Conformations of the stevastelins: A reassessment

Shameless self-promotion: my paper on the conformational analysis of cyclic antiviral peptides called stevastelins is now online on the Biopolymers site. Here's a brief overview.

The stevastelins are cyclic peptides that show promising antiviral activity against the vaccinia viral VHR phosphatase. These peptides are phosphorylated in vivo before they can inhibit their target protein. A group in Germany previously did a meticulous analysis of four diastereometric analogs of these peptides which included their synthesis, biological characterization and conformational analysis. However, the conformational analysis was done using force field conformational searches from a single force field, constrained by variables from the NMR data (coupling constant derived dihedral angles and NOESY derived distances). Using such a protocol, the group concluded that each of the four diastereomers exists as a single conformational family in solution.

The problem with constrained conformational searches (or constrained molecular dynamics for that matter) is that they constitute a rather self-fulfilling exercise, with the assumption that there is in fact a single conformation of the molecule under question. However, as I have often discussed on this blog, any molecule with a couple of rotatable bonds is going to exist as multiple conformers in solution, so an assumption of a single conformation would be fuzzy unless supported by more data. NMR by itself is of scant value in determining these conformations for thermodynamic and kinetic reasons. Plus, analyzing conformations using a single force field can be fraught with ambiguity, since every force field comes with its own set of parameters and convergence criteria. Especially trusting energies from force fields can be dangerous. In this case, the stevastelin peptides have 9 rotatable bonds each, so I thought it worthwhile to apply our previously developed and applied NAMFIS (NMR Analysis of Molecular Flexibility In Solution) methodology combining NMR variables with structures from extensive conformational searches to the enumeration of the conformational behavior of these interesting molecules.

The paper essentially describes the conformational variability obtained for each of the diastereomers. Many of the conformations are very similar to the previously postulated families, but some are quite different. There are also some striking observations that are corroborated; for instance, the use of a d-serine truly seems to 'lock' the peptides in a single conformation. Such a lock could be effected to counter the entropic penalty that a multiconformational ensemble of molecules might have to pay. The instructive general observation is that subtle changes in sterechemistry at one or two chiral centers can dramatically affect conformational behavior, a fact that continues to surprise and confound medicinal chemists. I also note that if the NMR data for the phosphorylated peptides were available, an interesting comparison of the conformational pool for the phosphorylated and unphosphorylated counterparts could be attempted. This would shed light on whether phosphorylation leads to less conformational variability or simply increases the proportion of a chosen subset of conformations of the peptides.

Comments, criticism and general feelings of chagrin are welcomed.

Jogalekar, A. (2010). Conformations of stevastelin C3 analogs: Computational deconvolution of NMR data reveals conformational heterogeneity and novel motifs Biopolymers DOI: 10.1002/bip.21504

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

∆G, ∆G†† and All That: Implications for NMR

Since we were on the subject of NMR and determining conformations, I think it would be pertinent to briefly discuss one of the more slippery basic concepts that I have seen a lot of chemistry students (naturally including myself) get plagued with; the difference between thermodynamics and kinetics. I find myself often besieged by a distinction between these two important ideas that encompass all of chemistry. Simply saying that thermodynamics is "where you go" and kinetics is "how you get there" is not enough of a light to always assuredly guide students through the sometimes dark corridors of structure and conformation.

Going beyond the fact that thermodynamics is defined by the equilibrium free energy difference (∆G) between reactants and products and that kinetics relates to the activation barrier (∆G^††) for getting from one to the other, I want to particularly discuss the importance of both these concepts for determining conformation by NMR spectroscopy.

