Field of Science


I am attending the Cambridge Healthtech Institute (CHI) conference on Structure-Based Design from June 7-8, along with a short kinase inhibitor design segment on June 6 at the World Trade Center in Boston. The conference includes some tasty-looking presentations and talks, with good emphasis on hit-to-lead optimization, not to mention some emphasis on luncheon workshops.

So if anyone wants to network or just bitch about the presentations (including my own poster) I will be more than happy to get in touch. I also plan to live-blog a little from there. Should be interesting.

AD, metals, and evolution

There always seems to be something new emerging in Alzheimer's disease research. In the last few years, at least one widely accepted hypothesis, the amyloid hypothesis, has come under scrutiny. In AD, it was thought that peptides aggregate into beta-amyloid fibrils (Aß) which causes toxicity to neurons.

However, many reports in the last two years are suggesting that it's not the aggregated beta-amyloid, but its soluble precursors that seem to be more toxic. There were some indications that this was true before too. For example, neuronal damage and memory impairment did not always correlate with Aß levels.

Another big hypothesis that has been widely prevelant in the AD community is that metals such as copper, aluminium, iron, and zinc, might correlate with Aß aggregation. New reports also suggest that these metals may in fact bind to the soluble and toxic forms of Aß, and help them to aggregate. One very interesting aspect of metabolism emerging from this research seems to be that it's not necessarily a larger amount of these metals from our environment or diet that's causing the problems, but an age-related breakdown in metal metabolism in the brain, which is an especially capacious reservoir of metals. Thus, perhaps we need not be especially worried about getting more copper or zinc from our diets.

One more thing that the metals might be doing is to help in free-radical generation, that might damage neurons. Again, there is still a correlation vs causation problem with this hypothesis, but free radicals are being increasingly found in vitro, when metals are added to Aß fibrils. While aluminium seems to have been largely a scare idea, zinc and copper are emerging as more plausible agents for free radical production and neuronal damage.

Based on evolutionary concepts, I cannot help but think that there may actually have been some protective function of amyloid in ancient human civilizations. Maybe it sequestered copper and provided a ready source when there was a lack of copper (or iron for that matter) in the diet. Or maybe it recruited copper to fight microorganisms and especially bacteria, at a time when there were no defenses against them except for natural ones. In this way, it could have dealt a double blow; denying essentials metals to the bacteria, and recruiting them to produce free radicals which would kill the little beasties.

In any case, AD research at the molecular level seems to be advancing rapidly, and we can expect some interesting discoveries in the years to follow.

1. A nice review on AD and amyloid, free radicals, and oxidative damage
2. A recent Angewandte Chemie paper on the influence of copper concentrations on Aß geometry, morphology, and aggregation.

The beastly behemoth

The damn thing took me more time in Chemdraw than I expected. If anyone wants the cdx file, I will be happy to mail it to them. That way it could save you some slave labour. Please be merciful and don't point out any mistakes in the least for now.

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Going back to the original structure determination papers for maitotoxin (Yasumoto, Kishi; 1993, 1994, 1996), I find that some of the NOE assignments are kind of fuzzy, because the spectrum was naturally incredibly crowded in the disputed region. Regular 2D NOESY produced a smatter of fizz.

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(Reference DOI: 10.1039/a901979k)

So they resorted to a fancier technique, PFG 3D NOESY, to pick out the cross peaks. The third axis in this NOESY is the C13 shifts, so that the protons are also separated on the basis of C13 shifts which differ more than proton chemical shifts (although still not by much, 3-4 ppm in some cases).

If I were asked if I believe in the assignments and the NOE correlations, I would probably say yes, but mainly on the basis of a process of elimination, and not on the basis of unambiguous confidence. But then I guess such is the nature of painstaking NMR structure determination of such complex natural products.

In any case, Spencer, Gallimore, and Nicolaou are definitely on to something, as described in the post before the last one, and we may learn something quite interesting about the NMR data or the biosynthesis.

Tortoise defends its territory from cat

There's also a sick video on Youtube named "Killer Bite" in which a warped mind has a video of his pet boa strangling and consuming two of his pet rabbits who are minding their own business, eating carrots and grass. I thought that the video was incredibly cruel, and it's quite clear that its sole purpose is entertainment, especially considering the ostentatious ominous sounding battle music in the background. The pervert has blocked comments, but if you find the video offensive, please do flag it as inappropriate.

