Connecting the functional effects of drugs to how they change PPAR gamma

将药物的功能效应与其改变 PPAR gamma 的方式联系起来

• 批准号: 8767700
• 负责人: Travis Shane Hughes
• 金额: $ 9万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8767700
• 关键词: 3T3-L1 Cells; Acute; Adipocytes; Adverse effects; Affect; Affinity; Agonist; Animals; Antidiabetic Drugs; Area; Arts; Atherosclerosis; Binding; Biology; Biophysics; Calorimetry; Cell Line; Cells; Cluster Analysis; Collaborations; Complex; DNA; Data; Development; Diabetes Mellitus; Diabetes prevention; Dimerization; Drug Design; Drug Prescriptions; Drug Targeting; Drug effect disorder; Entropy; Environment; FDA approved; Family; Florida; Fluorine; Fracture; Gene Expression; Genes; Genetic Transcription; Glyburide; Goals; Grant; Heart failure; Human; Inflammation; Knowledge; Length; Ligand Binding Domain; Ligands; Light; Link; Location; Maintenance; Manuscripts; Maps; Measurement; Measures; Mentors; Metformin; Methods; Modeling; Molecular Conformation; Motion; Movement; Names; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Magnetic Resonance; Nuclear Receptors; Osteogenesis; Output; PPAR gamma; PPARBP gene; Patients; Peptides; Peroxisome Proliferator-Activated Receptors; Pharmaceutical Preparations; Phase; Physics; Play; Prediabetes syndrome; Preparation; Production; Proteins; RXR; Research; Research Institute; Research Personnel; Role; Sampling; Site; Solutions; Structure; Surface; Techniques; Testing; Thermodynamics; Time; Titrations; Training; Weight Gain; Woman; Work; bone cell; bone loss; cost; diabetic; flexibility; functional outcomes; immune function; immunoregulation; improved; insulin sensitizing drugs; lipid biosynthesis; molecular dynamics; prevent; programs; public health relevance; receptor function; research study; shape analysis; simulation; transcription factor

DESCRIPTION (provided by applicant): Connecting the functional effects of drugs to how they change PPAR? The most effective drugs for treating and preventing Type II diabetes are those that bind to a protein named PPAR?. PPAR? is a transcription factor critical for the production and maintenance of adipocytes and bone cells and it affects immune function. Some PPAR? dependent effects can be beneficial to people with diabetes and some are not. The challenge is to develop PPAR? binding drugs that retain the unique and robust anti-diabetic effects but reduce the side effects of heart failure, weight gain and bone loss. Current research indicates that it may be possible to achieve separation of unwanted and wanted effects by targeting PPAR? with the right drug. This concept is supported by the fact that animal studies show some new PPAR? drugs activate distinct gene sets from currently prescribed drugs. However, how different drugs uniquely change PPAR? in order to produce these drug specific effects are unknown. Development of improved anti-diabetic PPAR? drugs is more likely when it is known how drugs change PPAR? and how these changes produce functional changes such as changes in gene expression. Understanding how ligands produce function in PPAR? and closely related proteins is the long term goal of the principle investigator (PI). This knowledge will aid in development of better drugs for the whole family of PPAR? like proteins (nuclear receptors) which are the target of ~13% of FDA approved drugs. Recently we have discovered that a large region of PPAR? exists in at least two conformations in solution alone or when bound to less efficacious drugs, however one PPAR conformation is detected when bound to a ligand that induces high transcription (Hughes et al. 2012). Importantly, these data are time consuming and expensive to obtain and only indicate that internal movement exists with little other detail. To better understand the link between PPAR?s internal motion and its function in cells we have developed low- cost, rapid NMR methods that can be used on large complexes and a NMR line shape analysis program which reveal in detail the range of conformations present (i.e. the conformational ensemble) at one site within the flexible region of PPAR?. These methods reveal conformational complexity that would be very difficult to observe using other NMR methods (manuscript in preparation) and allow characterization of a sufficient number of PPAR? drugs to draw statistically meaningful conclusions about any correlation between drug induced changes to PPAR? and changes in gene expression in cells. During the independent phase (aim 2) of the project the amount of NMR probe locations will be expanded (from the single current location) to get a more complete picture of PPAR?'s conformational ensemble in different areas. Additionally we will study PPAR? in the two main forms that it is found in the body 1) full length PPAR? (FL-PPAR?) and 2) the full-length heterodimer complex, which consists of PPAR?, RXR? and DNA. This work will reveal in unprecedented detail how ligands affect the range of related structures that comprise the conformational ensemble of PPAR?. However, they will not detect ligand-induced changes in PPAR?'s small fast movements (i.e. conformational entropy) that may be critical to how ligands produce effects in humans. To study these movements the PI will be trained in using molecular dynamics simulations. These simulations will be checked against experiment where possible using NMR. These data will be used to estimate the average regional change in the small fast internal movement of PPAR? (conformational entropy) that occurs when a drug binds PPAR. All of these measurements of drug-induced changes in PPAR? will be tested for correlation with functional outcomes such as dimerization with FL-RXR¿, recruitment of coregulator peptides and gene expression in adipocytes. In order to fully utilize these recent advances and to build the best possible model of how PPAR dynamics and conformation leads to function the PI needs protected time for training in molecular dynamics simulation. The advisory team, which includes Dr. Cheatham, will provide expert guidance in this area. During the mentored portion of the grant (aim 1) the PI will continue to receive guidance in NMR and protein molecular dynamics simulations from collaborators (Drs. Art Palmer and Mark Rance), in addition to the PI's primary mentor Dr. Kojetin. The PI will also receive training in the methods and analysis of PPAR? drug effects on target gene expression in cells from Dr. Griffin (co-mentor). The Scripps Research Institute in Florida (TSRI) has seven groups (Nettles, Griffin, Kojetin, Kameneka, Solt, Rousch and Smith) that study nuclear receptors. Four of these groups are currently using different approaches and methods for answering important questions about PPAR?. This makes collaboration natural and provides an excellent environment in which to receive the training necessary to sustain independent research in this area. The PI has degrees in physics and biology which has allowed him to quickly acquire expertise in many areas of nuclear magnetic resonance (NMR) of proteins and several other biophysical and biology techniques during his 3 years of training at TSRI and provides a breadth of training that will be essential for connecting the biophysics and thermodynamics of PPAR movement and structure to functional outcomes.

