Protein-protein interactions: Can't live without 'em, can't easily drug 'em

The many varieties of protein-protein interactions

Here's a pretty good survey of efforts to classify and drug protein-protein interaction (PPI) targets by a group from Cambridge University in Nature Review Drug Discovery. Most drug developers have been aware of the looming mountain of these ubiquitous interactions - there's at least 300,000 of them at last count and most likely many more - and have been trying to attack them in one way or another for more than a decade. There's also no doubt that many PPIs are involved in crucial ways in disease like cancer and inflammation. By any token PPIs are important.

As the review indicates though, attacking these interactions has been daunting to say the least. They present several challenges that seem to ask for both new scientific and institutional models of strategy. From the scientific standpoint PPIs present a nightmarish panoply of difficulties: proteins that change conformation when they comes together, water molecules that may or may not be involved in key interactions, the universal challenges of designing 'beyond rule of 5' drugs for such targets and the challenges of developing highly sensitive new biophysical techniques to detect ligand binding to begin with.

Consider protein flexibility, a factor which often is the nemesis of even 'regular', non PPI projects. Protein flexibility not only make crystallization hard and its results dicey, but it decidedly thwarts computational predictions, especially if the conformational changes are large. PPIs however regularly present cases in which the conformations of the unbound proteins are different from the bound ones, so at the very minimum you need crystal or NMR structures of both bound and unbound forms. This is even harder if one of the partners is a peptide, in which case it's likely going to undergo even more conformational changes when it binds to a protein target. The hardest case is two peptides such as Myc and Max, both of which are unstable and disordered by themselves, which stabilize only when they bind to each other. That's quite certainly an interaction forged in the fires of Mount Doom; good luck getting any kind of concrete structural data on that gem.

The screening challenges involved in studying PPIs are as if not harder than the structural challenges. NMR is probably the only technique that can reliably detect weak binding between proteins and ligands in as much of a 'natural' state as possible. although it presents its own difficulties like protein size and other technical challenges. SPR and FRET can help, but you are really testing the limits of these binding assays in such cases. Finding reliable, more or less universal methods that combine high throughput with good sensitivity has been an elusive goal, and most data we have on this front is anecdotal.

Finally, the medicinal chemistry challenges can never be underestimated, and that's where the institutional challenges also come in. In many PPI projects you are basically trying to approximate a giant block of protein or peptide by a small molecule with good pharmacological properties. In most cases this small molecule is likely going to fall outside the not-so-hallowed-anymore Lipinski Rule of 5 space. I should know something about this since I have worked in the area myself and can attest to the challenges of modeling large, greasy, floppy compounds. These molecules can cause havoc on multiple levels: by aggregating among themselves, by sticking non-specifically to other proteins and by causing weird conformational changes that can only be 'predicted' when observed (remember that old adage about the moon...). Not only do you need to discover new ways of discovering large PPI inhibitors that can make it across cell membranes and are not chewed up the moment they get into the body, but you also need new management structures that encourage the exploration of such target space (especially in applications like CNS drug discovery: in oncology, as happens depressingly often, you can get away with almost anything). If there's one thing harder than science, it's psychology.

In spite of these hurdles which are reflected in the sparse number of bonafide drugs that have emerged from PPI campaigns, the review talks about a number of successful pre-clinical PPI projects involving new modalities like stapled peptides, macrocycles and fragment-based screening that at the very least shine light on the unique properties of two or more proteins coming together. One of the more promising strategies is to find an amino acid residue in one partner of a PPI that acts like an 'anchor' and provides binding energy. There have been some successful efforts to approximate this anchor residue with a small molecule, although it's worth remembering that nature designed the rest of the native protein or peptide for a reason. 

Another point from the review which seems to me like it should be highlighted is the importance of academia in discovering more novel features of PPIs and their inhibitors. In a new field even the basics are not as well known, it seems logical to devote as many efforts to discovering the science as to applying it. At the very least we can bang our collective heads against the giant PPI wall and hope for some cracks to emerge.

