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

描述（由申请人提供）：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。在这些颗粒形成后，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有很多了解，但胶体聚集体的生物学后果仍然基本上未被探索。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，这个项目调查胶体聚集体对药物分布的影响。本研究将验证Janssen等人提出的疏水药物在肠道内聚集，并被肠道内专门的颗粒吸收细胞吸收到淋巴中的理论。Janssen等人提出，这种机制解释了非核苷类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的假设，我们将在Sprague Dawley大鼠身上检测几种已知的疏水聚集药物的吸收和生理分布。如果发现疏水药物以聚集体的形式存在于淋巴中，将深刻影响目前对药理学、药物分布和生物利用度的认识。本项目研究胶体聚集体的潜在细胞毒性。在进入“先导”状态之前，候选药物和工具要进行毒性测试。在这里，分子被故意在远高于其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。因为聚集体已被证明具有溶血作用，它们可能导致假阳性毒性结果。特性良好的聚集物将在高于或低于其临界聚集浓度（CAC）的细胞毒性试验中进行测试。如果发现聚集体具有细胞毒性，而它们的可溶性单体对偶物是无害的，它可以防止由于检测条件带来的聚集引起的细胞毒性假阳性结果而不必要地放弃先导化合物。本项目探讨了聚集体形成的热力学和基于聚集体的酶抑制动力学。如果疏水效应驱动聚集体形成，将观察到热容（Cp）的大变化。如果测量到一个小的Cp，它表明了一个完全不同的聚集体形成机制。利用2-内酰胺酶和几种表征良好的聚集物进行动力学分析，利用经典酶学方法对观察到的以聚集物为基础的抑制的孵育效应进行动力学研究。从这些实验中获得的知识可能阐明控制聚集体形成和随后的酶抑制的方法。这些见解可以用来防止由聚集体引起的药物副作用，以及优化药物吸收和生物利用度的条件。

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.