Time-dependent receptor-target interactions in molecular interaction networks

分子相互作用网络中时间依赖性受体-靶标相互作用

• 批准号: 7733247
• 负责人: Sandra Smith-Gill
• 金额: $ 25.09万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733247
• 关键词: ATP phosphohydrolase; Accounting; Address; Adverse effects; Affinity; Animal Structures; Antibodies; Antigens; Antineoplastic Agents; Attention; Behavior; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Factors; Calorimetry; Cancer Biology; Cell Survival; Client; Clinical Trials; Competitive Binding; Complex; Coupled; Coupling; Cysteine; Data; Dependence; Depsipeptides; Development; Docking; Dolastatin 10; Dolastatin 15; Dolastatin Compound; Drug Delivery Systems; Drug Interactions; Exhibits; Experimental Designs; Family; Goals; Heat shock proteins; In Vitro; Inhibitory Concentration 50; Investigation; Kinetics; Laboratories; Laboratory Study; Lead; Length; Ligand Binding; Ligands; Measures; Methodology; Microtubules; Modeling; Molecular; Patients; Peptides; Personal Communication; Pharmaceutical Preparations; Proteins; Protocols documentation; Range; Reporting; Residencies; Screening procedure; Signal Transduction; Specificity; Structure-Activity Relationship; Study models; Surface; Surface Plasmon Resonance; System; Technology; Testing; Thermodynamics; Time; Time Study; Tissues; Titrations; Tubulin; Vinca; analog; base; cancer cell; chemotherapeutic agent; cytotoxicity; experience; in vivo; inhibitor/antagonist; insight; instrument; marine organism; mutant; novel; polymerization; prototype; receptor; research study; residence; study characteristics

Surface plasmon resonance (SPR), using BIAcore2000 instruments, is utilized by our laboratory for studying protein-ligand interactions. For many years our laboratory has studied the interaction between antigens and antibodies and we have developed a novel experimental design using the surface plasmon resonance to resolve time-dependent kinetic behavior which is consistent with the formation of a complex, first as an encounter complex and changing into a more stable docked complex, evidencing at least two-states. This is the same model which recently has been proposed for time-dependent binding of the 90kDa heat shock protein (Hsp90) to inhibitors of the genaldenamycin (GA) family. Our previous experience with the antibody-antigen system therefore informs the study of new, uncharacterized systems such as Hsp90-GA interaction. The unique protocols we have developed in the long-term project on antibody-protein interactions will be the basis of several new initiatives addressing targeted Receptor-Ligand interactions important to cancer biology. The development of reliable methodology to time-dependent residency times is important to understanding pharmacological profiles of drugs many drugs. We have begun experiments to examine the time-dependent complex formation using SPR, and to develop general methodology for identifying time-dependent binding in other drug-target interactions. The first initiative is the use of SPR to identify time-dependent drug-target interactions leading to long residence times and selectvity of inhibitors. In vitro drug-target interaction assays, classically quantified in terms of binding parameters such as IC50 or Kd., do not always predict in vivo efficicacy. Hsp90 interaction with the natural product GA is an example, where cellular activity is much higher than in vitro affinities would predict. Hsp90 is emerging as an important target for chemotherapeutic agents. The GA binds to Hsp90, inhibits its ATPase activity, and decreases levels of many of its client proteins implicated in cancer cell survival. Derivatives of this natural product are currently in multiple clinical trials. Although binding assays show affinities of the analogues to be in the micromolar range, they exhibit cellular effects in low nanomolar ranges. Time-dependent formation of a tight complex is one recently proposed mechanism to explain this phenomena, and may account for the accumulation of the drug in tissues and its pharmacological