However, what was even more appalling than the video were the comments. Some said "O it's ok, this is exactly what happens in nature". Well, once in a while lions also kill human beings in the wild. Should we feed convicts or normal human beings to lions and say that that too is "natural"? Others said that "It's the rabbits who were too stupid...why didn't they dodge the boa?". I don't even want to respond to these gits. Also, rabbits are not exactly the 'natural' food of boas (not that feeding birds would have been any more humane). Who knows if the boa was deliberately starved before letting him loose on the rabbits, which is perfectly possible.

In any case, quite apart from the fact that nature is indeed red in tooth and claw, this kind of deliberate setup shows an extraordinary apathy and condescension towards nature and animals. This pompous human being needs to be summarily condemned.

Even when such a thing is natural, there are limits. I always recount a story about Charles Darwin which indicates that it is possible to be an objective observer of nature as well as a humane person. Darwin was treading through some forest in South America, when he saw a wasp repeatedly stinging a spider and flying away. The spider was desperately trying to get back at the wasp, flailing and steadily faltering. Darwin the dedicated naturalist could have documented the entire episode in his diary dispassionately, but he did not; he put the spider out of its misery by instantly killing him.

MD? least in this case

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I have often talked on this blog about the fallacy of deriving single average conformations for molecules from average NMR data. How can a molecule with ten rotatable bonds (and frequently more) have any single or dominant conformation in solution? And yet, one repeatedly comes across publications dealing with single average conformations derived for organic molecules.

Peptides, being favourite targets for solution conformational analysis, are often subjected to such analysis. One comes across many instances where people want an alpha helix, or a 3/10 helix, or a gamma turn. They then design a peptide which they think is constrained to form that motif. They get their average NMR data (coupling constants and distances from NOE data), and then do a constrained molecular dynamics run, and see their favourite motif on the screen. But of course, what you put in is what you get out. If you are constraining the molecule to fit the NMR data in the first place, it's not going to stray away from it, and not surprisingly you see what you want to see (Isn't this a general theme and trap for so many of be tempted to see what we want to see?).

In general, molecular dynamics is not a very efficient method of searching conformational space. Plus, every MD program uses some force field, and force fields are well-known to give fairly accurate geometries if well parametrized, but not good energies. So there is no guarantee that all the "low-energy" conformations of any molecule will be sampled. But in theory, if one could actually search all of conformational space with a good force field, then one would explore the whole ensemble of structure that a molecule adopts in solution. Even with faster computers though, that goal is going to unrealizable for a general situation.

However, MD can be a good way of exploring conformational space if the molecule is highly constrained in the first place. In such a case, it can serve as a good way of deconvoluting the average NMR data. And this is prcisely what Nikiforovich and Marshall, two researchers who do seem aware of the general problem of assigning multiple conformations, have done (J. Med. Chem, DOI: 10.1021/jm070084n). They have taken a cyclic pentapeptide, Pro-Ala-Ala-Ala-Ala, and done long 100 nanosecond (which is a huge amount of time for a MD run) on the molecule. Most importantly, their MD runs are unconstrained. After running the MD with two different force fields and different starting velocities, they consistently see the same results; a major conformer without a gamma turn, and a minor one (about 6%) with a gamma turn, a usually forbidden motif. They also gratifyingly note that the average distances from their ensemble of MD structures match well with the NMR data.

This analysis was feasible and realistic because of a couple of reasons; firstly, the molecule is small (although it still has 8-10 rotatable bonds) and one can actually do a 100 ns MD run on this. Secondly, they used many different conditions that converged to the same two conformational profiles, and thirdly they used unconstrained MD to remove any bias. I am not completely satisfied with it though, because the molecule still does have 10 rotatable bonds and is quite flexible. But it is a ring, and some of the bonds are not as flexible, so in general it is constrained and I think the conclusions are quite believable.

For once, it gave me a satisfied feeling to see an analysis that gave results that are quite likely to be true, complete, and sweet.

P.S. Garland Marshall also has one of the more memorable quotes in science that I have heard:
"Nature does not shave with Ockham's Razor!"

The mighty Maito

Totally Synthetic has already touched upon the recent structure debate about Maitotoxin in which the latest piece is penned by Nicolaou in Angew. Chem. But since I am planning to present the controversy for a group meeting, I thought it may be worth summarising the main points.

