The Uncertainty Principle for climate (and chemical) models

A recent issue of Nature had an interesting article on what seems to be a wholly paradoxical feature of models used in climate science; as the models are becoming increasingly realistic, they are also becoming less accurate and predictive because of growing uncertainties. I can only imagine this to be an excruciatingly painful fact for climate modelers who seem to be facing the equivalent of the Heisenberg uncertainty principle for their field. It's an especially worrisome time to deal with such issues since the modelers need to include their predictions in the next IPCC report on climate change which is due to be published next year.

A closer look at the models reveals that this behavior is not as paradoxical as it sounds, although it's still not clear how you would get around it. The article especially struck a chord with me I see similar problems bedeviling models used in chemical and biological research. In case of climate change, the fact is that earlier models were crude and did not account for many fine-grained factors that are now being included (such as the rate at which ice falls through clouds). In principle and even in practice there's a bewildering number of such factors (partly exemplified by the picture on top). Fortuitously, the crudeness of the models also prevented the uncertainties associated with these factors from being included in the modeling. The uncertainty remained hidden. Now that more real-world factors are being included, the uncertainties endemic in these factors reveal themselves and get tacked on to the models. You thus face an ironic tradeoff; as your models strive to mirror the real world better, they also become more uncertain. It's like swimming in quicksand; the harder you try to get out of it, the deeper you get sucked in.

This dilemma is not unheard of in the world of computational chemistry and biology. A lot of the models we currently use for predicting protein-drug interactions for instance are remarkably simple and yet accurate enough to be useful. Several reasons account for this unexpected accuracy; among them cancellation of errors (the Fermi principle), similarities of training sets to test sets and sometimes just plain luck. Error analysis is unfortunately not a priority in most of these studies, since the whole point is to publish correct results. Unless this culture changes our road to accurate prediction will be painfully slow.

But here's an example of how "more can be worse". For the last few weeks I have been using a very simple model to try to predict the diffusion of druglike molecules through cell membranes. This is an important problem in drug development since even your most stellar test-tube candidate will be worthless until it makes its way into cells. Cell membranes are hydrophobic while the water surrounding them is hydrophilic. The ease with which a potential drug transfers from the surrounding water into the membrane depends among other factors on its solvation energy, on how readily the drug can shed water molecules; the smaller the solvation energy, the easier it is for drugs to get across. This simple model which calculates the solvation energy seems to do unusually well in predicting the diffusion of drugs across real cell membranes, a process that's much more complex than just solvation-desolvation. 

One of the fundamental assumptions in the model is that the molecule exists in just one conformation in both water and the membrane. This assumption is fundamentally false since in reality, molecules are highly flexible creatures that interconvert between several conformations both in water and inside the membrane. To overcome this assumption, a recent paper explicitly calculated the conformations of the molecule in water and included this factor in the diffusion predictions. This was certainly more realistic. To their surprise, the authors found that making the calculation more realistic made the predictions worse. While the exact mix of factors responsible for this failure can be complicated to tease apart, what's likely happening is that the more realistic factors also bring more noise and uncertainty with them. This uncertainty piles up, errors which were likely canceling before no longer cancel, and the whole prediction becomes fuzzier and less useful.

I believe that this is what is partly happening in climate models. Including more real-life factors in the models does not mean that all those factors are well-understood. You are inevitably introducing some known unknowns. Ill-understood factors will introduce more uncertainty. Well-understood factors will introduce less uncertainty. Ultimately the accuracy of the models will depend on the interplay between these two kinds of factors, and currently it seems that the rate of inclusion of new factors is higher than the rate at which those factors can be accurately calculated.

The article goes on to note that in spite of this growing uncertainty the basic predictions of climate models are broadly consistent. However it also acknowledges the difficulty in explaining the growing uncertainty to a public which has become more skeptical of climate change since 2007 (when the last IPCC report was published). As a chemical modeler I can sympathize with the climate modelers. 

But the lesson to take away from this dilemma is that crude models sometimes work better than more realistic ones. Perhaps the climate modelers should remember George Box's quote that "all models are wrong, but some are useful". It is a worthy endeavor to try to make models more realistic, but it is even more important to make them useful.
Image source

Anticancer drugs form colloidal aggregates and lose activity

Over the last few years, one of the most interesting findings in drug screening and testing at a preclinical level has been the observation that many drugs form colloidal aggregates under standard testing conditions and nonspecifically inhibit target proteins which they otherwise would not affect. This are large aggregates, a hundred nanometers or more in diameter, and they cause proteins to stick and partially unfold, creating the illusion of inhibition. This leads to false positives, especially in high-throughput screening protocols. And these false positives can be absolutely rampant.

