根据Ian Le Guillou的说法，一旦被认为不可药物，化学家就开始掌握蛋白质之间的相互作用
蛋白质很少单独起作用。从紧密结合的复合物到经过的短暂接触，蛋白质之间的相互作用在细胞功能中起着核心作用。 据一个估计，超过65万种蛋白质间相互作用 在人体中。这种压倒性的相互作用代表了新治疗药物几乎未开发的靶标资源。
数十年来，这些互动were considered to be ‘undruggable’。蛋白质与蛋白质相互作用（PPI）的高分辨率结构在1980年代和90年代表明，界面是大而平坦的无特征表面。这与典型的药物靶标（例如酶和受体）中的小口袋完全不同，小分子可以轻易结合。
A typical enzyme binding to its substrate is often taught at school as being like a lock and key. To find a drug to block this interaction, we simply need a new molecule that is sufficiently ‘key-shaped’ to jam the lock. By contrast, our knowledge of PPIs made them seem more like handshakes – two flat surfaces coming together firmly. But our understanding has improved to reveal that it is more complicated than that. There is a secret handshake feel to these interactions, with each side responding to the other and hidden points of contact that are of great significance.
通过在蛋白质-蛋白质界面上突变成千上万的单个氨基酸，生物化学家发现一小部分残基主要负责结合. These ‘hot spots’ were much more relevant to the strength of the interaction than the size of the interaction surface. This improved understanding made PPIs more tractable as targets, and the need for new, effective drugs certainly made them attractive. However, this is still easier said than done.
Although PPIs may no longer be viewed as undruggable, new approaches are needed to deal with this new class of targets. Inhibiting PPIs will need different libraries, assays and perspectives. Given the variety and complexity of PPIs, a range of techniques may be needed to inhibit different targets. As new approaches are developed, many researchers are turning to PPIs to address problems and therapeutic areas that were not previously manageable with traditional small molecule inhibitors.
Most enzymes act on multiple targets in the cell and rely on their protein binding partners to be selective. An inhibitor that blocks the enzyme’s active site would prevent its activity across all of its targets, potentially causing unintended side effects. ‘I think that PPIs give you a bit more flexibility. You can target them to block subsets of enzymes’ activities in a way that’s pretty challenging to do with traditional small molecule enzyme inhibitors,’ says Louise Walport from the Crick Institute in London, UK.
‘I think that PPIs is where peptides have their particular niche because they’re just that bit bigger than small molecules, so they can pick up more small interactions along these featureless surfaces than you can with a small molecule,’ she says. By their cyclic nature there is less entropic cost when they bind to the protein and it helps to make them more stable against being broken down by the body.
由日本东京大学的Shiro Hiroaki Suga创建的RaPid系统可以产生1014 different cyclic peptides – more than the number of stars in the universe. The advantage of this approach is that Walport has been able to find ‘low nanomolar, sometimes picomolar, binders straight out of a screen with no modification, not having done any optimisation’.
The challenge, however, is that these molecules are typically too large to enter the cell, meaning that this approach can be used only for extracellular proteins – or a lot of work is needed to adapt the inhibitor to allow it to penetrate the membrane. One notable exception to this is the natural product and immunosuppressant cyclosporine. It is a cyclic peptide of 11 amino acids, but able to enter the cell. ‘No one really understands it. We’d love to be able to make a cyclosporine. It has this kind of flipping mechanism where it can have its hydrophobic face out for a while and then it goes through the membrane and then it flips round – it’s magic,’ says Walport.
One of the main advantages of this approach is to study the PPI in a natural environment, says Tavassoli. ‘With assays in vitro, the protein isn’t dynamic – it’s locked in a single state in biochemical buffer. So the hidden pockets and the dynamic nature of the protein, which is one of the core components of its being, is lost.’
塔瓦索利研究转录因子，即可以激活或抑制基因的蛋白质。通过使用转基因大肠杆菌，塔瓦索利（Tavassoli）可以检测是否存在阻止PPI抑制特定基因的化合物。在生命死亡分析中，大肠杆菌 将其置于含有抗生素的溶液中，PPI负责封闭该基因以产生抗生素抗性。如果大肠杆菌 存活，则必须表示该化合物已成功抑制PPI。
Through taking advantage of the natural flexibility of the proteins, even very small molecules can interrupt the large interaction surfaces in PPIs. Tavassoli has even found examples where two or three amino acids can block PPIs. One of these even had a 5000?2 相互作用表面–大约是二肽的100倍。
‘It’s completely surprising, but you can make sense of it,’ says Tavassoli. ‘The fact that we’ve got di-peptides, two amino acids, that disrupt this complex – that tells me that these things can’t be working by just getting in between the interacting proteins and disrupting them.’