1. In 2006, Spencer and Gallimore speculated (DOI: 10.1002/anie.200504284) that the original structure of Maitotoxin may be wrong for the J/K ring junction, where the stereochemistry is opposite to what it is at all the other ring junctions, not to mention at all the ring junctions for lots of other similar "polyether ladders"

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2. Their reasoning was based on biosynthetic considerations. According to their analysis, all the other rings of Maitotoxin and those of other polyether ladders can be envisaged to arise from sequential trans epoxide openings, either all (R,R) or all (S,S). For the J/K ring of Maitotoxin, there would be a deviation from this path, since formation of those two rings would necessitate two consecutive epoxides, an (R,R) followed by an anomalous (S,S) (or vice versa). This is not in line with their hypothesis, since they reason that a single epoxidase enzyme would be most convenient for forging all (R,R) or (S,S) epoxides. Suddenly having only one expoxide that's different from all the others would put nature in a fix, and maybe require a separate epoxidase for just that one epoxide, not a comfortable situation according to Ockham's Razor. Which of course says that the simplest explanation is the most plausible...or in the Bishop's original words, "entities should not be unnecessarily multiplied". In this case, those words would be pretty accurate, because one would need an "unnecessary multiplication" of epoxidases, one for forging one epoxide, and another for all the others.

3. Enter Nicolaou (DOI: 10.1002/anie.200604656). He first bypasses all the biosynthetic epoxidase stuff, and goes straight to a simpler fact; Spencer and Gallimore's structure does not match the NOE data! Then he comes up with two other structures, one of which is the original one and naturally satisfies all the NOE data, and the other one which satisfies more NOE data than the Spencer structure, but still retains Spencer's hypothesis about the J/K ring stereochemistry. However, even this structure does not match all the NOE data. The structure choice he makes by using quantum chemical NMR C13 chemical shift calculations, in which the difference between calculated and observed chemical shifts is the least for the original structure. In the end, Nicolaou comes up with an alternative biosynthesis pathway involving a C-glycoside type structure, that does not need this pair of anomalous epoxides. Naturally, this biosynthetic pathway is for the original structure.

There are several possibilities here;
1. Either the NMR data and therefore Nicolaou's analysis is wrong OR
2. Nature does not follow Ockham's Razor, and the biosynthesis of the J/K ring junction does not have to be in line with that for all the other rings including those of dozens of other polyether ladders. That is, there actually could be two different epoxidases, or some totally different mechanism for the formation of the J/K rings.

To me, both these possibilities seem likely. However, we must not forget that the structure of Maitotoxin was derived very painstakingly over a long period of time by Yasumoto, Kishi and others. Unless there is evidence to the contrary, I don't see any reason to doubt their NOE data. In fact, I am a bit surprised; in their drive towards biosynthetic explanations, did Spencer and Gallimore simply forget to see whether their proposed structure matches the NOE data or not? Clearly, if the NOE data is correct, it does not. At the same time, it's also clear that the signals in this particular region strongly overlapped in the NMR spectrum.

On the other hand, Ockham's Razor is really a human invention. "Simple" and "complicated" are frequently functions only of our minds. Nature in the past has proven to be too clever for human beings to always be able to pin it into some predetermined or standard pattern. What we called complicated before has often turned out to be the simplest and most elegant explanation in hindsight (relativity? quantum mechanics??). So Maitotixin may well again provide us with the message that Nature loves exceptions as well as unity.

So either the NOE data will be revised and/or we will discover a new mode of biosynthesis. In any case, we are the ones who gain.

Sad and totally unexpected...

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Two years ago, we were working on determining the stereochemistry of some sphingolipid analogs. To this end, we corresponded several times with Prof. Luigi-Gomez Paloma from Salerno, Italy to seek his help in implementing a pulse sequence to determine 2 and 3 bond HMBC coupling constants. We were already aware of his pioneering efforts to compute stereochemistry by comparing experimental and calculated coupling constants using the mPW1PW91 density functional. Later, he also worked on calculating and comparing C13 NMR shifts for finding stereochemistry.

While the project fell through, he was quite helpful. I became acquainted with his work, and was quite happy to see Scott Rychnovsky use the same technique and functional in his work on hexacyclinol. While K C Nicolaou does not explicity say it, I suspect he used the same functional in his recent revision of the structure of maitotoxin. For reference, I still have a mail or two from Prof. Gomez-Paloma.

So it can be imagined what I must have felt when, completely unexpectedly, I saw a simple sentence on top of Nicolaou's recent paper.
"In memory of Luigi-Gomez Paloma"
Wow. That was unexpected and an unexpected way of getting to know it. Sad news.

