Evolving Libraries of Bivalent Compounds

描述（由申请人提供）：制备6个文库，每个文库至少15种化合物，使得文库中的每种化合物以1 - 2 g的量存在。 库中的化合物都被设计成半转模拟物，它们的侧链药效团将被设计成对应于在蛋白质-蛋白质相互作用的热点处最常见的天然氨基酸。 我们已经开发出一种方法，可以将单价化合物连接成二价分子。该方法不需要偶联剂，适用于具有未保护侧链官能团的化合物。二价分子库可以简单地通过将单价分子与碳酸钾混合（微量移液管）溶液而由单价分子形成。因此，15个单价化合物的一个文库可以组装成105个二价化合物（总文库大小= 105 + 15 = 120）。然而，可以制备的二价化合物的数量随着单价化合物的数量（n）的增加而急剧增加{二价化合物的数量= n（n-l）/2}。90个（对应于提出的6组15个化合物）单价结构单元的库可以产生4005个二价化合物。 从逻辑上讲，很难制造组装远超过4，000种二价化合物所需的单价化合物的量;表征问题变得非常耗时，并且不容易保存这么多样品的库存。因此，我们提出了“进化库”的概念，以保持数量可控（1,000 - 3,000），同时仍然探索多样性空间的有利区域。 这是基于形成每组约100 - 200个的二价化合物，选择导致二价命中的单价化合物，添加这些化合物的类似物以探索结构活性关系，以及从先导单价化合物、其类似物和一组新的单价化合物形成二价化合物的第二文库。 产生的所有二价化合物将被荧光标记。这将使得通过直接结合测定筛选文库成为可能;这些测定与通常用于蛋白质-蛋白质相互作用靶标的竞争性结合测定相比具有相当大的优势。首先，不需要标记的蛋白质。这一点很重要，因为许多标记的蛋白质是如此昂贵，以至于高通量筛选变得不可能。其次，可以检测到弱结合剂，因为它们不与天然配体竞争。 这种方法的另一个主要优点是标记的化合物是从可能用作药理学探针的测定中选择的。 这种方法可以很容易地适应包括其他类型的标记，甚至第三个小分子。 二价分子是有价值的，因为它们可以在蛋白质-蛋白质界面结合两个热点。提出了优化将两个小分子连接在一起以实现这一目标所需的接头尺寸的策略。随着二价化合物库的形成，这些信息将在不需要额外努力的情况下出现。

DESCRIPTION (provided by applicant): Six libraries of at least 15 compounds each will be prepared such that every compound in the library is present in 1 -2 g amounts. The compounds in the library are all designed to be ¿-turn mimics, and their side chain pharmacophores will be designed to correspond to the natural amino acids that are found most frequently at hot spots for protein-protein interactions. We have developed a method that allows monovalent compounds to be joined to give bivalent molecules. This method does not require coupling agents and is applicable to compounds with unprotected side chain functional groups. Libraries of bivalent molecules can be formed from monovalent ones simply by mixing (micropipette) solutions of the two with potassium carbonate. Thus one library of 15 monovalent compounds could be assembled into 105 bivalent ones (total library size = 105 + 15 = 120). However, the number of bivalent compounds that can be made increases steeply as the number of monovalent compounds (n) increases {number of bivalent compounds = n(n - l)/2}. A library of 90 (corresponding to the 6 sets of 15 compounds proposed) monovalent building blocks could give 4005 bivalent compounds. Logistically, it is difficult to make the amounts of monovalent compounds required to assemble much more than 4,000 bivalent compounds; characterization issues become prohibitively time consuming and inventories of this many samples are not easy to keep. Consequently, we propose the concept of "evolving libraries" to keep the numbers manageable (1,000 - 3,000) while still exploring favorable regions of diversity space. This is based on formation of the bivalent compounds in sets of about 100 - 200 each, selection of the monovalent compounds that led to bivalent hits, addition of analogs of these to explore structure activity relationships, and formation of a second library of bivalent compounds from the lead monovalent compounds, their analogs, and a new set of monovalent compounds. All the bivalent compounds produced will be fluorescently labeled. This will make it possible to screen the libraries via direct binding assays; these have considerable advantages over the competitive binding assays that are often used for protein-protein interaction targets. First, labeled proteins are not required. This is important because many labeled proteins are so expensive that high throughput screens become impossible. Second, weak binders can be detected because they are not in competition with the native ligand. Another major advantage of this approach is that labeled compounds are selected from the assays that might be used as pharmacological probes. The approach could easily be adapted to include other types of label, or even a third small molecule. Bivalent molecules are valuable because they can bind two hot spots at a protein-protein interface. Strategies are proposed to optimize the linker dimensions required to join two small molecules together to achieve this. This information will emerge with little extra effort as the libraries of bivalent compounds are formed.