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

酶在活细胞内催化化学反应，非常迅速和精确。治疗（或杀死）生物体的一种方法是使用一种被称为酶抑制剂的小分子来减缓或停止酶的活性。酶抑制剂必须具有很强的选择性（所以它们只影响目标酶）并且它们必须与酶紧密结合，这样它们就不会被酶的正常底物推掉。通过了解如何抑制一种酶，我们通常可以了解这种酶是如何工作的。酶抑制剂的设计和合成也是制药业的核心。许多药物是酶抑制剂，如青霉素、高血压药等。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，工业界用来寻找新的酶抑制剂的主要方法是非常快速地测试数以百万计的化合物，看看是否能找到有希望的化合物。然后将其修改为更有效，并具有成为药物所需的特性。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“基于片段”的方法依赖于寻找片段的方法。有几种方法可以做到这一点，我们建议使用其中一些方法，其中一些方法已经建立起来，而另一些方法则更新颖。这些方法使用的技术包括核磁共振光谱、质谱和量热法。除了虚拟筛选之外，所有这些都依赖于显示片段与酶结合的某种方式，虚拟筛选使用计算方法来尝试识别看起来应该与酶结合的片段。一旦我们确定了与目标酶结合的片段，我们就会尝试将它们培养成更大的分子。一种通用的方法是混合很多片段，让它们以各种可能的组合相互反应（称为动态组合化学），然后让酶选择最有效地结合它的那一个。这可能是酶作为一个模板，把两个单独结合的片段结合在一起。如果使用大量的酶，它可能会导致更多的关键化合物与它结合形成，使这种化合物被确定为主要产物。确定最佳结合化合物的一种更优雅的方法是在酶晶体存在的情况下形成化合物混合物，这种方法使用的有价值的酶较少。如果一个化合物与酶结合，它的身份可以通过解与酶结合的分子的晶体结构来推断。我们将通过尝试制造两种参与制造维生素B5（泛酸）的酶的抑制剂来发展这些想法。有证据表明，这些酶的抑制剂可能对结核病有用。另一个以这些酶为目标的原因是它们结合NADPH和ATP分子，这些分子也被其他酶所使用，所以我们可能会学到一些有用的关于如何抑制这些酶的一般信息。

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.