修饰肽进行详细的分子动力学模拟，以建立我们对肽结构改变如何影响结合的理解。总的来说，这项工作将提供针对重要癌症调节蛋白的新分子，新的计算方法将加速选择性肽结合物的发现，并更好地理解螺旋肽相互作用的生物物理决定因素。

DESCRIPTION (provided by applicant): Protein-protein interactions regulate all cellular processes and are attractive targets for therapeutic inhibition. The long-term goal of the proposed work is to accelerate the discovery of modified helical peptides that can be used as protein-protein interaction inhibitors in research, diagnosis and therapy. The short-term goals are to develop new, integrated computational and experimental methods that will deliver potent and selective inhibitors of Bcl-2 proteins. Anti-apoptotic Bcl-2 proteins are important in many cancers, where their over- expression counteracts cell-death signaling. Bcl-2 proteins provide resistance to chemotherapy, making them high-priority oncology targets. Many Bcl-2 protein interactions involve a well-conserved binding groove that engages short alpha helices of ~20 residues, called BH3 helices, in partner proteins. Synthetic peptides that mimic BH3 helices can inhibit anti-apoptotic function and lead to cell death. However, there are multiple members of the Bcl-2 family, and not all BH3 peptides are equally effective inhibitors of all Bcl-2 proteins. An important goal is to discover high-affinity and selective inhibitors for each family member. Another challenge is that engineered peptides are highly susceptible to proteases and have trouble crossing cell membranes, limiting their utility as reagents. Recent work has shown that chemical modifications that stabilize helices can improve their properties. The specific aims of this proposal are organized around tightly coupled computational and experimental techniques that will deepen our understanding of what makes a good helical-peptide inhibitor and help us discover useful molecules more efficiently. The first step will be to use computational structure- based methods to design peptides predicted to bind tightly and selectively to Bcl-2 family members Bfl-1 and BHRF1. This information will be used to design combinatorial libraries of ~107 peptides focused on high-priority candidates. Libraries will be screened for molecules with desired properties in a yeast-surface display procedure that will provide feedback about the quality of the computational library design methods. The best peptides from yeast display will be further characterized using biophysical measurements in solution and x-ray crystallography. Computational model building and analysis will help establish determinants of binding affinity and specificity. Finally, the best peptides resulting from these procedures will be further optimized using chemical techniques that introduce stabilizing crosslinks into helices. Current insights into what makes good vs. poor crosslinking modifications are limited. In this work, detailed molecular dynamics simulations of modified and unmodified peptides will be carried out to build our understanding of how altered peptide structure affects binding. Overall, this work wil deliver new molecules that target important cancer-regulating proteins, new computational methods that will speed the discovery of selective peptide binders, and a better understanding of the biophysical determinants of helical-peptide interactions.