Here are two of his pioneering papers. 1 (coupling constants) and 2 (C13 shifts). Prof. Gomez-Paloma was a talented organic chemist and an expert in NMR. He was definitely most instrumental in giving synthetic chemists the confidence to use quantum chemical NMR calculations to determine stereochemistry of natural products.

The last humane act?

I have always had mixed feelings about capital punishment. On one hand, I think it causes unnecessary suffering to a lot of people and reflects a kind of hubris in judging people on our part. On the other hand, I find it hard to argue the value it has for deterrence; one problem in assessing deterrence is that because it necessarily involves people who might have been deterred because of possible retribution, it's always difficult to pinpoint how valuable it has been because we never get to know the wannabes who never were.

In any case, one thing which we all can agree upon is that whatever capital punishment is meted out, it needs to be humane. Electrocution, death squads, hanging, and gas chambers in my opinion always have been grotesque and cruel methods, a blot on our humaneness. I used to think that lethal injection is "better" than these methods, but somehow could not shun a dissenting thought about how humane it really is.

Now, a morbid sounding but important editorial from the freely available PLOS journal PLOS Medicine offers a critique of lethal injection that should be carefully pondered by policy makers and officials as well as by common citizens. It basically questions the use of the procedure and points out that especially the first step- considered to be the key step in making the procedure humane- may not be so foolproof and benign after all.

This first step is the induction of anesthesia by an injection of thiopental which is administered to the condemned man in order to supposedly ease the next two steps; an injection of pancuronium which paralyzes the muscles, including the respiratory ones, and then a final injection of potassium chloride that causes cardiac arrest. In the absence of anesthesia, the victim would feel an extreme asphyxiation and muscle spasms.

The PLOS editorial first points towards an article published in 2005 in the distinguished journal Lancet, whose conclusions if true are horrible and alarming to say the least. Let the abstract speak for itself:
"Anaesthesia during lethal injection is essential to minimise suffering and to maintain public acceptance of the practice. Lethal injection is usually done by sequential administration of thiopental, pancuronium, and potassium chloride. Protocol information from Texas and Virginia showed that executioners had no anaesthesia training, drugs were administered remotely with no monitoring for anaesthesia, data were not recorded and no peer-review was done. Toxicology reports from Arizona, Georgia, North Carolina, and South Carolina showed that post-mortem concentrations of thiopental in the blood were lower than that required for surgery in 43 of 49 executed inmates (88%); 21 (43%) inmates had concentrations consistent with awareness. Methods of lethal injection anaesthesia are flawed and some inmates might experience awareness and suffering during execution."
What the journal is saying is clear; the procedure seems to be administered sloppily, with basically no concern for the dying man. The fact that the concentrations of thiopental in the blood were lower even than those in surgery patients sounds outrageous, because ideally, the thiopental administered to condemned men is supposed to be at a dosage much higher than that for surgery patients, to ensure the kind of anesthesia that would be considered deadly for surgery patients. But this study shows that it is even lower than in surgery patients, which seems to indicate negligence bordering on criminal behaviour.

What is even more frightening is the second article referenced in the editorial, published in the same issue. This more clearly indicates that the level of anesthesia would depend on how long the procedure took, and if the procedure took very long because of whatever reason, the anesthesia would not be enough and the victim could actually be aware. Even more significantly and morbidly, the main thrust of this article is that the three lethal compounds do not always act in a strict sequential manner. Thus, both potassium chloride and thiopental may not have done their job before pancuronium starts acting. The implication is horribly obvious; the victim undergoes death by chemical asphyxiation, being aware of it all the time. However, because he was under anesthesia, he would not even be able to indicate his suffering.
The fact is that very little research has been done in standardising the protocols of lethal injection, and more importantly, intensive research has not been thought to be necessary. All in all, it is a great travesty if true. The whole point of lethal injection was to make the procedure humane, and such kind of negligence of simple protocols might show that in fact it's supposed to be the opposite.

However, in the end, the PLOS editorial makes a compelling point:
"As editors of a medical journal, we must ensure that research is ethical, and there is no ethical way to establish the humaneness of procedures for killing people who do not wish to die..."
That is, we have a kind of paradox here, namely that even in the absence of research, it would at the same time be considered unethical to conduct research and "standardize" the conditions for lethal injection by performing experiments, admittedly even on animals.

