Mechanism of Slow Onset Enzyme Inhibition and Drug Target Residence Time

缓慢起效的酶抑制机制和药物靶标停留时间

• 批准号: 8727068
• 负责人: PETER J TONGE
• 金额: $ 29.28万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8727068
• 关键词: Affinity; Agonist; Anti-Bacterial Agents; Antibiotics; Area; Binding; Cells; Complex; Computing Methodologies; Coupled; Dependence; Development; Dose; Drug Exposure; Drug Kinetics; Drug Targeting; Enzyme Inhibition; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Equilibrium; Excision; Failure; Foundations; Francisella tularensis; Future; Goals; Growth; Hour; In Vitro; Inhibitory Concentration 50; Kinetics; Knowledge; Lead; Life; Measurement; Measures; Methods; Metric; Microbiology; Molecular; Mycobacterium tuberculosis; Outcome; Oxidoreductase; Pharmaceutical Preparations; Pharmacodynamics; Phase II Clinical Trials; Proteins; Pyridones; Regimen; Safety; Series; Site-Directed Mutagenesis; Staphylococcus aureus; Structure; System; Therapeutic; Thermodynamics; Time; Toxic effect; Translating; Whole Organism; X-Ray Crystallography; analog; antimicrobial; base; chemical synthesis; clinically relevant; design; drug candidate; drug discovery; drug efficacy; improved; in vivo; inhibitor/antagonist; innovation; insight; mathematical model; meetings; microbial; molecular dynamics; phenyl ether; protein function; residence; stem; time use

DESCRIPTION (provided by applicant): Many drug candidates fail due to lack of efficacy in Phase II clinical trials. This failure occurs in all therapeutic areas and primarily stems from poo in vivo efficacy as well as lack of safety (toxicity). We hypothesize that the use of drug-target residence time (tR) measurements, together with other thermodynamic estimates of compound potency, would improve the ability to predict drug efficacy in vivo. Since much of our appreciation for the importance of tR is anecdotally based on the observation that many drugs dissociate slowly from their targets, demonstration of correlations between tR and in vivo drug activity within specific compound series would, when coupled with knowledge of drug pharmacokinetics, allow mathematical models to be created that predict drug pharmacodynamics. This goal is innovative and will create a paradigm shift in how information on the interaction of lead compounds (inhibitors, agonists, antagonists) with their targets is both gathered and used. To meet this goal, we will use a combination of X-ray crystallography, site-directed mutagenesis, chemical synthesis, and computational methods. In particular, time-independent molecular dynamics (MD) simulations will help unravel the specific atomic-level interactions that are probed by the binding kinetics measurements, and will provide dynamic information to fill in the gaps in time between the stable states observed in the crystal structure. This will be accomplished using the FabI enzymes from Mycobacterium tuberculosis (mtFabI) and Staphylococcus aureus (saFabI), both of which are clinically relevant drug targets. In addition, while both enzymes are inhibited by the diphenyl ether compound class, and a second related series based on pyridones, through the same two-step slow-onset induced-fit kinetic mechanism, the structural changes that accompany enzyme inhibition differ. Thus our goal is to determine whether we can first understand and then rationally modulate residence time in two distinct enzymes whilst keeping the compound class constant. This will provide a platform for translating our knowledge to other systems. In Aim 1 we will elucidate the mechanism for the time-dependent inhibition of mtFabI. Time-indendent MD simulations and X-ray crystallography will be used to determine the structure of the transition state leading to the final enzyme-inhibitr complex (E-I*) and to identify key interactions critical for time-dependent inhibition. Inhibitors with increased tR values will be designed. This will provide a detailed understanding of an induced-fit binding mechanism. In Aim 2 we will determine the structural basis for the time-dependent inhibition of saFabI. This will be accomplished using kinetic and structural approaches. Additional analogues will be synthesized to interrogate our understanding of slow-onset saFabI inhibition. In Aim 3 we will delineate the relationship between tR, post-antibiotic effect (PAE) and in vivo activity. The PAE is the persistent suppression of microbial growth following drug exposure and removal, and is a well-known and frequently observed phenomenon in microbiology with widely stated implications for antimicrobial pharmacokinetics and the development of improved dosing regimens. The contribution of tR to PAE and, ultimately, in vivo antibacterial activity will be evaluated in S. aureus. The PAE measurements on live cells will provide a bridge between in vitro and in vivo estimates of drug activity. These studies will provide a foundation for using residence time in drug discovery. At a broader level, our studies will provide insight into the time dependence of conformational changes in proteins and how these relate directly to protein function, and will provide a platform for exploring the structural basis for time-dependent enzyme inhibition in other systems. Demonstrating the importance of tR will lead to a paradigm shift in lead compound optimization.