描述（由申请人提供）：将药物的功能效应与它们如何改变PPAR联系起来？治疗和预防II型糖尿病最有效的药物是那些与名为PPAR？的蛋白质结合的药物。PPAR？是一种对脂肪细胞和骨细胞的产生和维持至关重要的转录因子，并影响免疫功能。一些过氧化物酶体激活剂？依赖性效应对糖尿病患者有益，有些则不然。挑战在于开发PPAR？结合药物，保留独特和强大的抗糖尿病作用，但减少心力衰竭，体重增加和骨质流失的副作用。目前的研究表明，它可能是可能的，以实现分离不想要的和想要的效果，通过靶向过氧化物酶体增殖物激活受体？正确的药物。这一概念是支持的事实，动物研究表明，一些新的过氧化物酶体增殖物激活受体？药物激活的基因组与目前处方的药物不同。然而，不同的药物如何独特地改变PPAR？为了产生这些药物的具体效果是未知的。开发改进的抗糖尿病的过氧化物酶体激活受体？药物是更有可能的，当它是已知的药物如何改变过氧化物酶体增殖物受体？以及这些变化如何产生功能变化，如基因表达的变化。了解配体如何在PPAR中发挥作用？和密切相关的蛋白质是主要研究者（PI）的长期目标。这些知识将有助于开发更好的药物，为整个家庭的PPAR？像蛋白质（核受体），这是约13%的FDA批准的药物的目标。最近，我们发现，一个大区域的过氧化物酶体增殖物激活受体？在单独的溶液中或与不太有效的药物结合时，存在至少两种构象，然而，当与诱导高转录的配体结合时，检测到一种PPAR构象（Hughes et al. 2012）。重要的是，这些数据是耗时和昂贵的获得，只表明内部运动的存在，几乎没有其他细节。为了更好地了解PPAR之间的联系？的内部运动及其在细胞中的功能，我们已经开发出低成本，快速NMR方法，可用于大型复合物和NMR线形分析程序，详细揭示了构象的范围内存在的一个网站（即构象系综）的灵活区域的PPAR？。这些方法揭示了构象的复杂性，这将是非常难以观察使用其他NMR方法（手稿准备），并允许表征足够数量的PPAR？药物诱导的变化与PPAR之间的任何相关性得出有统计学意义的结论？以及细胞中基因表达的变化。在项目的独立阶段（目标2），NMR探头位置的数量将扩大（从单一的当前位置），以获得更完整的图像？不同区域的构象集合。此外，我们还将研究PPAR？在两个主要形式，它是在身体1）全长过氧化物酶体增殖体？（FL-PPAR？）和2）全长异二聚体复合物，由PPAR？组成，RXR？和DNA这项工作将以前所未有的细节揭示配体如何影响组成PPAR？构象系综的相关结构的范围。然而，他们不会检测配体诱导的变化，在过氧化物酶体增殖物激活受体？的小的快速运动（即构象熵），这可能是至关重要的配体如何在人类中产生的影响。为了研究这些运动，PI将接受使用分子动力学模拟的培训。在可能的情况下，将使用NMR对照实验检查这些模拟。这些数据将被用来估计平均区域变化的小快速内部运动的过氧化物酶体增殖物激活受体？当药物结合PPAR时发生的构象熵。所有这些药物引起的PPAR变化的测量？将测试与功能结果的相关性，例如与FL-RXR的二聚化，辅调节肽的募集和脂肪细胞中的基因表达。为了充分利用这些最新的进展，并建立最好的模型， 在分子动力学模拟中，为了训练PI，需要保护时间。包括Cheatham博士在内的咨询小组将在这一领域提供专家指导。在资助的指导部分（目标1），PI将继续接受合作者（Art Palmer博士和Mark Rance博士）在NMR和蛋白质分子动力学模拟方面的指导，此外还有PI的主要导师Kojetin博士。PI还将接受PPAR方法和分析方面的培训。药物对Griffin博士（共同导师）细胞中靶基因表达的影响。位于佛罗里达的斯克里普斯研究所（TSRI）有七个研究核受体的小组（Nettles、Griffin、Kojetin、Kameneka、Solt、Spinch和Smith）。其中四个小组目前正在使用不同的方法和途径来回答有关PPAR？的重要问题。这使得合作自然，并提供了一个良好的环境，在其中接受必要的培训，以维持在这一领域的独立研究。PI拥有物理学和生物学学位，这使他能够在TSRI的3年培训期间快速获得蛋白质核磁共振（NMR）和其他几种生物物理学和生物学技术的许多领域的专业知识，并提供了广泛的培训，这对于将生物物理学和PPAR运动和结构的热力学与功能结果联系起来至关重要。