Development of fragment-based approaches to build chemical tools for biology

酶催化活细胞内的化学反应，非常迅速和非常精确。治愈(或杀死)生物体的一种方法是通过使用一种名为酶抑制剂的小分子来减缓或停止酶的速度。酶抑制剂必须非常有选择性(因此它们只影响目标酶)，并且它们必须非常紧密地结合到该酶上，这样它们就不会被酶的正常底物推开。通过了解如何抑制酶，我们通常可以了解酶是如何工作的。酶抑制剂的设计和合成也是制药业的核心。许多药物都是酶抑制剂，如青霉素、降压药等。因此，发现新的酶抑制剂的方法非常重要也就不足为奇了。在过去的十年里，工业界寻找新的酶抑制剂的主要方法是非常快地测试数百万种化合物，看看是否能找到有前景的化合物。然后对它们进行改造，使其更有效，并具有作为药物所需的特性。然而，现在人们对发现酶抑制剂的一种完全不同的方法越来越感兴趣。这包括找到一个与酶结合的相当小的分子(称为片段)，然后将其作为锚点来构建更大、更有效的抑制剂。这些“基于碎片”的方法依赖于寻找碎片的方法。有几种方法可以做到这一点，我们建议使用其中的一些方法，其中一些是成熟的，另一些是更新颖的。这些方法使用的技术包括核磁共振光谱、质谱学和量热法。所有这些都依赖于某种方式来显示片段与酶的结合，除了虚拟筛选，在虚拟筛选中，使用计算方法来尝试和识别看起来应该与酶结合的片段。一旦我们确定了与我们的目标酶结合的片段，我们就会尝试将它们培养成更大的分子。一种常见的方法是混合大量的片段，让它们在每一种可能的组合中相互反应(称为动态组合化学)，然后让酶选择最有效地与其结合的片段。这种酶可能起到了模板的作用，将分别与其结合的两个片段结合在一起。如果使用大量的酶，可能会导致更多与其结合的关键化合物的形成，从而使这种化合物被确定为主要产品。识别最好的结合化合物的一种更优雅的方法是在酶的晶体存在的情况下形成化合物的混合物，这种结合化合物使用的有价值的酶更少。如果一种化合物与酶结合，则可以通过解算与其结合的分子的酶的晶体结构来推断其同一性。我们将通过尝试制造两种酶的抑制剂来发展这些想法，这两种酶与制造维生素B5(泛酸)有关。有证据表明，这些酶的抑制剂可能对防治结核病有用。针对这些酶的另一个原因是它们结合了称为NADPH和ATP的分子，这两种分子也被其他酶使用，所以我们可能会了解一些关于如何抑制这些其他酶的有用的一般信息。

Enzymes catalyse chemical reactions within living cells, very quickly and very precisely. One way to cure (or kill) an organism is to slow down or stop an enzyme by using a small molecule called an enzyme inhibitor. Enzyme inhibitors have to be very selective (so they only affect the target enzyme) and they have to bind very tightly to that enzyme, so that they are not pushed off by the enzyme's normal substrate. By understanding how to inhibit an enzyme we can often learn something about how the enzyme works. The design and synthesis of enzyme inhibitors is also central to the pharmaceutical industry. Many medicines are enzyme inhibitors e.g. penicillin, pills for high blood pressure etc. So it is not surprising that ways to discover new enzyme inhibitors are very important. In the last ten years the main approach industry has used to find new enzyme inhibitors is to test millions of compounds very quickly to see if promising compounds can be found. These are then modified to be more potent and have the properties they need to be a medicine. However there is now growing interest in a completely different approach to discovering enzyme inhibitors. This involves finding a quite small molecule (called a fragment) that binds to the enzyme, and then using this as an anchor point to build up bigger more potent inhibitors. These 'fragment-based' approaches depend on methods of finding fragments. There are several ways to this, and we propose to use a number of these, some of which are well established and others which are more novel. The methods use techniques including NMR spectroscopy, mass spectrometry, and calorimetry. All rely on some way of showing the fragment binds to the enzyme, with the exception of virtual screening, where computational approaches are used to try and identify fragments that look like they should bind to the enzyme. Once we have identified fragments that bind to our target enzymes we will try and grow them into bigger molecules. One general way to do this is to mix a lot of fragments and let them react with each other in every possible combination (called dynamic combinatorial chemistry), and let the enzyme select out the one that binds to it most efficiently. It may be that the enzyme acts as a template to bring together two fragments that bind to it separately. If a lot of enzyme is used, it may lead to more of the key compound that binds to it being formed, allowing this compound to be identified as the major product. A more elegant way to identify the best binding compound, which uses less of the valuable enzyme, is to form the mixture of compounds in the presence of a crystal of the enzyme. If a compound binds to the enzyme, its identity can be deduced by solving the crystal structure of the enzyme with the molecule bound to it. We will devlop these ideas by trying to make inhibitors of two enzymes involved in making vitamin B5 (pantothenate). There is evidence to suggest that inhibitors of these enzymes may be useful against tuberculosis. Another reason to target these enzymes is that they bind molecules called NADPH and ATP, which are also used by other enzymes, so we might learn some useful general information about how to inhibit these other enzymes.