There are two reasons why determining conformations in solution can become a particularly challenging endeavor. The first reason is thermodynamics. Again consider the all-important relation ∆G = -RTlnK which makes the equilibrium constant exquisitely sensitive to small changes in free energy (∆G). As mentioned before, an energy difference of only 1.8 kcal/mol between two conformations means that the more stable one exists to the extent of 96% while the minor one exists to the extent of only 4%. In practice such energy differences between conformers are seen all the time. A typical scenario for a flexible molecule in solution will posit a complex distribution of conformers being separated from each other by tiny energy differences ranging from say 0.5-3 kcal/mol. Again, the above exponential dependence of equilibrium constant K on ∆G means that the concentration of minor conformers which are higher in energy than the more stable ones by only 3 kcal/mol will be so tiny (~0.04%) as to be virtually non-existent. NMR typically cannot detect conformers which are less than 2-3% percent in solution (and it's too much to ask of NMR to do this), but such populations exist all the time.

Thus, thermodynamics is often the bane of NMR; in this case the technique is plagued by its low sensitivity

If thermodynamics is the bane, kinetics may be the nemesis. Rotational barriers between conformations (∆G^††) can be even tinier compared to thermal energy available to jostle molecules around at room temperature. For example, the classic rotational barrier for interconversion in ethane (whose origins are still debated by the way) is only 3 kcal/mol. Energy available at room temperature is about 20 kcal/mol which will make the ethane conformations interconvert like crazy. So even for energy barriers that are several kcal/mol, conformational interconversion is usually more than adequate to observe averaging of conformations and consequently all associated parameters- most importantly chemicals shifts and coupling constants- in NMR. The resolution time of NMR is on the order of tens of milliseconds, while conformational interconversion is on the order of tens of microseconds or less. Now in theory one can go to lower temperatures and 'freeze out' such motions. In many such experiments, line broadening at lower temperatures is observed, followed by separation of peaks at the relevant temperature. But consider that even for a barrier as high as 8-10 kcal/mol, NMR usually gives distinct, separate signals for the different conformers only at -100 degrees celsius. For barriers like those in ethane, the situation would be hopelessly challenging. As an aside, that means that sharp, well-defined resonances at room temperature do not indicate lack of conformational interconversion but can simply mean that conformational interconversion is fast compared to the NMR time scale.

Thus, kinetics is also often the bane of NMR; in this case the technique is plagued by low resolution time

Now there may be situations in which either thermodynamics or kinetics is favourable for carrying out an NMR conformational study. But for the typical flexible organic molecule, both these factors are usually pitted against the technique; rapid interconversion because of low rotational barriers, and low thermodynamic energy differences between conformers. Given this fact, it probably should not sound surprising to say that NMR is not that great a technique. However, as is well known to every chemist, its advantages far outweigh its drawbacks. Conformational studies comprise but one important aspect of countless NMR applications.

Nonetheless, when conformational studies are attempted, it should always be kept in mind that thermodynamics and kinetics have both conspired to make NMR an unattractive method for our purposes. Thermodynamics leads to low populations. Kinetics leads to averaging of populations. And yet the average information gained from NMR is invaluable and can shed light on individual solution conformations when combined with a deconvolution technique like NAMFIS or molecular dynamics. On the other hand, fitting the average data to a single conformation for a flexible molecule is inherently flawed and unrealistic. No one who has tried to take pictures of a horse race with a low-shutter speed camera should believe that NMR by itself is capable of teasing apart individual conformations in solution.

For determining conformations then, NMR alone does provide a wealth of data locked inside a safe. Peepholes in the door may illuminate some aspects of the system. But you need a key, best obtained from other sources, that will allow you to open the door and savor the treasures unearthed by NMR in their full glory.

Article on NAMFIS in IIT-D magazine

A short holiday break and a rather protracted bout of the flu have kept me from blogging. So I will link to an article of mine that just got published in the magazine of the Chemical Society of the Indian Institute of Technology (IIT), Delhi. The article is written for the layman and talks about the importance of realizing that flexible molecules have multiple conformations in solution. Such conformations cannot be determined by NMR alone due to their rapid interconversion.

In the article, I describe NAMFIS (NMR Analysis of Molecular Flexibility In Solution), a joint computational-NMR approach which can derive a Boltzmann population for flexible molecules in solution. This information can be very useful for deducing, for example, the protein-bound conformation of a drug. But it can also be useful under other circumstances where determining conformation is important, such as for organic molecules assembling on a surface. Comments, criticism and questions are of course always welcome.