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: 7965707
• 负责人: Sandra Smith-Gill
• 金额: $ 17.43万
• 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/7965707
• 关键词: Address; Affinity; Antibodies; Antigens; Behavior; Binding; Binding Sites; Biological; Biological Assay; Budgets; Calorimetry; Cancer Biology; Competitive Binding; Complex; Dependence; Development; Diagnostic; Docking; Drug Delivery Systems; Experimental Designs; Family; Future; Goals; Heat shock proteins; Isomerism; Kinetics; Laboratories; Laboratory Study; Lead; Length; Ligands; Measures; Methodology; Modeling; Molecular; Muramidase; Patients; Pharmaceutical Preparations; Proteins; Protocols documentation; Publications; Reagent; Reporting; Research; Residencies; Signal Transduction; Specificity; Surface Plasmon Resonance; System; Temperature; Testing; Therapeutic; Thermodynamics; Time; Time Study; Tissues; Titrations; Tryptophan; Ultracentrifugation; base; clinically significant; conformational conversion; dimer; experience; in vitro activity; inhibitor/antagonist; insight; instrument; light scattering; mutant; novel; programs; 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. 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 which show 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-GA 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 In addition, we have recently found that uncomplexed single chain Fv fragments (scFv of anti-lysozyme antibodies undergo a concentration- and time-dependent, temperature-modulated change in affinity which we hypothesize represents a conformational transition. It was initially observed in scFv which had 6-19F-tryptophan incorporated into the 6 tryptophans of the binding site, and we developed a novel SPR protocol to study the temperature, time, and concentration dependence of the change. We then demonstrated that this occurs in unsubstituted scFv, and may be evidence of a conformational isomerism. Alternatively, it could be evidence of a concentration-dependent transient dimerism, but dynamic light scattering experiments indicate the protein is in a monomeric state, and the kinetics are not consistent with dimer binding. We expect by the end of this month to resolve this question using ultracentrifugation. These results are of clinical significance, because it may be a general phenomena of scFv, rather than specific to the antibody we are studying, and scFv reagents are used clinically for both diagnostic and therapeutic purposes. Thus, it is necessary to understand the effects of storing a scFv reagent at high concentrations and then subsequent dilution upon administration upon subsequent binding behavior when administered to a patient. Future Plans I expect to bring this research program to closure by September 30, 2009, which is the close of the FY09 budget period. This will entail completing experiments in progress, including the recent initiatives outlined above, and submitting results for publication.

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。最近，我们使用模型抗体系统表明，与原始抗原相比，与原始抗原相比，在长期竞争性结合测定中检查时，具有明显较低的结合亲和力的突变抗原实际上是一种更好的抑制剂：与原始抗原进行检查：结合抑制与与抑制剂前孵育时间的长度相关。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-GA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，开发可靠的方法来研究这种特征可能会产生很大的影响，我们最近发现，毫无复杂的单链FV片段（抗溶酶抗体的SCFV经历了浓度和时间依赖于温度的亲和力的变化，我们在SCFF中构成了6-19，这是SCFF的浓度变化。结合位点，我们开发了一种新的SPR协议来研究变化的温度，时间和浓度依赖性，然后证明这是在未取代的SCFV中发生的，并且可能是构象异构体的证据，这可能是浓度依赖性散射实验的证据。我们期望在本月底之前，将使用超离心分析解决这个问题。这些结果具有临床意义，因为它可能是SCFV的一般现象，而不是针对我们正在研究的抗体，并且SCFV试剂在临床上用于诊断和治疗目的。因此，有必要理解在高浓度下以高浓度存储SCFV试剂的效果，然后在对患者施用后随后的结合行为后随后稀释。我希望未来的计划将这项研究计划在2009年9月30日之前结束，这是09财年预算期的结束。这将需要完成正在进行的实验，包括上面概述的最新举措，并提交成果进行发布。