What's striking is the sheer ubiquity of this phenomenon which has been observed with all kinds of drugs under all kinds of conditions; while the initial observation was limited to isolated protein-based assays, the phenomenon has also been seen in simulated gastric fluids and in the presence of many different kinds of proteins like serum albumin which are found inside the body. The colloid spirit seems to emphatically favor a shotgun approach.

Now a team led by the brother-sister duo Brian and Molly Shoichet (UCSF and Toronto) has found something that should give drug testers further pause for thought; they see some bestselling anticancer drugs forming colloids (shown above) in cell-based assays to an extent that actually diminishes their activity, leading not to false positives but to false negatives. They test seven known anticancer drugs in cell assays both under known colloid forming conditions along with conditions that break the colloids up. This is not as easy as it sounds since it involves adding a detergent which would usually be too toxic to cells; fortunately in this case they find the right one. Another interesting finding is the re-evaluation of a popular dye used to study "leaky" cancer blood vessels; unlike the previously proposed mechanism, the current study seems to suggest that the dye too forms large aggregates and nonspecifically inhibits the protein serum albumin.

The testing essentially reveals that the drugs when they form colloids basically show activity that's so low as to be negligible and equivalent to the controls. That's a self-(un)proclaimed false negative. Now anybody who deals with error analysis knows that false negatives are fundamentally worse than false positives since by definition they cannot even be detected. The present study raises the pertinent question; how many promising drugs might we be missing because they form aggregates and lower the observed response in cells? And since the colloid forming phenomenon has been shown to be so ubiquitous, could it possibly be influencing the mechanism of action of all kinds of drugs inside the body? And in what ways? It's a fascinating question, and one of those that continues to make basic research in drug discovery still so interesting.
Image source and credit: ACS

P-glycoprotein: The vacuum cleaner that makes Sir James weep

There has been a lot of discussion during the last decade about the continuing attrition in the pharmaceutical industry and the absence of novel drugs. Several factors including layoffs, narrow-minded management practices, outsourcing etc. have been held responsible for this trend which only promises to exacerbate in the near future. All eminently sensible points. But one thing should be clear; drug discovery remains hard because we still just don't understand a lot of the basic science very well. This is something that should always be on the mind of anyone who wants to hold  non-scientific factors responsible for drug failures. The fact is that there still remain very basic challenges that drug discoverers have to surmount. And by scientific challenges I am not talking about cutting edge, futuristic, overhyped strategies like gene therapy and nanotechnology that haven't yet borne fruit. I am talking about fundamental challenges here, problems that have been realized for years and yet not solved.

A review in this week's issue of Journal of Medicinal Chemistry has an account of something that greatly contributes to one of these challenges; getting compounds into cells. It's a basic problem in developing any drug. You may have a molecule that looks miraculous in the test tube but which utterly fails once you put it into a living organism. Many factors can contribute to this lack of translation but one of the most basic reasons is simply that the compound is not getting inside the cell. Recall that the cell membrane is expressly designed to keep things out, which is a good thing for evolution but a bad thing for drug designers. The membrane is composed of phospholipids with all kinds of proteins and other biomolecules embedded within it. Drugs can get across this membrane by simple passive diffusion, although in some cases they may be shuttled across by special helper proteins. In general any foreign substance will have to be hydrophobic enough to get past this membrane. But even otherwise it will have to satisfy some simple properties; it can't be too big and charged for instance. And it can't be too hydrophobic otherwise it won't dissolve in the aqueous medium surrounding the membrane in the first place.

But hydrophobicity is where your troubles only begin. Cells have an assortment of watchdog proteins whose purpose is to keep out unwanted substances. In the modern world, "unwanted substances" includes pretty much all drugs. The J. Med. Chem. review focuses on one of these watchdogs - very likely their king - which plagues drug designers all the time; the P-glycoprotein efflux multi-drug transporter (Pg). The fancy name only hides the fact that it's essentially a simple pump embedded within the membrane, designed to throw drugs out. It's the ultimate bouncer; even drugs that have the right mix of hydrophobic and hydrophilic character quake and rapidly exit when they encounter PgP. In fact the protein was discovered when it was found that some cancers were becoming resistant to certain drugs; what was happening was that these drugs were being pumped out or "effluxed". Even worse, the presence of these drugs was increasing the expression of the protein. Later it was found that a wide variety of drugs bind to and increase the expression of Pgp, reducing their effective concentration inside the cell; it's still one of the principal mechanisms of resistance in some kinds of cancer. 

