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

• 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
• 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

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，用于治疗骨骼肌疾病， 细胞内钙调节异常，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（用荧光供体标记的特定SR蛋白）的设计开始 和受体），用于小分子的高通量筛选（HTS）。一项关键的创新是我们最近 基于荧光寿命（FLT）检测蛋白质结构变化的HTS方法， 荧光共振能量转移（FRET）。我们将FRET生物传感器工程与 独特的FLT检测产生了前所未有的灵敏度，特异性，速度和 基于蛋白质结构的药物发现机制研究的精确性。我们之前已经验证过了 通过应用于心肌。我们现在专注于骨骼肌，针对两个关键的SR 参与钙调节的蛋白质，钙释放通道（RyR 1）和钙泵（SERCA 1a）。 目标1：靶向RyR 1泄漏减少。我们的生物传感器是基于两个调节蛋白之间的FRET （FKBP12.0和CaM）与RyR 1结合。在试验性筛选中，我们已经证明，这种基于FLT的FRET检测可以 检测恢复异常RyR 1功能的小分子，其中Ca通道将Ca从SR泄漏到 细胞质，诱发肌病。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期望的钙渗漏的逆转。 目标2：靶向SERCA 1a激活。我们寻求一种互补的解决方案来对抗钙泄漏增强 SERCA 1a活性将Ca泵回SR管腔。该方法还针对因素（例如，突变或 氧化），损害SERCA 1a活性。我们将在之前的基础上使用两种互补的方法 使用SERCA 2a（心脏）的研究，使用在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SERCA 1a的不同结构域），以筛选 小分子文库，用于检测与SERCA结合、改变酶结构和激活Ca 运输(B)我们将使用分子间生物传感器，供体在SERCA 1a上，受体在SERCA 1a上 调节剂肌磷脂（SLN），以检测通过解偶联抑制作用激活酶的化合物， SLN。我们将使用肌纤维和肌肉的测定来评估候选药物的效力和功效， 体外和体内小鼠模型，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， 由大卫托马斯（SERCA 1a，FLT-FRET），Razvan Cornea（RyR 1，生物传感器工程）和Dawn Lowe领导 （骨骼肌功能分析）。我们还将与具有互补专业知识的合作者一起， 药物化学（Aldrich）和肌纤维Ca测定（Launikonis），以及两位具有独特专业知识的顾问 在肌肉Ca调节紊乱的动物模型上（Dirksen和汉密尔顿）。