The compound library that Tavassoli uses, known as Siclopps, generates 3.2 million different cyclic peptides. While the hits may bind a thousand times less tightly than the larger cyclic peptides from the RaPid library, Tavassoli is not too concerned by this. ‘If you want to compete with the substrate of an enzyme, you are going to have to get quite a lot of your inhibitor in there, or it’s going to have to be super-duper potent for it to be effective,’ he explains. ‘But, compare that to the amount of a given protein that’s present in the cell, which is several log-orders less. Potentially you’re going to have to get a lot less of your compound into the cell and equally it doesn’t have to be quite as potent.’
Protein–protein interactions can activate biological pathways as well as repress them, so it’s not always the case that we would want to inhibit an interaction. Luc Brunsveld from Eindhoven University in the Netherlands is using his background in supramolecular chemistry to devise new compounds that can stabilise protein–protein interactions. ‘Stabilisation of protein assemblies is much more like supramolecular thinking than inhibition. Inhibition is classical med chem where you make a molecule that binds to something. But for stabilisation, you talk about bringing multiple things together and the underlying mechanisms,’ he says.
Rather than using a large library of compounds, Brunsveld favours a more structure-based approach. Using the crystal structure of the protein complex, he is looking for opportunities to design a molecule that will stabilise the complex. ‘We want to see, if you can get two proteins together, do they form a novel composite binding pocket for small molecules? That is where the individual proteins don’t have a clear pocket but the coming together of them forms a new binding pocket,’ he says.
This approach starts with a very small molecule, or fragment, that binds very weakly, and adapting it based on where the fragment binds. There are several techniques that Brunsveld uses to study how they bind to the protein complex, such as soaking them into crystals of the protein complex and using x-ray crystallography to see where it binds or designing the fragments so that they react with the protein in a particular site to form a covalent disulfide bond. ‘For PPI stabilisation, you can’t say there’s a general mechanism; it depends very much on the type of proteins you look at,’ he says.
This complicated interaction raises questions about how the different binding sites are regulated in the cell and what is the impact of binding to one site over another. For investigating this type of complex, inhibition is unlikely to be successful, says Brunsveld. ‘We see in those multi-valence complexes that inhibition is a big challenge, because as soon as you inhibit one of the nine sites then the others will take over and you will hardly lose affinity [between the two proteins]. But if you specifically stabilise one of the nine binding sites, then that one interaction really becomes dominant and you get huge shifts in the stability of the complex.’
This means that if the different binding sites are important for different processes, then stabilising one will effectively inhibit the others. This provides a very different mechanism for altering biological pathways compared to inhibition. ‘Every PPI can be different; you really need to adapt to it and understand the underlying mechanisms that are acting there,’ says Brunsveld.
Protacs在20年前被首次提出, but better understanding of the dynamic nature of these complexes has helped researchers to design better compounds. Alessio Ciulli from the University of Dundee, UK, says that the field initially pictured these complexes being like dumbbells. ‘We thought that were these two heads and then a line in the middle, and so conceptually we didn’t think that the proteins were touching. But of course, the dumbbell is flexible – it can twist and turn. Once these proteins are brought into proximity, then they can form very tight interactions,’ he says.
这种紧密联系的蛋白质-蛋白质相互作用对于通常通常与彼此几乎没有关系的两种蛋白质而言似乎是违反直觉的。但是，它可以用于增加对特定蛋白质的选择性。 Ciulli的靶标之一是一种叫做BRD4的蛋白质，它与另外两种蛋白质BRD2和BRD3非常相似，因此很难用小分子抑制剂特异性地靶向。 Ciulli开发了一种Protac，它以相同的亲和力与BRD2，BRD3和BRD4结合，但是降解过程仅对BRD4具有高度选择性。
‘The reason is that the binding pocket where the [Protac] binds is exquisitely conserved. There’s no difference between the targets. In contrast, the surface around the binding pocket is much less conserved. That’s the region that forms the new protein–protein contacts with the ligase and that’s what gives us specificity,’ says Ciulli. ‘This has provided proof of concept that this is an added advantage of Protacs: that you can discriminate across highly conserved homologues of targets in ways that you can’t do simply with inhibitors.’
最近的研究表明BRD4特别与侵袭性前列腺癌有关。选择性靶向BRD4但不靶向类似蛋白的药物可能会在患者中产生较少的副作用。正如蛋白质是动态的一样，Protac中的接头链也是如此。 Ciulli能够确定三元Protac配合物的第一个晶体结构，并将其用于设计更类似于主动构象的循环形式. ‘This is the first demonstration of this idea of locking the Protac in a bioactive conformation by forming a macrocycle,’ he says. ‘We saw that the compound was extremely active. Interestingly, as a result of that, we lost a lot of binary binding [between the Protac and the target protein]. Despite that, it was as potent as the uncyclised one. So it clearly demonstrated that cyclisation had done something extremely favourable in the process.’
‘I think what really attracts me is the fact that your traditional approaches just haven’t been working against these targets,’ says Tavassoli. ‘And so people have had to innovate and invent new things, which if you just thought about them you would think that they wouldn’t be suitable, and yet it has taken this sort of outside the box thinking to drive the field forward.’
Ian Le Guillou是法国巴黎的科学作家