A year ago I co-authored a J. Med. Chem. paper that proposed a protein-bound conformation for discodermolide using a combination of NMR data and molecular modeling techniques. We followed up with a paper published last week in JACS in which we applied similar techniques to dictyostatin. In a nutshell, the two studies revealed surprising and unexpected dissimilarity in the solution and protein-bound 3D conformations of the molecules; similarity which is belied by their superficial 2D structures. While dictyostatin presents a diverse family of conformations, discodermolide sustains a remarkably constant conformation in very diverse environments (solid-state, solution, and in the protein binding site) that is enforced primarily by steric factors.
I would like to describe the work in the latest paper separately, but for now I am intrigued by another aspect of the problem. In both cases we proposed protein-bound conformations of two medicinally relevant molecules, but in both cases our conformations were not unique. In case of dictyostatin there is at least one alternative proposed conformation while in case of discodermolide there are no less than two. Of course we think that our proposed conformation better satisfies the data (otherwise we wouldn't have published the papers!), but the fact is that we are now presented with a puzzle. Which of the proposed conformations is correct and what technique would best resolve the quandary? The answer is unambiguous: x-ray crystallography on dictyostatin and discodermolide bound to tubulin should tell us what the correct conformation is.
Max Perutz once said that one of the most attractive qualities of science is that there is usually only one right answer, unlike politics where the answer depends on the viewpoint. I think this example illustrates that quality. The question is well-defined. We now have several competing proposals for the protein-bound conformations of two important molecular targets, but we know that there must be only one bound conformation in the solid-state, one right answer. Which conformation among these is it? Or is it a totally different one which has slipped through the cracks? The question is important not only because it would reveal the mode of action of a potentially novel class of anticancer drugs, but also because it could be very useful to organic chemists who could then modify the structures of the drugs based on their bound conformations to improve their potency and other properties.
In case of discodermolide, one molecule, three proposed conformations. But only one true conformation to rule them all. Which is it? In one sense the gauntlet has been thrown in front of crystallographers and the goal should be tantalizing for them, especially because there is a single right answer. The task will undoubtedly be difficult. Until now only the tubulin-binding drug taxol has succumbed to x-ray crystallography while the drug epothilone has lent itself to electron diffraction. Both dictyostatin and discodermolide are flexible molecules that won't yield to protein co-crystallization easily. And yet the solution would almost certainly result in publication in a top journal and new directions for synthetic chemists. Most importantly, it would be the definitive validation of a scientific puzzle that is currently unresolved.
But this train of thought brings to my mind another idea. Wouldn't it be great if we could have an exclusive website where experimentalists post results that theorists have to explain and theorists post results that experimentalists have to validate? The interplay between theory and experiment has of course been the bedrock of science since antiquity. But all too often, the right kind of puzzle is not clearly communicated by one group to another. Sure, if you work in a particular field, you will probably be up to speed on the literature in your field. But the sheer deluge of information ensures occasional omission, and sometimes you may also be interested in potential challenges from other areas which cannot be easily communicated to you. For instance, the dictyostatin/discodermolide puzzle may be interesting to scientists who don't have anything to do with tubulin but who are simply eager to test a new structure determination method that can be applied to such complicated molecules. As we all know, solutions to scientific puzzles can emerge from unexpected corners, and scientists sometimes may find surprises from other fields that pique their curiosity. For example, the spectacular harnessing of physics-based methods in chemistry and biology is well-known.
Yet scientists in one field cannot possibly keep track of all other fields whose developments may be attractive to them. For instance a physicist who may be developing a promising new electron diffraction technique, potentially applicable to tubulin and discodermolide, is usually not going to be aware of literature in this area. In such cases, it would be tremendously useful to have a website whose express purpose is to serve as a bridge between theorists and experimentalists. The website would be divided into the traditional fields of science along with interdisciplinary sections. Every week, a theorist or experimentalist would pose a puzzle from his or her field whose unambiguous solution he or she believes would be amenable to experimental techniques. The puzzle would be tagged with the names of all possible fields to which it could be relevant. People could vote up or down a problem which they find particularly enticing and tractable. Experimentalists from different disciplines can then take a look at the problem. The right answer could come from left field, from quarters which were completely unexpected for the scientist who posed the question. There would still be some querying that would be necessary, but the specific nature of the website would necessitate far less wading through literature from other fields than what's usually required. Similarly, experimentalists could post curious, unexplained results that would tickle theorists' grey cells.
The website could perhaps be called "The Thexperiment Cafe" or something less obnoxious. It would be a place where theorists and experimentalists rendezvous and challenge each other with specific puzzles. It could bypass the usual exhaustive literature searching and serve as a rapid delivery vehicle for problems whose solutions are unambiguous (or even ambiguous!) and which could benefit members from each camp. Experimentalists and theorists could be one big, happy family. And science will always win.
"there is usually only one right answer", but one must keep in mind that you might have asked the wrong question, or that the "one answer" might the same in practice as multiple answers.
ReplyDeleteFor example, you make the implicit assumption that the structure & binding site in the crystal form is the relevant binding site for the anticancer activity. This is usually the case, but not always, so the "one right answer" to the question "where does it bind in the crystal" isn't necessarily the "one right answer" to the question "where does it bind for therapeutic effectiveness". (Indeed, there's even the implicit assumption that it's the binding to tubulin that's actually causing the anticancer activity, as opposed to some secondary effect.)
The other issue is the potentially erroneous assumption that there's only one binding site. There could be multiple binding modes all sampled in solution in different proportions. You may get a single binding mode in protein crystallography, but seeing only one, low energy mode at 77 K doesn't mean that there aren't other, significantly populated states at 300 K. The answer to "where does it bind" could be a list of multiple sites. (Again, the "active" mode doesn't have to be the low energy mode.)
... isn't science fun?
RM: Good points. Interestingly, the question about multiple binding modes is addressed in the paper so you might want to take a look.
ReplyDeleteIn case of tubulin-binding agents I think there's a fair amount of confidence and a vast amount of literature about the mode of action and the resulting effect. As you mentioned yourself, the crystal structure is usually close to the bioactive structure, unless there is evidence to the contrary. We are also quite aware that the active conformation is not the low-energy one; in fact in our past studies that has never been the case. In case of disctyostatin too it was a minor solution conformation (and therefore a relatively high-energy one) that turned out to be the bioactive conformation.
In any case, I agree that asking the right question is important. But given all these caveats, a website for exchange between theorists and experimentalists could be fruitful even if the answers are ambiguous. The idea was really about saving time and paring down search space.