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然是困难的。 挑战性利用天然产物和其他复杂分子库进行高通量筛选（HTS） 仍然是基本方法。然而，HTS在许多方面都不令人满意：成本过高， 效率、猖獗的误报和阻碍“点击率领先”发展的“点击率”的复杂性。片段 基于药物发现（FBDD）的构想很好地克服了这些局限性，但可以说没有 正如所希望的那样。FBDD的有限影响是因为大多数碎片“命中”分子是非常弱的粘合剂 并且不能通过当前的分析方法检测到。因此，FBDD的巨大潜力丧失了。这里是 将开发一种方法，该方法可以可靠地检测微弱但特异性的结合，目的是帮助 重振和加强早期小分子药物的发现。 结合的可靠检测要求配体和蛋白质浓度至少为约0.5 μ mol/L。 解离常数，这对于弱粘合剂在实践上和经济上是不现实的。移除的策略 这个基本的屏障很简单。反胶束（RM）的水核用于限制单个蛋白质分子 和足够高浓度的碎片以克服不利的结合熵。NMR光谱 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 NMR FBDD的首次应用突出了其极大扩展小型药物发现的潜力。一条规则- 白细胞介素-1 β（IL-1β）的三（Ro 3）片段筛选显示：1）弱但特异性结合可有效地与IL-1 β结合， 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 可行的; 3）观察到高命中率; 4）表面覆盖范围非凡，提供前所未有的连接性 潜在的; 5）高度期望的更极性的粘合剂被照亮。 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表面覆盖率是否典型？Ro 3和五规则（Ro 5）的碎片命中亲和力分布如何？ 图书馆一般？对于一个最佳的RM，有哪些有用的碎片的化学特性可供选择 核磁共振筛选库？如何有用的是非常弱的结合命中铅发展？Ro 5库是否 提供命中亲和力和表面覆盖率的更好折衷？实施风险管理最有效的方法是什么 核磁共振筛选？RM NMR筛选定量可靠吗？该项目将解决这些和其他 技术挑战，阻碍了创建一个更充分地实现卓越洞察力的战略， FBDD范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.