描述（由申请人提供）：许多候选药物由于在II期临床试验中缺乏疗效而失败。这种衰竭发生在所有治疗区域，主要源于体内功效以及缺乏安全性（毒性）。我们假设使用药物目标停留时间（TR）测量以及其他复合效力的热力学估计值将提高预测体内药物疗效的能力。由于我们对TR的重要性的大部分欣赏是基于这样的观察结果，即许多药物与其靶标相结合，因此在特定化合物系列中TR和体内药物活性之间的相关性证明将与对药物药物的知识相结合，允许创建数学模型，从而预测药物药物药物学模型。这个目标是创新的，将在铅化合物（抑制剂，激动剂，拮抗剂）与目标的相互作用的相互作用方面产生范式转变 收集和使用。为了满足这一目标，我们将使用X射线晶体学，定向诱变，化学合成和计算方法的组合。特别是，与时间无关的分子动力学（MD）模拟将有助于揭示由结合动力学测量值探测的特定原子水平相互作用，并将提供动态信息以填补晶体结构中观察到的稳定状态之间的时间间隔。这将使用来自结核分枝杆菌（MTFABI）和金黄色葡萄球菌（Safabi）的Fabi酶来完成，这两种酶都是临床上相关的药物靶标。此外，虽然这两种酶都被二苯基醚化合物类别抑制，而第二个基于吡啶酮的相关系列通过相同的两步缓慢发表诱导的动力学机制，但伴随酶抑制差异的结构变化。因此，我们的目标是确定我们是否可以首先理解，然后以两个不同的酶为合理地调节停留时间，同时保持化合物类别的恒定。这将提供一个将我们的知识转化为其他系统的平台。在AIM 1中，我们将阐明MTFABI时间依赖性抑制的机制。时间引导MD模拟和X射线晶体学将用于确定导致最终酶抑制剂复合物（E-I*）的过渡状态的结构，并确定对时间依赖性抑制至关重要的关键相互作用。将设计具有增加TR值的抑制剂。这将提供对诱导拟合结合机制的详细理解。在AIM 2中，我们将确定时间依赖性抑制萨法比的结构基础。这将使用动力学和结构方法来完成。将合成其他类似物，以询问我们对缓慢发表的萨法比抑制的理解。在AIM 3中，我们将描述TR，抗生素后效应（PAE）和体内活性之间的关系。 PAE是药物暴露和去除后对微生物生长的持续抑制作用，并且是微生物学中众所周知且经常观察到的现象，对抗菌药代动力学和改进的剂量方案的发展具有广泛陈述的影响。 TR对PAE的贡献，最终将在金黄色葡萄球菌中评估体内抗菌活性。活细胞上的PAE测量将在药物活性的体外和体内估计之间提供一个桥梁。这些研究将为在药物发现中使用停留时间提供基础。在更广泛的层面上，我们的研究将洞悉蛋白质构象变化的时间依赖性以及它们如何直接与蛋白质功能相关，并为探索其他系统中时间依赖性酶抑制的结构基础提供一个平台。证明TR的重要性将导致铅化合物优化的范式转移。