Progress was only hindered by not knowing the structure of the protein (a part of which is illustrated above) which was only recently and partially solved by x-ray crystallography, and even then it's not really helping. The protein's structure and interior are exquisitely hideous to say the least; 12 transmembrane segments composed of 1280 amino acids, a mammoth internal cavity of 6000 Å³ and a wondrously complex mechanism of compound binding and extrusion during which the protein undergoes a massive conformational challenge. As it snakes its way through the lipid bilayer and wraps itself around drugs, the precision of this molecular machine would be wholly admirable if it were not for the eminent heartburn that it causes drug discoverers. 

The constant extrusion of drugs by Pgp means that you may have to increase the dosage of your drugs (or saturate the protein with another drug) to maintain high blood levels, but that's just skimming the surface of the Pgp world of pain. Since its original discovery the protein has turned into a minor nemesis for drug designers and it's become a part of a notorious list of proteins called "anti targets" that can lead to side-effects and lack of efficacy (we encountered one of these anti targets before - the hERG channel protein). And that's not only because Pgp is ubiquitously expressed in the intestine and liver where most drugs are metabolized. Nor is it because of its special role in the blood-brain barrier which creates additional problems for CNS drugs. It's because when it comes to Pgp, scientists may not have a clue about how to possibly solve the problem. Usually when you encounter an unwanted protein that binds to your drugs, you try to add a modification to your drug to block this binding. In many cases, structure-activity relationship (SAR) can help you pin down some trends; you remove a basic nitrogen atom here, you get rid of a double bond there, you add a fluorine to that ring. If you know what kinds of molecular features a rogue antitarget protein likes, you can avoid those features in your drug.

But not so for Pgp. Pgp is, in the words of the review author, a "hydrophobic vacuum cleaner". And it's one that will put Sir James to shame. What kinds of molecules does it like as substrates? Here's a description from Kerns and Di's book "Drug-like Properties":

"The substrate specificity for Pgp is very broad. Compounds ranging from a molecular weight of 250 to 1850 are known to be transported by Pgp. Substrates may be aromatic, non-aromatic, linear or circular. They can be basic, acidic, zwitterionic or uncharged. Some substrates are hydrophobic, others are hydrophilic and yet others are amphipathic."

The authors could have saved themselves all those words by simply saying something like "Pgp binds to and extrudes everything in the universe except possibly the human soul". As should be obvious, this kitchen sink description of every molecule of every kind is not exactly a guide for drug designers to rationally add modifications that would prevent Pgp binding. I was myself part of a project where the whole "rational" drug design process was going extremely well - well-defined changes in structure contributing to improved potency - until we found that the compounds were being generously ejected by Pgp. At this point our gung-ho approach screeched to a halt and we found ourselves transported from the sunlight of rational design into the night of Pgp-mediated chaos. Where before we had been confidently stepping across a brightly lit landscape, we now found ourselves groping around in the dark with our eyes closed. It was like falling down an abyss. There was no rational modification to our existing molecules that would ensure a Pgp-free existence. From then on it was largely about gut feelings, intuition and Hail Mary passes.

In reality, Pgp binding is sometimes considered so painfully complex to circumvent that the best strategy may actually be to wave a wand and temporarily forget about it. Counterintuitive as this seems, what this strategy means is that often the best way to prevent Pgp drug binding is to simply increase the passive diffusion of your compounds so much that it swamps any Pgp-enabled extrusion. Basically you just keep on bumping up the magnitude of one process until it can one-up the opposing process.

