Skeletal muscle protein structural dynamics and function drive applications to drug discovery

骨骼肌蛋白结构动力学和功能驱动药物发现的应用

基本信息

批准号：
10650572
负责人：
DAWN A LOWE
金额：
$ 65.22万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-01 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10650572
关键词：
ATP phosphohydrolase Animal Model Animal Testing Animals Back Binding Biochemical Biological Assay Biophysics Biosensor Ca(2+)-Transporting ATPase Calcium Calcium Channel Calmodulin Cardiac Cells Clinical Collaborations Cornea Cytoplasm Detection Disease Disease model Drug Industry Engineering Ensure Enzyme Uncoupling Enzymes FKBP1B gene Fiber Fluorescence Fluorescence Resonance Energy Transfer Future Generations Goals Healthcare Homeostasis Impairment In Vitro Label Libraries Link Membrane Modification Mus Muscle Muscle Proteins Muscle function Mutation Myocardium Myopathy Nature Pathology Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacotherapy Physiological Physiology Post-Translational Protein Processing Proteins Regulation Relaxation Research RyR1 SERCA2a Sarcoplasmic Reticulum Site Skeletal Muscle Specificity Speed Structure System Tacrolimus Binding Protein 1A Technology Testing Therapeutic Agents combat design drug candidate drug discovery drug efficacy drug testing enzyme structure genetic regulatory protein high throughput screening improved in vitro Assay in vivo innovation mouse model novel strategies novel therapeutics oxidation pre-clinical protein structure sarcolipin screening skeletal small molecule small molecule libraries small molecule therapeutics structural biology therapeutic development therapeutic target therapeutically effective translational impact uptake

项目摘要

Our goal is to develop small-molecule drugs for treatment of skeletal muscle disorders related to dysregulation of intracellular calcium, focusing on specific proteins in the sarcoplasmic reticulum (SR). Each Aim starts with the design of fluorescent biosensors (specific SR proteins labeled with fluorescent donor and acceptor), to be used in high-throughput screening (HTS) of small molecules. A key innovation is our recently developed HTS approach based on fluorescence lifetime (FLT) detection of protein structural changes by fluorescence resonance energy transfer (FRET). Our combination of FRET biosensor engineering with unique FLT detection has produced an unprecedented combination of sensitivity, specificity, speed, and precision in protein structure-based studies of mechanism for drug discovery. We previously validated this approach through applications to cardiac muscle. We now focus on skeletal muscle, targeting the two key SR proteins involved in Ca regulation, the Ca release channel (RyR1) and the calcium pump (SERCA1a). Aim 1: Targeting RyR1 leak reduction. Our biosensor is based on FRET between two regulatory proteins (FKBP12.0 and CaM) bound to RyR1. In pilot screens, we have shown that this FLT-based FRET assay can detect small molecules that restore aberrant RyR1 function, in which the Ca channel leaks Ca from the SR into the cytoplasm, inducing myopathies. We will carry out larger-scale screening, to identify new drug candidates, then use cellular and in vivo muscle assays to test the reversal of undesirable calcium leak in fibers and mice. Aim2: Targeting SERCA1a activation. We seek a complementary solution to combat Ca leak – enhancing SERCA1a activity to pump Ca back into the SR lumen. This approach also targets factors (e.g., mutation or oxidation) that impair SERCA1a activity. We will use two complementary approaches, building on our previous studies with SERCA2a (cardiac), with fluorescent biosensors expressed in live cells. (A) We will use an intramolecular FRET biosensor (donor and acceptor attached to different domains of SERCA1a), to screen a small-molecule library to detect compounds that bind to SERCA, alter enzyme structure, and activate Ca transport. (B) We will use an intermolecular biosensor, with donor on SERCA1a and acceptor on the SERCA1a regulator sarcolipin (SLN), to detect compounds that activate the enzyme by uncoupling the inhibitory effects of SLN. We will evaluate potency and efficacy of drug candidates, using assays on myofibers and muscles, both in vitro and in vivo in mouse models including pre-clinical longitudinal drug testing. We have assembled a multi-PI team with complementary expertise and decades of successful collaboration, led by David Thomas (SERCA1a, FLT-FRET), Razvan Cornea (RyR1, biosensor engineering), and Dawn Lowe (skeletal muscle functional analysis). We will also be joined by collaborators with complementary expertise in medicinal chemistry (Aldrich) and myofiber Ca assays (Launikonis), and two consultants with unique expertise on animal models of disorders in muscle Ca regulation (Dirksen and Hamilton).

我们的目标是开发小分子药物来治疗与以下疾病相关的骨骼肌疾病细胞内钙的失调，重点关注肌浆网 (SR) 中的特定蛋白质。每个目标都从荧光生物传感器的设计开始（用荧光供体标记的特定 SR 蛋白）和受体），用于小分子的高通量筛选（HTS）是我们最近的一项关键创新。开发了基于荧光寿命 (FLT) 检测蛋白质结构变化的 HTS 方法我们将 FRET 生物传感器工程与荧光共振能量转移 (FRET) 相结合。独特的 FLT 检测实现了前所未有的灵敏度、特异性、速度和我们之前验证了基于蛋白质结构的药物发现机制研究的准确性。我们现在专注于骨骼肌，针对两个关键的 SR。参与 Ca 调节、Ca 释放通道 (RyR1) 和钙泵 (SERCA1a) 的蛋白质。目标 1：以减少 RyR1 泄漏为目标，我们的生物传感器基于两种调节蛋白之间的 FRET。（FKBP12.0 和 CaM）与 RyR1 结合在试点筛选中，我们已经证明这种基于 FLT 的 FRET 测定可以。检测恢复异常 RyR1 功能的小分子，其中 Ca 通道将 Ca 从 SR 泄漏到我们将进行更大规模的筛选，以确定新的候选药物，然后使用细胞和体内肌肉测定来测试纤维和小鼠中不良钙泄漏的逆转。目标 2：针对 SERCA1a 激活，我们寻求一种补充解决方案来对抗 Ca 泄漏 - 增强。 SERCA1a 活性将 Ca 泵回 SR 腔。这种方法还针对因素（例如突变或）。氧化）会损害 SERCA1a 活性，我们将在之前的基础上使用两种互补的方法。使用 SERCA2a（心脏）的研究，使用活细胞中表达的荧光生物传感器 (A) 我们将使用分子内 FRET 生物传感器（供体和受体连接到 SERCA1a 的不同域），以筛选用于检测与 SERCA 结合、改变酶结构并激活 Ca 的化合物的小分子库 (B) 我们将使用分子间生物传感器，供体位于 SERCA1a 上，受体位于 SERCA1a 上。调节肌磷脂 (SLN)，通过解偶联抑制作用来检测激活酶的化合物 SLN。我们将评估候选药物在肌纤维和测定肌肉上的效力和功效。小鼠模型的体外和体内，包括临床前纵向药物测试。我们组建了一支多 PI 团队，拥有互补的专业知识和数十年的成功合作，由 David Thomas（SERCA1a、FLT-FRET）、Razvan Cornea（RyR1、生物传感器工程）和 Dawn Lowe 领导（骨骼肌功能分析）。我们还将邀请具有互补专业知识的合作者加入。药物化学 (Aldrich) 和肌纤维钙测定 (Launikonis)，以及两名具有独特专业知识的顾问肌肉钙调节障碍的动物模型（德克森和汉密尔顿）。