effects (Gooljarsingh et al, Proc natl Acad Sci 103:7625,2006). In parallel experiments, we have recently shown, using our model antibody system, that a mutant antigen with apparently lower binding affinity is actually a better inhibitor in SPR of antibody binding than the original antigen when examined in a long term competitive binding assay: binding inhibition correlates with length of preincubation time with the inhibitor. We have developed unique protocols for measuring binding kinetics and thermodynamics and thermodynamics whichshow that the underlying mechanism is the slow, time-dependent formation of a very tight complex by the mutant inhibitor. This is the first reported demonstration of time-dependent inhibition efficicacy. We are planning experiments to also demonstrate this by isothermal titration calorimetry (ITC), which will require the development of additional novel ITC protocols. This experimental design can now be applied to drug-target interactions such as the Hsp90-GS interaction. The time-dependence of binding leading to long residence time of such inhibitors is likely to significantly impact their pharmalogical profile in patients, such as pharmacological effect and target selectivity. Thus development of reliable methodology to study this characteristic is likely of high impact A second new initiative is study of the interaction of anti-tubulin drugs with tubulin. Microtubule-targeted peptides and depsipeptides are receiving considerable attention as possible anti-cancer agents, and new synthetic analogues have fewer side effects than natural compounds such as dolstatin-15 isolated from marine organisms. They inhibit binding of vinblastin to tubulin, and bind to what has been termed the Vinca domain of tubulin. Extensive biochemical, cellular, and whole animal structure activity relationship (SAR) data for dolastatin-10 and dolastatin-15, in combination with modeling studies, have led to a preliminary model of the dolastatin binding site to the Vinca domain on tubulin (Hamel & Covell, Curr. Med. Chem. 2:19,2002). Potent cytotoxicity has not been observed in the absence of a strong tubulin-binding activity. However, the absence of a quantitative binding assay has limited the precision with which a pharmacaphore can be defined. SPR has high potential for investigation of tubulin-based assays. The compound SJ-81, a synthetic analogue of dolstatin-10, has been shown to inhibit tubulin polymerization in cellular and biochemical assays (Tarasova & Hamel, personal communication). Attachment of a linker to the amino terminus does not interfere with binding. Previous attempts to develop an SPR tubulin-binding assay, by capturing tubulin with anti-tubulin and using SJ-81 as the analyte did not yield results (personal communication, E. Hamel & R.Fischer). A maleimidocapronic linker has been has covalently attached to the amino terminus of SJ-81. This can be readily covalently coupled to a cysteine-derived surface Biacore chip, producing a stable interaction surface for binding assays. By covalently coupling the stable drug to the chip and using tubulin as the analyte, we can better control the tubulin state, and binding of the larger analyte will be more readily observable by the SPR technology. New methodology for determining binding kinetics and thermodynamics will be developed as necessary to study to biological and physical factors controlling tubulin polymerization. Tubulin protein will be provided by E. Hamel (SRB,TP, NCI) whose expertise will also inform the experimental design of the SPR experiements. Development of a quantitative binding assay will provide a basis for more detailed SAR studies, characterization of the ligand binding site(s) on tubulin, and more precise definition of a pharmacaphore. Understanding the details of the interaction may also give insight into a better experimental design for screening multiple drug interactions with immobilized tubulin. Finally, it will serve as a prototype for investigation of the molecular details of novel anti-tubulin agents with tubulin.