The present review in J. Med. Chem. provides some respite from this depressing existence. The author describes several case studies where strategies like tying up hydrogen bond donors, getting rid of them, reducing basicity or reducing polar surface area helped to design out Pgp binding. These are valuable examples, but they are anecdotal nonetheless and may not work for other molecules with similar functionalities. Well-defined rational approaches to Pgp binding are still lacking and the complex mechanism of the Pgp-drug binding precludes designing specific Pgp inhibitors even if the structure is known. Reviews like the present one provide useful guidelines, but for the foreseeable future at least, Pgp will stand in splendor as one among a handful of scientific challenges that continue to make drug discovery just so damn difficult.
Image source: Wikipedia

"Arsenic bacteria": If you hadn't nailed 'im to the perch 'e'd be pushing up the daisies

Rosie Redfield (who blogs on this network) has just published an official, careful and decisive rebuttal to the "arsenic bacteria" fiasco in collaboration with a group at Princeton. The paper which will appear in Science is under embargo for now, but there is a copy available at that bastion of free publication arXiv. Readers may remember Redfield as the scientist who offered the most meticulous preliminary criticism of the original paper by Felisa Wolfe-Simon and others. Wolfe-Simon and the rest of the arsenic group refused to engage in debate with Redfield and other critics at the time, citing the "non-official" nature of the offered criticism and asking for publication in a more formal venue. Looks like they finally got their wish.

The abstract could not be clearer:

"A strain of Halomonas bacteria, GFAJ-1, has been reported to be able to use arsenate as a nutrient when phosphate is limiting, and to specifically incorporate arsenic into its DNA in place of phosphorus. However, we have found that arsenate does not contribute to growth of GFAJ-1 when phosphate is limiting and that DNA purified from cells grown with limiting phosphate and abundant arsenate does not exhibit the spontaneous hydrolysis expected of arsenate ester bonds. Furthermore, mass spectrometry showed that this DNA contains only trace amounts of free arsenate and no detectable covalently bound arsenate."

It's a fairly short paper but there are many observations in it which quite directly contradict the earlier results. The strain of bacteria that was claimed to grow only when arsenic was added to the medium was found to not grow at all. In fact it did not budge even when some phosphate was added, growing only after the addition of other nutrients. Trace element analysis using several techniques detected no arsenate in DNA monomers and polymers. This is about as definitive an argument as can be published indicating that the claims about the bacteria using arsenic instead of phosphorus in their essential biomolecules were simply incorrect. Much credit goes to Redfield who patiently and probingly pursued the counterargument, undoubtedly at the expense of other research in her lab. In addition she did open-science a great service and described all the ongoing research on the blog. She sets a standard for how science should be done, and we should hope to see more of this in the future.

Sociologically the episode is a treasure trove of lessons on how science should not be done. It checks off some standard "don'ts" in the practice of science. Don't fall prey to wishful thinking and confirmation bias that tells you exactly what you wanted to hear for years. Don't carry out science by press conference and then refuse to engage in debate in public venues. And of course, don't fail in providing extraordinary evidence when making extraordinary claims. If the original paper had been published cautiously and without hullabaloo, it would have become part of the standard scientific tradition of argument and counterargument. As it turned out, the publicity accompanying the paper made it a prime candidate for demolition by blogs and websites. If nothing it provided a taste of how one needs to be extra careful in this age of instant online dissemination. There's also some "do's" that deserve to be mentioned. The researchers did reply to criticism later and make their bacterial strains available to everyone who wanted to study them in a gesture of cooperation, but their earlier behavior left a bad taste in everyone's mouth and detracted from these later acts.

When the original paper came out, many of us were left gaping with eyes wide open at visions of DNA, ATP, phosphorylated proteins and lipids swirling around in a soup of arsenic, carrying out the exact same crucial biological processes that they were carrying out before without skipping a heartbeat. We just had a gut feeling that this couldn't be quite right, mainly because of the sheer magnitude of the biochemical gymnastics an organism would have to undergo in order to retool for this drastically different environment. Gut feelings are often wrong in science, but in this case it seems they made perfect sense.

What next? As often happens in science, I suspect that the defenders of the original paper will not outright capitulate but will fight a rearguard retreat until the whole episode drops off everyone's radar. But this paper here, it clinches the case for normal biochemistry as well as anything could. Good old phosphorus is still one of life's essential elements, and arsenic is not.