And this is the real heart of the matter which the PLOS editors are getting at, that no forcible killing can be humane. Admittedly, whether we believe in their stance on abolishing the death penalty or not, this is an extremely important matter, and it does not seem to be doing humanity's moral record any good. Personally, even if it might sound macabre, I think that the only possible humane way to kill a human being may be to bring him within ten meters of an atomic explosion, with instant flash incineration. The ludicrousness of this thought itself indicates how thorny and immoral the shades of this issue are.

These studies highlight the inherently oxymoronic nature of the whole matter. Humane killing? Can any method of killing someone against his or her will be considered humane? Let's face it, capital punishment can never be humane. Something to think about.

Disgusting damn popups

Can someone tell me how to get rid of this disgusting damn popup that started showing up suddenly without any input on my part? My blog's reputation is being catapulted from non-existent to bad!

How do you choose a good crystal structure for docking?

The first step in much of SBDD, including docking, is the selection of a good crystal structure if it exists. The crystal structure is used as the starting point for seeking new leads and optimizing them. Consider any docking method evaluation paper in J. Med. Chem. and one will come across a benchmarking set of protein structures that are used as starting models for testing the docking protocols.

Now crystal structures are frequently as close as you can get to "reality", but even they are models and should be treated with some skepticism. But the more obvious question for such a study when multiple crystal structures of a protein are available is, which crystal structure among those should you use?

The short answer to this question is, choose one with good resolution (preferably 2.0 A or less), which does not have missing portions, and which is preferably also unencumbered by the presence of a whole lot of counterions, stabilizing molecules, and other ligands.

But is that really all? Maybe not. Recently, I was playing around with docking some molecules into kinase crystal structures. I was trying to see if docking scores can correlate with the selectivity for one related protein over the other. Usually they don't, but I was going to look at similar proteins and similar structures, so I though it may be worth a shot. I was particularly looking at cyclin-dependent kinases (CDKs) which share a lot of homology especially in their ATP binding pocket. CDK2 is probably the most well-characterised CDK among the CDKs, and there are at least four to five different high-resolution CDK2 structures in the PDB. Also, I was more keen on using CDK2, because it was one of the proteins used for benchmarking the docking program.

So I decided upon two structures, both of high resolution. One had ATP docked into it, the other one had Staurosporine. I took an inhibitor which was known to be selective for another CDK over CDK2. First I docked it into that other CDK, and into the CDK2 structure that had ATP bound to it (without the ATP of course). I noted that the score for the other CDK was higher (which actually means more negative, since it is supposed to reflect the free energy of binding). That was consistent with the experimental data, which showed that the inhibitor was in fact more selective for the other kinase. But then, I docked it into the other CDK2 structure, and now the score was much better than for the other kinase. So the two docking runs gave two opposite results for the same protein. One predicted that the inhibitor would be less selective for CDK2, and the other one predicted that it would be more selective.

Now one of the things this says is that you cannot trust docking scores much. But this still was weird, because the question persists; which CDK2 structure should I use if I am going to do some SBDD and selectivity studies? I don't know the answer to this question, but I took a look at the two structures to try to figure out. In the one with the ATP, the adenine region of ATP nicely made two hydrogen bonds with the hinge region of the kinase, and so did my inhibitor which was supposed to be an ATP mimic. In the other one however, the backbone carbonyl that was supposed to form the hydrogen bond to the inhibitor was rotated by almost 90 degrees upwards. It did not form a bond with stauroporine, and it did not have to, because staurosporine does not "look" like ATP. And needless to say, it could not hydrogen bond with my inhibitor too. That's why the docking score was much worse.

What's the solution for circumventing such a problem? One quick answer that comes to my mind is; if you are docking a ligand that is "similar" to ATP, use the protein structure that has ATP bound to it. However, "similarity" can be a tricky concept, and should be considered carefully. Also, it may be slightly easy for kinase inhibitors, because there are literally hundreds of very typical planar, heterocyclic amino-pyrimidine based kinase inhibitors that share some very obvious similarity to ATP (or not...)

But probably the best message to take home from this from a computational standpoint is that rigid protein docking not surprisingly can get you into some bad trouble. Not allowing the protein to move means that you are going to preconstrain the protein based on its preconstrained conformation in the crystal. To test this thought, I did an induced-fit docking run on both structures with the inhibitor. Gratifingly, both the runs converged on the same protein-ligand structure.

Choosing a PDB x-ray structure may not be as easy as we think, and may have to be done critically. And more importantly as usual, what we put in is what we get out. Rigid docking is ok if there's only one crystal structure, and then only because there's no other choice. But in other circumstances, always allow the protein to move. That's closer to nature.