Core D5: Phage display synthetic toxin pipeline

核心D5：噬菌体展示合成毒素管道

• 批准号: 7922839
• 负责人: Steve A N Goldstein
• 金额: $ 23.6万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7922839
• 关键词: Acetylcholine; Affinity; Amino Acid Transporter; Amino Acids; Bacteria; Binding; Biochemical Genetics; Biological Factors; Cell surface; Chemicals; Chloride Channels; Cloning; Computing Methodologies; Disease; Hormone Receptor; In Vitro; Ion Channel; Laboratories; Lead; Ligands; Link; Locales; Location; Maps; Membrane; Membrane Proteins; Methodology; Methods; Modification; Molecular; Molecular Conformation; Optics; Pain; Pathway interactions; Peptide Synthesis; Peptides; Phage Display; Physiology; Potassium; Property; Pump; Research; Rest; Role; Scorpions; Site; Snails; Snakes; Sodium Channel; Specificity; Spiders; Structure; Technology; Tissues; Toxin; Variant; Work; base; design; improved; in vivo; novel; operation; receptor; receptor function; scaffold; sensor; tool; voltage

The Membrane Protein Structural Dynamics (MPSD) Consortium seeks to achieve mechanistic understanding of membrane protein operation by linking structure, dynamics and function. Membrane proteins change their conformation to operate. Our purpose is to study different conformational states associated with function and to map the pathway that links operating and resting conformations. Naturally-occurring peptide toxins have become an integral part of research on many membrane proteins. This core employs a new, high-throughput methodology that exploits the structural robustness of natural peptide toxin scaffolds and the power of phage display technology. The purpose is to produce novel synthetic toxins that bind to specific membrane receptors in site and/or state-dependent manner with high affinity and selectivity. This method extends the proven strategy of using high-affinity peptide ligands to study membrane proteins beyond a handful of natural toxins that have been isolated. Peptide toxins isolated from spiders, scorpions, snails and snakes have been potent analytic tools to advance understanding of channels, pumps, transporters, and hormone receptors in vitro and in vivo revealing the roles of these membrane proteins in physiology and their mechanisms of action [1]. In the wild, toxins act to immobilize prey; they are potent (pM-nM affinity) and broadly effective on a wide spectrum of membrane targets. Many toxins lock target receptors in unique functional states. Natural toxins are small (-10-80 amino acids) and are constructed on resilient structural scaffolds that tolerate wide residue diversity to yield products with markedly different properties. Laboratory synthesis of peptide toxins using bacteria or by de novo chemical methods has proven straightfoHA/ard in most cases. These strategies improve yield compared to isolation of natural products and, significantly, allow incorporation of useful modifications such as residue alterations to improve target specificity or affinity, to alter impact on receptor function, or to attach cargo for delivery to specific cellular and molecular locations [2, 3]. Natural toxins and their synthetic variants have been used to identify membrane receptor subtypes in different tissue and subcellular locales [4]; distinguish roles in physiology and disease [5]; delineate molecular mechanisms [6]; immunopurify target receptors [7]; to treat pain via blockade of ion channels [8]; and, of unique relevance here, to define receptor structure as a function of conformational state using biophysical [9], optical [3] and computational methods [10]. Even though the predicted diversity of the natural peptide "toxome" extrapolated from biochemical and genetic studies is vast (> 11 million), specific targets are not identified for most of the hundreds of toxins that have been isolated and studied. Those toxins that bind to known receptors are often of low affinity or cross-react with related targets. This state-of-affairs is easily understood: neither their purpose in the wild nor non-directed searches for target receptors favor isolation of specific, high-affinity toxins. Here, these problems are avoided by cloning toxins based on their functional attributes.

膜蛋白结构动力学（MPSD）联盟试图通过连接结构，动力学和功能来实现对膜蛋白操作的机械理解。膜蛋白会改变其构象作用。我们的目的是研究与功能相关的不同构象状态，并绘制连接工作和静止构象的途径。 天然肽毒素已成为许多膜蛋白研究的组成部分。该核心采用了一种新的高通量方法，可利用天然肽毒素支架的结构鲁棒性和噬菌体展示技术的力量。目的是产生新型的合成毒素，这些毒素与位点和/或状态依赖性的特定膜受体具有高亲和力和选择性结合。该方法扩展了使用高亲和力的验证策略 肽配体研究膜蛋白超出了少数天然毒素，这些蛋白质已被分离出来。 从蜘蛛，蝎子，蜗牛和蛇中分离出来的肽毒素已成为有效的分析工具，可以在体外和体内促进对通道，泵，转运蛋白和激素受体受体的了解，从而揭示了这些膜蛋白在生理学及其作用机制中的作用[1]。在野外，毒素起作用固定猎物；它们是有效的（PM-NM亲和力），并且在各种膜靶标上广泛有效。许多毒素将目标受体锁定在独特的功能状态下。自然的 毒素很小（-10-80氨基酸），是在弹性结构支架上构建的，可耐受宽的残基多样性，以产生具有明显不同特性的产品。 在大多数情况下，使用细菌或从头化学方法的实验室合成肽毒素或通过从头化学方法进行了证明。与天然产物的隔离相比，这些策略提高了产量，并显着纳入了有用的修饰，例如残留变化以提高目标特异性或亲和力，以改变对受体功能的影响，或将货物附加到特定的细胞和分子位置[2，3]。 天然毒素及其合成变体已用于鉴定不同组织和亚细胞层中的膜受体亚型[4]；区分生理和疾病中的作用[5]；描绘分子机制[6]；免疫促靶受体[7];通过阻断离子通道治疗疼痛[8]；并且，在这里具有独特的相关性，可以使用生物物理[9]，光学[3]和计算方法[10]定义受体结构作为构象状态的函数。 尽管从生化和遗传研究中推断出的天然肽“毒素”的预测多样性是广泛的（> 1100万），但对于已经分离和研究的数百种毒素中的大多数尚未确定特定靶标。那些与已知受体结合的毒素通常具有低亲和力或与相关靶标的交叉反应。这种伴侣很容易被理解：它们在野外的目的既不是针对靶受体的非指导搜索，这有利于隔离特定的高亲和力毒素。在这里，通过基于其功能属性克隆毒素来避免这些问题。