An almost-Nobelist's lesson to his daughter

Last year's physics Nobel Prize was awarded to a group of three people who discovered one of the most significant recent facts about our universe; the fact that its expansion is accelerating. It turns out that two out of the three laureates had gotten their Ph.D. with Robert Kirshner at Harvard, who among other things has written the excellent book "The Extravagant Universe". Kirshner was involved in a big way with the supernova project that discovered the acceleration; he trained dozens of students apart from the two prize winners. He would almost certainly have won the prize had it not been restricted to three scientists. One might have expected him to feel at least a few pangs of regret about not winning. But even if he did, his response to his daughter which she published in this week's Science is worth reading:

One morning this past October, I woke up to find an email from my father. Reading the subject line, I immediately burst into tears. My father, Robert Kirshner, is an astronomy professor at Harvard University. The subject of his e-mail was, “My Students won the Nobel Prize!”...I was worried because I knew my father to be incredibly competitive...But as it turns out, his response to not winning is the lesson I really value. When I spoke to him that morning, he amazed me: He was proud of the people he has worked with and taught; he was generous-spirited; he was funny; and he had perspective. What a relief! It turns out that a guy who spent his life trying to understand the immensity of the universe could put into perspective the relative importance of which particular earthling took home the ribbons and the medals and got to bow to the King of Sweden. It turns out that what was really important to him was the work itself, the wonder of this extraordinary universe, the honor and the fun of trying to figure things out, and maybe, just a little bit, the thrill of the chase. I admire all the terrific scientists who contributed to this greater understanding of the universe we live in, but in particular I admire my father, whose expansive understanding of what really matters taught me something of astronomical importance.

That's a lesson which, while contrary to the common human emotions of jealousy and vanity, seems to be alive and well in scientists like Kirshner and it's worth always keeping in mind. Science is inherently a community enterprise; even the science done by supposed loners like Newton and Einstein would not have been possible had it not built on a body of work extending back several centuries. The culmination of this body of work is what's real. The prizes are incidental.

The protein makes the poison: Dancing fruit flies and terfenadine

"Chemophobia" is the name of the exasperating phenomenon in which every material substance is branded as a "chemical" and made to look dangerous irrespective of context. Since everything in the universe is supposedly material, by definition chemophobia extends to everything. The media in particular has eagerly latched on to this idea, forgetting that almost everything (not just chemicals but life, liberty and the pursuit of happiness) is dangerous in the wrong quantities and context and harmless in the right ones. 

Matt Hartings at Sciencegeist had the excellent idea for us bloggers to do our part in dispelling chemophobia. He wants us to write about our favorite toxic chemical compounds. This will not only give us an opportunity to explore the many incarnations of toxicity but will also help inform the public about the highly context-specific safety and toxicity of chemicals.

My fellow bloggers have done a great job so far in documenting the various facts and myths about toxic molecules (you can find summaries on Matt's blog). A resounding theme in their posts is that "the dose makes the poison". It's an idea which goes back to Paracelsus in the 15th century and sounds intuitively true (consider the widespread injunction against gluttony), but which seems surprisingly recalcitrant to being universally accepted. This dose-specific toxicity especially makes its appearance in medicine, with unfortunate reports of celebrities fatally overdosing on prescription drugs regularly appearing in the news media. Strangely, the same media which readily accepts the fact that prescription drugs are safe as long as they are not abused in large quantities abandons its critical attitude when talking about "chemicals" in our food and clothing.

Dose-specific toxicity is indeed of paramount importance in medicine, but if you delve deeper, the common mechanism underlying the toxicity of many drugs often has less to do with the specific drugs themselves and more to do with the other major player in the interaction of drugs with the human body - proteins. Unwarranted dosages of drugs are certainly dangerous, but even in these cases the effect is often mediated by specific proteins. Thus in this post, I want to take a slightly different tack and want to reinforce the idea that when it comes to drugs it's often wise to remember that "the protein makes the poison". I want to reinforce the fact that toxicity is often a function of multiple entities and not just one. In fact this concept underlies most of the side-effects of drugs, manifested in all those ominous sounding warnings delivered in rapid fire intonations in otherwise soothing drug commercials.

What do I mean by "the protein makes the poison"? Almost every drug demonstrates its effects by binding to specific proteins which may be involved in particular diseases, and the goal of pharmaceutical research is to find molecules that target and inhibit or activate these proteins. There is of course much more to a drug than just inhibition of a protein, but that's the fundamental challenge. This goal was delineated during the turn of the twentieth century in Paul Ehrlich's notion of a "magic bullet", a compound that would hit only the rogue protein and nothing else. We are still trying to implement Ehrlich's program and in the process have discovered how hideously complicated the process is.

