Investigation of the Mechanism and Biological Impact of Protein-based Inhibition

基于蛋白质的抑制机制和生物学影响的研究

• 批准号: 8060128
• 负责人: Kristin Therese Ziebart
• 金额: $ 4.28万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2011-11-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8060128
• 关键词: Accounting; Address; Adjuvant; Adverse drug effect; Affect; Behavior; Binding; Biochemical; Biological; Biological Assay; Biological Availability; Buffers; Cells; Colloids; Drug toxicity; Enzymatic Biochemistry; Enzyme Inhibition; Generations; HIV; Heating; In Vitro; Intestines; Investigation; Kinetics; Knowledge; Lactamase; Lead; Liquid substance; Lymph; Marketing; Measures; Methods; Oral; Pharmaceutical Preparations; Pharmacology; Physiological; Plasma; Prevalence; Proteins; Proxy; Reagent; Research; Research Personnel; Research Project Grants; Simulate; Small Intestines; Source; Sprague-Dawley Rats; Structure of aggregated lymphoid follicle of small intestine; Testing; Thermodynamics; Toxic effect; Toxicity Tests; absorption; base; bile salts; cytotoxic; cytotoxicity; drug candidate; drug discovery; drug distribution; high throughput screening; in vivo; inhibitor/antagonist; insight; non-nucleoside reverse transcriptase inhibitors; particle; prevent; research study; theories; tool

DESCRIPTION (provided by applicant): Many organic molecules, biological reagents, and some drugs form sub-micrometer sized colloidal aggregates in biochemical buffers at micromolar concentrations. Upon formation of these particles, non- specific, picomolar enzyme inhibition is observed. Although much is known about the in vitro behavior of colloidal aggregates, the biological ramifications of colloidal aggregates remain essentially unexplored. This project addresses key biological and mechanistic aspects of colloidal aggregates. Most ambitiously, this project investigates the impact of colloidal aggregates on drug distribution. The research will test the theory proposed by Janssen et al. that hydrophobic drugs aggregate in the gut, and are absorbed into the lymph by specialized particle-absorbing cells in the intestinal tract. Janssen et al. proposed that such a mechanism accounts for the higher than expected efficacy of non-nucleoside reverse transcriptase inhibitors and other hydrophobic drugs [Ref. 6]. To test Janssen's hypothesis, the absorption and physiologic distribution of several hydrophobic, known aggregating drugs will be examined in Sprague Dawley rats. If hydrophobic drugs are found to exist in the lymph as aggregates, it will profoundly affect current understanding of pharmacology, drug distribution and bioavailability. This project investigates the potential cellular toxicity of colloidal aggregates. Prior to advancing to "lead" status, candidate drugs and tools are tested for toxicity. Here, molecules are intentionally tested at concentrations much higher than their EC50 values, and at these concentrations many compounds aggregate. Because aggregates have been shown to be hemolytic, they may be responsible for false-positive toxicity results. Well-characterized aggregators will be tested in cell toxicity assays above and below their critical aggregation concentration (CAC). If aggregates are found to be cytotoxic, while their soluble monomeric counterparts are innocuous, it could prevent lead compounds from being unnecessarily abandoned based on false-positive cellular toxicity results that arise from aggregation brought about by the assay conditions. The thermodynamics of aggregate formation and the kinetics of aggregate-based enzyme inhibition are explored in this project. If the hydrophobic effect drives aggregate formation, a large change in heat capacity ( Cp) would be observed. If a small Cp is measured, it suggests a radically different mechanism of aggregate formation. The kinetics of the incubation effect of observed with aggregate-based inhibition will be probed with classical enzymology in kinetic assays using 2-lactamase and several well-characterized aggregators. The knowledge acquired from these experiments may illuminate methods for controlling aggregate formation and subsequent enzyme inhibition. These insights could be exploited to prevent negative drug side effects caused by aggregates, as well as to optimize conditions for drug absorption and bioavailability. PUBLIC HEALTH RELEVANCE: This research project investigates whether certain drugs associate into particles called colloidal aggregates inside the body, and if so, how those particles affect the distribution of drug within the body, and if drug toxicity is due to formation of these particles. The project will also investigate the mechanism of particle formation. The results of this research may profoundly impact current practices and concepts related to drug discovery.

描述(申请人提供)：许多有机分子、生物试剂和一些药物在微摩尔浓度的生化缓冲液中形成亚微米大小的胶体聚集体。当这些颗粒形成时，观察到非特异性的皮摩尔酶抑制。虽然对胶体聚集体的体外行为已知很多，但胶体聚集体的生物学分支基本上仍未被探索。该项目涉及胶体聚集体的关键生物学和机械方面。最雄心勃勃的是，这个项目调查了胶体聚集体对药物分布的影响。这项研究将检验Janssen等人提出的理论。这些疏水药物聚集在肠道中，并被肠道中特殊的颗粒吸收细胞吸收到淋巴中。Janssen等人。提出，这种机制解释了非核苷逆转录酶抑制剂和其他疏水药物的疗效高于预期[参考文献]。6]。为了验证Janssen的假说，我们将研究几种已知的疏水性聚集性药物在SD大鼠体内的吸收和生理分布。如果发现疏水药物以聚集体形式存在于淋巴中，将深刻影响目前对药物的药理、药物分布和生物利用度的理解。本项目研究胶体聚集体的潜在细胞毒性。在进入“铅”状态之前，对候选药物和工具进行毒性测试。在这里，分子被有意地在远高于其EC50值的浓度下进行测试，在这些浓度下许多化合物聚集在一起。因为聚集体已经被证明是溶血的，它们可能是假阳性毒性结果的原因。特征良好的聚集器将在其临界聚集浓度(CAC)以上和以下进行细胞毒性测试。如果聚集体被发现具有细胞毒性，而它们的可溶性单体对应物是无害的，它可以防止基于假阳性的细胞毒性结果而不必要地放弃先导化合物，这些结果是由分析条件带来的聚集引起的。本项目对聚集体形成的热力学和基于聚集体的酶抑制动力学进行了探索。如果疏水效应驱动聚集体的形成，则会观察到热容(Cp)的较大变化。如果测量一个小的CP，它表明了一个完全不同的聚集体形成机制。在使用2-内酰胺酶和几个特征良好的聚合体的动力学分析中，将用经典的酶学来探索观察到的基于聚集体的抑制的孵育效应的动力学。从这些实验中获得的知识可能会对控制聚集体形成和随后的酶抑制的方法有所启发。这些见解可以被用来防止聚集体引起的负面药物副作用，以及优化药物吸收和生物利用度的条件。 与公共健康相关：这项研究项目调查某些药物是否在体内结合成称为胶体聚集体的颗粒，如果是，这些颗粒如何影响药物在体内的分布，以及药物毒性是否源于这些颗粒的形成。该项目还将研究颗粒形成的机制。这项研究的结果可能会对当前与药物发现相关的实践和概念产生深远的影响。