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尺寸的胶体聚集体。在形成这些颗粒后，观察到非特异性的皮摩尔酶抑制。尽管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分支仍然基本上未被探索。这个项目涉及胶体聚集体的关键生物学和机械学方面。 最雄心勃勃的是，这个项目研究了胶体聚集体对药物分布的影响。这项研究将测试Janssen等人提出的理论，即疏水性药物在肠道中聚集，并被肠道中专门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性药物的疗效高于预期的原因[参考文献6]。为了检验Janssen的假设，将在Sprague道利大鼠中检查几种疏水性、已知聚集性药物的吸收和生理分布。如果发现疏水性药物在淋巴中以聚集体的形式存在，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，分子被有意地在远高于其EC 50值的浓度下测试，并且在这些浓度下许多化合物聚集。由于聚集体已被证明具有溶血性，因此它们可能是导致假阳性毒性结果的原因。将在高于和低于其临界聚集浓度（CAC）的细胞毒性试验中检测充分表征的聚集剂。如果发现聚集体具有细胞毒性，而其可溶性单体对应物是无害的，则可以防止基于由测定条件引起的聚集引起的假阳性细胞毒性结果而不必要地放弃先导化合物。 本计画探讨聚集体形成的热力学与聚集体抑制酵素的动力学。如果疏水效应驱动聚集体形成，则将观察到热容（Cp）的大变化。如果测量到较小的Cp，则表明聚集体形成的机制完全不同。将在使用2-内酰胺酶和几种充分表征的聚集剂的动力学试验中，采用经典酶学对观察到的基于聚集体的抑制的孵育效应的动力学进行探索。从这些实验中获得的知识可以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。 公共卫生相关性：该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，如果是这样，这些颗粒如何影响药物在体内的分布，以及药物毒性是否是由于这些颗粒的形成。该项目还将研究颗粒形成的机制。这项研究的结果可能会深刻影响目前与药物发现相关的实践和概念。

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.