Myths about disrupting protein-protein interactions with small molecules

The first era of medicinal chemistry was finding small molecules to target proteins. It's still going strong and will continue to do so. The second era that promises a treasure trove at least in principle is finding small molecules for disrupting protein-protein (PP) interactions. So many important processes in our body are regulated by these crucial interactions that finding small molecules to modulate them could promise a bonanza of new therapies.

But disrupting PP interactions is fundamentally different from designing a small molecule to bind to a (mostly) well-defined active site on a protein. PP interaction surfaces are rather flat and expansive and depend on subtle interactions between amino acids that add up to provide substantial binding affinity. Designing a small molecule to disrupt such interactions is somewhat like disrupting the sliding of one door hinge against another by lodging a grain of sand between the two. However, the picture has been simplified somewhat in recent years by the identification of "hotspots"- key amino acid epitopes that provide the bulk of binding interaction. If one could design a small molecule that could provide this major contribution to the free energy of binding, one could have an effective drug that could target PP interactions. After all, a grain of sand can indeed inhibit hinge sliding if it's placed in the right position.

One of the big players in investigating small molecule PP interaction agents has been Jim Wells, formerly at Genentech and Sunesis and now at UCSF. He has a nice Nature review that traces recent successful examples of small molecule PP interaction antagonists. Wells considers six or seven successful stories involving important proteins playing key roles in health and disease. For example, disrupting the inhibition of the pro-apoptotic BAD and BAK by the anti-apoptotic Bcl-2 and Bcl-xl, disrupting the binding of interleukin IL-2 to its receptor, and disrupting the binding of HDM2 to the tumour suppressor p53.

But more importantly, Wells then address some widely held myths about PP interactions that seem to drive pessimism in the field. They are worth taking a look at:

Myth 1: It's very difficult to find a small molecule that would lodge between the rather disordered flat surfaces of two proteins.

While this is true, in practice as is exemplified by almost all the examples, the protein surface does not remain flat when a molecule binds to it. Loops and side chain adapt and form shallow pockets and dents to which the molecule can bind. The fallacy in believing the myth is to assume that small-molecule protein surface binding is a rigid body interaction. It's not, and there's a strong element of induced fit in the process. This is probably the single most important thing to keep in mind, that small molecules will form their own small pockets and bind well to initially flat protein surfaces.

Myth 2: Small molecules that disrupt PP interactions are too large to be drugs

This is part of the partly substantiated myth that large molecules usually don't become drugs, because of many factors including ROF problems. But many molecules disrupting PP interactions cited in the review are about 500-700 Da, perhaps a little large but not intractable as drugs. The authors also calculated the ligand efficiency which is the free energy of binding per non-hydrogen (heavy) atom for the ligands, and found that it was comparable to that of kinase or protease inhibitors. Clearly with sound med chem efforts, it won't be too difficult to have such drugs. Interestingly, since the molecules occupy about half the binding site that the parts of the native protein partner do, their ligand efficiency is almost twice.

Myth 3: Small molecules disrupting PP interactions won't be potent

Just not true. Almost all the molecules found in the cited examples had mid to low nanomolar Ki values, almost as good as the binding constants for the partner proteins.

Myth 4: Screening would not help find novel small molecule PP modulators

Again, not true. Most of the cited molecules were found by HTS. Interestingly, there may be even more wealth in HTS than we have now. As the authors explain, HTS hits are fundamentally going to be limited by chemotypes present in the libarries. After all we can do only as well as chemical space in existing libraries. Existing libraries contain many molecules targeted against kinases, GPCRs and other well-known targets for which common privileged structures have been deduced. But because of the diversity of PP interactions, it is improbable that common scaffolds will exist for disrupting them, and libraries will have to contain novel scaffolds to get better hits. Given this fact, it's impressive and encouraging that the existing libraries could come up with such potent structures for disrupting a few PP interactions. Remarkably, even with such different scaffolds, the ligand efficiency remains more or less constant for the cases studied.

Clearly the field of small-molecule-PP interactions is alive and kicking. In the next few years, hopefully computational, screening and NMR approaches will converge to discover novel agents for these important processes.

Wells, J.A., McClendon, C.L. (2007). Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature, 450(7172), 1001-1009. DOI: 10.1038/nature06526