Fragment Based Drug Design in HIV Protease

我们将应用基于片段的筛选来鉴定HIV蛋白酶（PR）中的小分子结合位点 从而设计出对多位点PR突变体保持功效的有效抑制剂。突变 在PR中产生抗性影响蛋白质-配体相互作用和蛋白质构象。高效 与多位点PR突变体的结合要求化合物能够与保守的结构特征结合， 固有地是柔性的，以非活性构象捕获PR，和/或利用诱导的适合构象变化。 基于片段的筛选是一种新颖的药物设计方法，其采用小的化学结构库， 不同的小分子（约500种化合物，MW约150），以识别弱但特异性的结合剂。抑制剂设计 需要随后的加工、分析和“片段”的连接。一个非常大的化学空间区域， 筛选，并且抑制剂（药物）衍生自各自具有高配体效率的较小片段。与 随着高通量X射线晶体学的发展，现在可以筛选许多小分子， 即片段，用于蛋白质晶体中的特异性结合位点。我们已经开发出良好的衍射晶体， 无配体野生型PR和对广谱抑制剂TL-3具有抗性的6个残基突变体（6X）。六 突变通常发生在耐药HIV株的临床分离株中。基于对68种化合物的筛选 我们已经鉴定了三种结合在PR二聚体中新位点的小分子片段。的 实验策略包含三个目标。(1)完成针对野生型PR的片段筛选，以及 用含有来自初始筛选的片段（F1）的共晶重复筛选。识别第二个 位点结合物（F2）将利用PR中诱导的适合构象变化，并提供支架， 开发连接的高亲和力抑制剂，即F1-F2。(2)针对6X PR重复两阶段碎片筛选。 与6X突变体结合的片段可能不同（f1），而在第二阶段结合的片段（f2）可能不同。 由于蛋白质的构象反应改变而不同。衍生片段文库，即FT，F2 ′，f1 ′， 将筛选在项目3中通过计算机模拟鉴定和/或合成的f2 '，以改善结合。结果将 提供了PR的化学亲和力的经验图片，以及它是如何被电阻改变的， 突变。(3)开发并测定一种有效的双位点结合抑制剂。综合的迭代设计周期 - 分析-结构-合成将在计划项目的范围内使用。结构 基于目的1和2中的结果，合成了与HIV PR结合的连接片段衍生物， 项目3，将确定。目的是发现具有新模式的高亲和力抑制剂，例如F1 ′-f2 ′ 的结合和紧密结合多位点的能力，耐药PR突变体。HIV PR抑制剂 以这种方式开发的药物应该有利于解决艾滋病毒耐药性的演变。

We will apply fragment based screening to identify small molecule binding sites in HIV protease (PR) leading to the design of potent inhibitors that maintain efficacy against multi-site PR mutants. Mutations giving rise to resistance in PR affect both protein-ligand interactions and protein conformation. Efficient binding to multi-site PR mutants requires that the compound be able to bind to conserved structural features, be inherently flexible, trap PR in an inactive conformation, and/or exploit induced fit conformational changes. Fragment based screening is a novel approach to drug design that employs a small library of chemically diverse small molecules (~500 compounds, MW ~150) to identify weak but specific binders. Inhibitor design entails subsequent elaboration, assay, and linkage of the 'fragments'. A very large area of chemical space is screened, and the inhibitor (drug) is derived from smaller fragments each having high ligand efficiency. With the development of high throughput X-ray crystallography, it is now feasible to screen many small molecules, i.e. fragments, for specific binding sites in protein crystals. We have developed good diffracting crystals of ligand free wild-type PR and a 6-residue mutant (6X) resistant to the broad spectrum inhibitor, TL-3. The six mutations commonly occur in clinical isolates of resistant HIV strains. Based on a screen of 68 compounds we have already identified three small molecule fragments that bind at novel sites in the PR dimer. The experimental strategy entails three Aims. (1) Complete the fragment screen against wild-type PR, and repeat the screen with cocrystals containing fragments (F1) from the initial screen. Identification of second site binders (F2) will exploit induced fit conformational changes in PR, and provide a scaffold on which to develop linked high affinity inhibitors, i.e. F1-F2. (2) Repeat the two-stage fragment screen against 6X PR. Fragments binding to the 6X mutant may differ (f1), while fragments binding in the second stage (f2) may differ due to altered conformational response of the protein. Derivative fragment libraries, i.e. FT, F2', f1', f2', identified in silico and/or synthesized in Project 3, will be screened for improved binding. The results will provide an empirical picture of the chemical affinity of PR, and how it is altered by resistance causing mutations. (3) Develop and assay a potent dual site binding inhibitor. An iterative design cycle of synthesis - assay - structure - synthesis will be employed within the context of the Program Project. Structures of linked fragment derivatives bound to HIV PR, based on the results in Aims 1 and 2, and synthesized in Project 3, will be determined. The goal is to discover high affinity inhibitors, e.g. F1'-f2', having novel modes of binding and the ability to bind tightly to multi-site, drug resistant PR mutants. Inhibitors to HIV PR developed in this fashion should be beneficial in addressing the evolution of drug resistance in HIV.