我们的实验室使用BIACORE2000仪器的表面等离子体共振（SPR）用于研究蛋白质 - 配体相互作用。多年以来，我们的实验室研究了抗原与抗体之间的相互作用，我们使用表面等离子体共振来开发了一种新型的实验设计，以解决时间依赖性的动力学行为，这与复合物的形成是一致的，首先是遭遇复合物，并变成更稳定的更稳定的结对结合的复合物，至少是两层架。这是最近提出的用于90KDA热休克蛋白（HSP90）与Genaldennycin（GA）家族​​抑制剂的时间依赖性结合的模型。因此，我们以前在抗体 - 抗原系统的经验为新的未表征系统（例如HSP90-GA相互作用）提供了信息。我们在抗体 - 蛋白质相互作用的长期项目中开发的独特方案将是一些针对针对性受体配体相互作用对癌症生物学重要的新计划的基础。将可靠的方法发展为时间依赖性居住时间对于理解许多药物的药理特征很重要。我们已经开始进行实验，以检查使用SPR的时间依赖性复合物的形成，并开发用于识别其他药物目标相互作用中时间依赖性结合的通用方法。第一个倡议是使用SPR来识别时间依赖的药物目标相互作用，从而导致长期停留时间和抑制剂的选择。在结合参数（例如IC50或KD。）上经过经典量化的体外药物目标相互作用分析并不总是预测体内效率。 HSP90与天然产物GA的相互作用是一个例子，其中细胞活性远高于体外亲和力的预测。 HSP90成为化学治疗剂的重要靶标。 GA与HSP90结合，抑制其ATPase活性，并降低与癌细胞存活有关的许多客户蛋白的水平。该天然产品的衍生物目前正在多次临床试验中。尽管结合测定显示类似物的亲和力在微摩尔范围内，但它们在低纳摩尔范围内表现出细胞效应。紧密复合物的时间依赖性形成是最近提出的解释这种现象的机制，可能解释了该药物在组织及其药理作用中的积累（Gooljarsingh等人，Proc Natl Acad Sci 103：7625,2006）。在并行实验中，我们最近使用模型抗体系统表明，与原始抗原的SPR结合抗原显然是一种更好的抗原抗原，在长期竞争性结合测定中检查时，与原始抗原相比，具有更好的抑制剂：结合抑制与抑制时间的长度相关。我们开发了用于测量结合动力学和热力学和热力学的独特方案，这些方案表明，基本机制是突变抑制剂对非常紧密的复合物的缓慢，时间依赖性的形成。这是第一次报道的时间依赖性抑制效率。我们正在计划实验，还通过等温滴定量热法（ITC）来证明这一点，这将需要开发其他新型的ITC协议。现在，该实验设计可以应用于诸如HSP90-GS相互作用之类的药物目标相互作用。导致这种抑制剂长期停留时间的结合的时间依赖性可能会显着影响其药理效应和靶向选择性等患者的药物特征。因此，开发可靠的研究这种特征的方法可能具有很高的影响，第二个新计划是研究抗微管蛋白药物与微管蛋白的相互作用。 靶向微管的肽和深度肽正在接受相当大的关注，这是可能的抗癌剂，而新的合成类似物的副作用少于天然化合物，例如从海洋生物体中分离出的dolstatin-15。它们抑制vinblastin与微管蛋白的结合，并与所谓的小管蛋白结构结合。多拉斯汀-10和Dolastatin-15的广泛的生化，细胞和整个动物结构活性关系（SAR）数据与建模研究结合使用，导致了多拉斯汀蛋白结合位点与小管蛋白上的Vinca结构域的初步模型（Hamel＆Covell。在没有强大的小管蛋白结合活性的情况下，尚未观察到有效的细胞毒性。然而，缺乏定量结合测定限制了可以定义药物的精度。 SPR具有很高的研究基于小管蛋白的测定的潜力。复合SJ-81是Dolstatin-10的合成类似物，已显示出抑制细胞和生化测定中的小管蛋白聚合（Tarasova＆Hamel，个人交流）。连接器与氨基末端的附着不会干扰结合。先前通过用抗微管蛋白捕获小管蛋白并使用SJ-81作为分析物没有产生结果来开发SPR微管结合分析的尝试（个人通讯，E。Hamel＆R.Fischer）。已在SJ-81的氨基末端共价连接了一个MaleImidocapronic连接器。可以很容易地将其共价耦合到半胱氨酸衍生的表面biacore芯片，从而产生稳定的相互作用表面进行结合测定。通过将稳定药物与芯片耦合并使用微管蛋白作为分析物，我们可以更好地控制微管蛋白态，而SPR技术可以更容易地观察到较大的分析物的结合。必须开发用于确定结合动力学和热力学的新方法，以研究控制小管蛋白聚合的生物学和物理因素。小管蛋白将由E. hamel（SRB，TP，NCI）提供，其专业知识还将为SPR体验的实验设计提供信息。定量结合测定法的开发将为更详细的SAR研究，小管蛋白上的配体结合位点的表征以及药物paphore的更精确的定义提供基础。了解相互作用的细节还可以深入了解更好的实验设计，以筛选与固定小管蛋白的多种药物相互作用。最后，它将用作研究带微管蛋白的新型抗微管蛋白的分子细节的原型。