The thing is, in spite of much progress we still understand woefully little about the human body. When we design a drug to inhibit one protein, it has to contend with the thousands of other proteins in the body which perform crucial functions. Making a drug that binds to a protein is essentially like designing a key to fit a lock. Even if you think you have a perfect key that fits only one lock, the number of locks with similar structures is so large that it's very likely for parts of the key to fit other locks. And if these other locks or proteins play fundamental roles in normal physiological processes, you may be in trouble. In fact there's a name for this group of unwanted proteins - antitargets - and there are entire books written on how to avoid them.

Ideally you have to contend with every other protein when your goal is to target only one, but somewhat fortunately, the history of drug research has found out a handful of key proteins which seem to be often hit, leading to side-effects. In this post I will focus on two, and I will illustrate both through the example of the drug terfenadine (illustrated on top). Interestingly, the story of terfenadine reinforces the idea about both dosage and protein-specific toxicity.

Terfenadine was introduced in 1985 as an anti-allergy drug. Things seemed to be going well for its maker Hoechst Marion Roussel until 1990 when troubling reports emerged of a serious and potentially lethal side effect. This side effect was a perturbation of the heart's rhythm. It can be of several types, all of which are usually lumped under the title of "arrhythmias". In particular, terfenadine caused two phenomena with the impressive names of QT prolongation and torsades de pointes.

The heart is a pump, but it's also a kind of electrical motor with its own electrical cycle. This cycle is governed by the influx and outflux of various ions into heart cells; most commonly, sodium, potassium, calcium and chloride. The cycle shows up as peaks and troughs in electrocardiograms (ECGs). Each peak is alphabetically labeled, and the interval between the trough Q and the peak T is particularly important. It turns out that several drugs including terfenadine prolong this interval, essentially throwing the heart's rhythm out of sync. It is not hard to see that the consequences of disturbing this very fundamental rhythm of life can be catastrophic; the heart can stall, go into cardiac arrest and kill the unfortunate victim. QT prolongation can also be part of a larger indication called torsades de pointes, characterized by a specific shape of the ECG.

But what's responsible for this effect at the molecular level is a unique protein called human ether a-go-ho (hERG). The protein is an ion channel conducting potassium ions through heart cells, thus its crucial significance in maintaining heart rhythm should not be surprising. Terfenadine and several other drugs (most notably some antidepressants and antipsychotics) bind to this protein with high affinity and can even block it. The amusing name of the protein points to an amusing origin. The protein was a product of the human analog of genes discovered in fruit flies by researchers at the University of Wisconsin who were studying mutations in these genes. They found that the mutant flies' legs started to shake when they were anesthetized, making the insects look like entomological versions of Elvis. Another scientist at the City of Hope remembered where he had seen humans doing a similar dance; at the Whisky a Go Go nightclub in West Hollywood. It was the ultimate in anthropomorphization. Here's what part of the protein looks like.

When the FDA found out about the dangerous side effects that terfenadine mediated through the hERG ion channel, they sent a letter to doctors who were prescribing the drug and issued a black box warning. In 1997 the FDA finally withdrew terfenadine; after all there were several anti-allergy medications out there and there was no need to market an especially dangerous one. Since then, testing potential drugs against hERG is a mandatory part of seeking FDA approval and there is much research dedicated to finding out specific molecular features of drugs which may turn out to be hERG blockers; one common determinant seems to be the presence of a positively charged basic nitrogen atom. There are entire lists of drugs including marketed ones that can cause QT problems to varying extents under different conditions.

So there it is, toxicity mediated not just by a particular "chemical" but by its interaction with a particular protein. But the story does not end there. Toxicity problems with terfenadine seemed to occur - you guessed it - only at high dosages. The dose indeed made the poison. But there was an added twist. Some patients experienced hERG blockage only when they were taking other drugs, most notably the antibiotic erythromycin. Surprisingly this also happened when they were drinking quantities of, of all things, grapefruit juice. Grapefruit juice has also turned out to be important in the effects of other popular drugs like statins for heart disease.

