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: 8545198
• 负责人: PETER J TONGE
• 金额: $ 28.19万
• 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/8545198
• 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（申请人提供）：许多候选药物因二期临床试验缺乏疗效而失败。这种失败发生在所有治疗领域，主要源于粪便体内功效以及缺乏安全性（毒性）。我们假设，使用药物靶标停留时间（tR）测量以及化合物效力的其他热力学估计，将提高预测体内药物功效的能力。由于我们对 tR 重要性的认识很大程度上是基于对许多药物缓慢与其靶点解离的观察，因此证明特定化合物系列中 tR 与体内药物活性之间的相关性，当与药物药代动力学知识相结合时，将允许建立预测药物药效学的数学模型。这一目标是创新性的，将在如何获取有关先导化合物（抑制剂、激动剂、拮抗剂）与其靶标相互作用的信息方面产生范式转变。 收集并使用。为了实现这一目标，我们将结合使用 X 射线晶体学、定点诱变、化学合成和计算方法。特别是，与时间无关的分子动力学（MD）模拟将有助于揭示通过结合动力学测量探测到的特定原子级相互作用，并将提供动态信息以填补晶体中观察到的稳定状态之间的时间间隙结构。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，这两种酶都是临床相关的药物靶点。此外，虽然这两种酶均受到二苯醚化合物类和基于吡啶酮的第二相关系列的抑制，但通过相同的两步缓慢启动诱导拟合动力学机制，伴随酶抑制的结构变化有所不同。因此，我们的目标是确定我们是否可以首先了解然后合理地调节两种不同酶的停留时间，同时保持化合物类别恒定。这将为将我们的知识转化为其他系统提供一个平台。在目标 1 中，我们将阐明 mtFabI 时间依赖性抑制的机制。时间相关的 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*) 的过渡态结构，并确定对时间依赖性抑制至关重要的关键相互作用。将设计具有增加的 tR 值的抑制剂。这将提供对诱导拟合结合机制的详细理解。在目标 2 中，我们将确定 saFabI 时间依赖性抑制的结构基础。这将通过动力学和结构方法来实现。将合成额外的类似物来询问我们对缓慢起效的 saFabI 抑制的理解。在目标 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，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给药方案的开发具有广泛的影响。 tR 对 PAE 的贡献以及最终的体内抗菌活性将在金黄色葡萄球菌中进行评估。对活细胞的 PAE 测量将在药物活性的体外和体内估计之间架起一座桥梁。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，我们的研究将深入了解蛋白质构象变化的时间依赖性以及这些变化如何与蛋白质功能直接相关，并将为探索其他系统中时间依赖性酶抑制的结构基础提供平台。证明 tR 的重要性将导致先导化合物优化的范式转变。