Improving Fragment Based Drug Discovery and the Development of Tools for Chemical Biology through Nanoscale Encapsulation and NMR Spectroscopy

尽管在药物发现方面取得了巨大的技术进步，但小分子药物的从头开发仍在进行中 很有挑战性。利用天然产物和其他复杂分子文库的高通量筛选(HTS) 仍然是最基本的方法。然而，HTS在许多方面都不能令人满意：超高的成本，糟糕的 效率、猖獗的误报和复杂的“点击”，阻碍了“点击到领先”的发展。碎片 基于药物的发现(FBDD)被巧妙地构思来克服这些限制，但可以说并没有 如所愿地表演。FBDD的影响有限是因为大多数碎片“击中”的分子都是非常弱的结合剂 用目前的检测方法是无法检测到的。因此，FBDD的巨大潜力就失去了。在这里，一个 要开发一种方法，能够可靠地检测到微弱但特定的结合，目标是帮助 重振和加强早期小分子药物发现。 准确的结合检测要求配体和蛋白质的浓度至少在 离解常数，这对于弱粘结剂来说在实践上和经济上都是不现实的。要移除的策略 这一基本障碍很简单。反胶束(Rm)的水核被用来限制单个蛋白质分子。 以及浓度足够高的碎片，以克服不利的结合熵。核磁共振波谱 然后允许以合理的成本进行结合亲和力的位点分辨检测和定量。 RM核磁共振FBDD的首次应用突出了它极大地扩大小型药物发现的潜力。一条规则-- 白细胞介素1β(IL-1β)的三选一(Ro3)片段筛选表明：1)弱而特异的结合可有效地 在结构背景下检测；2)实现所需的高蛋白质和配体浓度是经济的 可行；3)观察到高命中率；4)表面覆盖率非凡，提供了前所未有的连通性 潜力；5)高期望的更多的极性粘合剂被照亮。 现在，更充分地实现FBDD的巨大前景的大门已经打开，但关键问题仍然存在： IL-1β表面覆盖范围是典型的？Ro3和五规则(Ro5)的碎片命中亲和力的分布是什么 更广泛地说，图书馆？要选择最佳RM的有用碎片的化学特征是什么 核磁共振筛选文库？对于Lead开发来说，非常弱的约束性点击有多大用处？Ro5库是否 提供更好的热门亲和力和表面覆盖率的折衷方案？实施RM最有效的方法是什么 核磁共振筛查？核磁共振核磁共振筛查定量可靠吗？该项目将解决这些问题和其他问题 阻碍创建更充分实现以下卓越洞察力的战略的技术挑战 FBDD模式，并释放其最初预期的潜力。

Despite tremendous technical advances in drug discovery, de novo development of small molecule drugs is still challenging. High-throughput screening (HTS) with libraries of natural products and other complex molecules remains the bedrock approach. However, HTS is unsatisfactory in many ways: extraordinary cost, poor efficiency, rampant false positives and a complexity of “hits” that hinders hit-to-lead development. Fragment based drug discovery (FBDD) was brilliantly conceived to overcome these limitations, but has arguably not performed as hoped. The limited impact of FBDD is because most fragment “hit” molecules are very weak binders and are undetectable by current assay methods. The enormous potential of FBDD is therefore lost. Here, an approach is to be developed that can reliably detect weak but specific binding with the goal of helping to reinvigorate and enhance early phase small molecule drug discovery. Faithful detection of binding requires that the ligand and protein concentrations be at least on the order of the dissociation constant, which is practically and financially unrealistic for weak binders. The strategy to remove this basic barrier is simple. The water core of the reverse micelle (RM) is used to confine a single protein molecule and fragments at high enough concentrations to overcome the unfavorable binding entropy. NMR spectroscopy then permits site-resolved detection and quantification of binding affinity at reasonable cost. The first application of RM NMR FBDD highlights its potential to greatly expand small drug discovery. A rule- of-three (Ro3) fragment screen of interleukin-1β (IL-1β) shows that 1) weak yet specific binding can be efficiently detected in a structural context; 2) achieving the required high protein and ligand concentrations is economically feasible; 3) a high hit rate is observed; 4) surface coverage is extraordinary and gives unprecedented connectivity potential; 5) highly desired more polar binders are illuminated. The door is now open to more fully realize the tremendous promise of FBDD but critical questions remain: Is the IL-1β surface coverage typical? What is the distribution of fragment hit affinities of Ro3 and rule-of-five (Ro5) libraries more generally? What are the chemical characteristics of useful fragments to choose for an optimal RM NMR screening library? How useful are the very weakly binding hits for lead development? Does the Ro5 library offer a better compromise of hit affinity and surface coverage? What is the most efficient way to carry out RM NMR screening? Is RM NMR screening quantitatively reliable? This project will address these and other technical challenges that stand in the way of creating a strategy that more fully enables the brilliant insights of the FBDD paradigm and unleashes its originally anticipated potential.