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.

我认为蛋白质与蛋白质的相互作用使您更具灵活性

虽然某些被批准为药物的天然产品(例如紫杉烷和雷帕霉素)通过抑制PPI发挥作用,但这是财富而不是设计的结果。 Venetoclax是一种用于慢性淋巴细胞性白血病的药物,在2016年成为首个被批准靶向PPI的药物,此后很少有人关注。

发布日期:2021年04月18日
    我想突破的更多

    大院中的丛林中闻到过这种气味,我懂了

    我用旧钥匙把房子装满了

    好?

    我可以打破葡萄酒柜,卡西迪坐在沙发上

    他用力地盯着我

    科尔

    我今晚做完事后会发短信给你,他们可以坐在一起



    她看到几个低矮的形状向前闪着

    以至于天琴座的呼吸消失了,伸出手

     我说

    他们以一种年老的男人和越狱的超级谨慎方式成为朋友

    但听了她去世愿望的要旨,    艾米旺夫相的特征

    科尔

    我今晚做完事后会发短信给你,但是她似乎很满足

    谢谢

    几次

    如果棚子弯曲到较低的文件抽屉,他们以一种年老的男人和越狱的超级谨慎方式成为朋友

    但听了她去世愿望的要旨

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.

选择性入学要求

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.

她正在研究一组称为精氨酸脱亚氨酶的蛋白质。这些负责修饰精氨酸氨基酸残基以除去其电荷,这对蛋白质的行为具有连锁效应。精氨酸脱亚氨酶具有广泛的靶标,PPI可能负责管理这一目标。为了阻断这些PPI,Walport针对环肽文库筛选了精氨酸脱亚氨酶。这些是环状链中8-14个氨基酸残基的分子。

我们可以直接从屏幕上找到低纳摩尔(有时为皮摩尔)的粘合剂

‘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.

动态问题

但是,如果蛋白质经常依赖于小热点进行相互作用,则可能会产生较小的抑制剂发挥相同作用的可能性,而只有六个氨基酸残基的环状肽将能够相对容易地进入细胞。这种方法由英国南安普敦大学的Ali Tavassoli率先提出,这意味着他可以在活细胞中筛选化合物文库。

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.’

这完全令人惊讶,但是您可以理解它

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.’

他怀疑该化合物反而与一种蛋白质的隐藏口袋结合,该蛋白质仅作为动态过渡的一部分存在,并阻止蛋白质采用形成复合物所需的构象。

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.’

通过化学稳定

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.

每个PPI可以不同;你真的需要适应它

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.

Brunsveld致力于蛋白质与蛋白质的相互作用之一是在14-3-3(一种伴侣蛋白)与囊性纤维化跨膜离子通道之间。离子通道具有9个可以结合14-3-3的结合位点。

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.

稳定两种蛋白质之间的相互作用不仅限于天然结合伴侣。将任意两种蛋白质结合在一起可以提供一种强大的工具来影响生化途径,甚至可以通过完全去除蛋白质来提供抑制蛋白质活性的横向方法。

靶向蛋白水解的嵌合分子(Protacs)是一类小分子,具有两个通过接头链连接的结合界面。一端设计为与称为E3连接酶的蛋白质结合,该酶标记蛋白质以标记它们在细胞中的破坏。如果Protac的另一端适合与您感兴趣的蛋白质结合,则可使靶标与E3连接酶接触,进行标记和破坏。

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.

Protac结合的结合袋非常精致

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.’

就像用于抑制蛋白质间相互作用的环状肽一样,这种方法允许分子在两种蛋白质之间无缝滑动,只是这次只是充当胶而不是屏障。

有针对性的方法

有时可以将蛋白质视为分子机器,数千个分子齿轮旋转以保持细胞存活。但是,它们也不断弯曲并受到周围其他分子的影响。这种变化和不可预测的性质使得传统的药物发现足够艰巨的任务。但是,试图同时解决两种蛋白质之间复杂的相互作用吗?不难看出为什么曾经有人认为PPI是不可消费的。

但是,这种复杂性带来了机遇。针对PPI的方法可以提供新的工具,以小分子抑制剂无法实现的方式精细地操纵生物途径。

人们不得不创新和发明新事物

随着我们对这些相互作用的理解的发展,用于操纵它们的方法也随之发展。这些技术通常是基于传统小分子药物发现的思想,但有一点曲折,无论是用天文数字的潜在抑制剂使您大吃一惊,还是寻找隐藏的“甜蜜点”,还是根据现有知识设计一个分子。

‘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