"The Thexperiment Cafe": Bridging theory and experiment?

Discodermolide and dictyostatin are complex, flexible molecules that bind to the protein tubulin and promote the assembly of microtubules during cell division. This mechanism, similar to that of the bestselling drug Taxol, derails the precise timing of cell division and kills cells by causing apoptosis or cell death. Since cancer is quintessentially a disease of aberrant cell division, both molecules have emerged as potentially promising anticancer agents. Discodermolide and dictyostatin are of special interest not only because of their extraordinary potency, but especially because they seem to retain that potency against cells which have become resistant to taxol.

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.

Constancy of the discodermolide hairpin motif

Our paper on the conformational analysis of discodermolide is now up on the ACS website. The following is a brief description of the work.

Discodermolide (DDM) is a well-known highly flexible polyketide that is the most potent microtubule polymerization agent known. In this capacity it functions very similar to taxol and the epothilones. However the binding mode of DDM will intimately depend on its conformations in solution.

To this end we have performed multiple force field conformational searches on DDM and the first surprising thing we noticed was that all four force fields located the same global minimum for the molecule in terms of geometry. This is surprising because, given the dissimilar parameterization criteria used in different force fields, minima obtained for flexible organic molecules are usually different for different force fields. Not only that, but all the minima closely superimposed on the x-ray structure of DDM which we call the "hairpin" motif. This is also surprising since the solid state structure of such a highly flexible molecule should not generally bear resemblance to a theoretically calculated global minimum.

Next, we used our NAMFIS methodology that combines parameters from conformational searches to coupling constants and interproton distances obtained from NMR data to determine DDM conformations in two solvents, water and DMSO. We were again surprised to see the x-ray/force field global minimum structure existing as a major component of the complex solution conformational ensemble. In many earlier studies, the x-ray structure has been located as a minor component so this too was unexpected.

However, this same structure has also been remarkably implicated as the bioactive conformation bound to tubulin by a series of elegant NMR experiments. To our knowledge, this is the first tubulin binder which has a single dominant preferred conformation in the solid-state, as a theoretical global minimum in multiple force field conformational searches, in solution as well as in the binding pocket of tubulin. In fact I personally don't know of any other molecule of this flexibility which exists as one dominant conformation in such extremely diverse environments; if this happened to every or even most molecules, drug discovery would suddenly become easier by an order of magnitude since all we would have to do to predict the binding mode of a drug would be to crystallize it or to look at its theoretical energy minima. To rationalize this very pronounced conformational preference of DDM, we analyze the energetics of three distributed synthons (methyl-hydroxy-methyl triads) in the molecule using molecular mechanics and quantum chemical methods; it seems that these three synthons modulate the conformational preferences of the molecule and essentially override other interactions with solvent, adjacent crystal entities, and amino acid elements in the protein.

Finally, we supplement this conformational analysis with a set of docking experiments which lead to a binding mode that is different from the earlier one postulated by NMR (as of now there is no x-ray structure of DDM bound to tubulin). We rationalize this binding mode in the light of SAR data for the molecule and describe why we prefer it to the previous one.

In summary then, DDM emerges as a unique molecule which seems to exist in one dominant conformation in highly dissimilar environments. The study also indicates the use of reinforcing synthons as modular elements to control conformation.

Jogalekar, A., Kriel, F., Shi, Q., Cornett, B., Cicero, D., & Snyder, J. (2009). The Discodermolide Hairpin Structure Flows from Conformationally Stable Modular Motifs Journal of Medicinal Chemistry DOI: 10.1021/jm9015284

Discodermolide unraveled?

Drugs affecting microtubule dynamics are familiar chemical players in med chem by now. First came Taxol, then the epothilones, then discodermolide, and the list continues with peluroside, eleutherobin, and dictyostatin to name a few of the better known entities.

Like it is for other drugs, one of the major questions asked about these molecules is how they bind to their target. Taxol and epothilone have been subjected to immense SAR and analog preparation by some of the hard hitters in the synthetic arena. Their binding conformations have been postulated with reasonable confidence. The common pharmacophore hypothesis, tempting but misleading and not true in this case, has been convincingly questioned. But for discodermolide, the binding conformation is not yet known. Now, groups from Spain and the UK have applied the "INPHARMA" NMR methodology to probe the interaction of disco with tubulin.

Admittedly, INPHARMA is a nifty technique- here is the original reference. It relies on magnetization transfer to a protein proton from a proton of a molecule that binds in the active site. This magnetization is then again transfered from the protein proton to a proton of another molecule that binds to the same site. For this to happen, the rate constants for binding have to be much smaller than the relaxation times for the protons.
Thus, the magnetization transfer sequence for two ligands A and B that bind to the same active site is

H(A)------>Protein proton------->H(B)
Naturally, this happens if both H(A) and H(B) are close to the same protein proton. Thus you see cross peaks between two protons A and B of two different ligands, mediated by a protein proton. Information from many such cross peaks allows us to map the protein protons to the ligand protons that are near them. In the end, not only does a picture emerge of the binding conformation of both ligands separately, but this information also allows us to suggest a common pharmacophore for the two ligands. And Paterson has now used this technique for disco and epothilone.

I am sure the technique has to be done carefully and that it was, and I also don't doubt the postulated conformation of disco. Most of the paper is really interesting and it's a neat study. But what concerns me is the fact that the end result, the binding conformation of disco can be mapped onto the x-ray conformation of disco proposed earlier, as well as the solution conformation of dictyostatin. Where my mind snags is in accepting this conclusion, because a single or even one dominant conformation for a flexible molecule derived in solution is unrealistic. It's what is called a 'virtual' solution. It's virtual simply because it's an average conformation. And since the average is a juxtaposition of all possible individual conformations, it simply does not exist in solution by itself. It's like saying that the contiguous structure of fan blades seen when a fan is moving very fast actually exists. It does not, because it is an average, and the resolution time of our eye is not short enough to capture individual positions of the fan blades.

So I wonder how the binding conformation of disco could be mapped onto the conformations of one x-ray conformation and one single dominant conformation in solution. Now I am sure there is more to this story, and I am still exploring the paper, but for commonsense reasons, a little red light in my brain always turns on (or at least should turn on) when a single or dominant conformation for a highly flexible molecule in solution is postulated.

More cogitations to come soon.