What was going on? When terfenadine is administered, like any foreign molecule it has to first get through the gut wall and the liver to enter the bloodstream. And it's in the liver that it encounters a protein called cytochrome P450. This crucial protein is the great gatekeeper of the human body, denying entry to thousands of molecules which it deems to be poisonous. It is responsible for the metabolism of about 75% of all drugs. It served a necessary function during evolution when organisms had to keep potentially poisonous chemicals out, but it haunts drug discovery scientists in their dreams because of its ability to affect drug structures in unexpected ways. The centerpiece of P450 is an iron atom that oxidizes electron-rich bonds in molecules. Most of the times the protein induces an oxidation reaction in a drug that changes it to something else. As a further testament to the complexities of drug development, that "something else" itself can be toxic, beneficial or neutral. In case of terfenadine there was a stroke of good luck; cytochrome P450 was transforming the compound into another drug called fexofenadine. Chemists will recognize the small difference in the structures - a single carboxylate group at the terminal end. 

But as is often the case in the wonderful world of pharmacology, this tiny difference had momentous consequences; fexofenadine no longer bound to hERG with high affinity to cause QT-prolongation. What happened at high doses was that terfenadine saturated cytochrome P450 and some of it made its way into the bloodstream without being transformed into fexofenadine. Similarly the compounds in grapefruit juice preferentially bound to cytochrome P450, again allowing terfenadine to get past the protein. And this terfenadine which escaped the clutches of cytochrome P450 blocked hERG. One thing is clear here; it is chilling to contemplate the effects of terfenadine had it not been metabolized to fexofenadine by P450 in the first place.

This fascinating (at least for me) story of terfenadine drives home many important points regarding toxicity. Firstly, it takes two to tango, and toxicity is always a function of a drug and its target and not just of the drug alone. Secondly, we again had a case where "the dose made the poison". And thirdly, the reason this was true was because of a guardian angel, a protein which changed terfenadine into something else that was not toxic; a corollary of this point is that it takes a tiny change to turn a toxic compound into a non-toxic one.

There should be little more evidence needed to prove that toxicity is a many splendored, context-specific thing.

All images are from Wikipedia

Physics's PR problem: Moving beyond string theory and multiple universes

 I was reminded of this by a timely post by MJ at "Interfacial Digressions". As everyone knows, chemistry has a PR problem. Fear of "chemicals" runs rampant without context or qualification. In addition, unlike physics and biology, chemistry is not considered to be the science that answers profound questions about the origins of life or the future of the universe. Of course there's evidence to the contrary for each one of these thoughts - modern life would be impossible without chemistry and the origin of life can claim to be the ultimate grand chemical question - but it's been hard to convince the public of this truth. The acute PR problem for chemistry is illustrated by the fact that popular literature on chemistry does not sell half as well as that on physics; just count the number of chemistry versus physics books in your Barnes & Noble the next time you visit (if you are still obsessed with paper that is).

But I think physics also has a PR problem, and it's of a different kind than chemistry's. This statement should elicit gasps of indignation, since the Greenes, Hawkings and Kakus seem to be doing quite well; they are household names and every one of their books instantly gathers hundreds of positive reviews on Amazon. But there's still a problem and it's not one that is acknowledged by many of these leading popular expositors, at least partly because doing so would rob them of their next big NewYork Times bestseller and the accompanying profits. Look at the physics section in your B&N next time and you will understand what I am talking about.

The problem is that most of the popular physics that the public enjoys constitutes perhaps 10% of the research that physicists worldwide are engaged in. Again, count the number of physics books in your local bookstore, and you will notice that about 90% of them cover quantum mechanics, cosmology, particle physics and "theories of everything". You would be hard-pressed to find volumes on condensed matter physics, biophysics, the physics of "soft" matter like liquids and non-linear dynamics. And yes, these are bonafide fields of physics that have engaged physics's best minds for decades and which are as exciting as any other field of science. Yet if you ask physics-friendly laymen what cutting-edge physics is about, the answers will typically span the Big Bang, Higgs boson, black holes, dark matter, string theory and even time-travel. There will be scant mention if any of say spectroscopy, optics, polymers, magnetic resonance, lasers or even superconductivity.

Whether physicists admit it or not, this is a PR problem. Laymen are being exposed to what is an undoubtedly exciting but tiny fraction of the universe of physics research. For eager readers of the popular physics literature, the most exciting advances in physics are encapsulated between the Higgs boson and the Big Bang and that's all they think exists in heaven and earth. In my opinion this does a great disservice to the majority of physicists around the world who work on other, equally exciting topics. Just consider one major academic physics department, say Stanford, and you get an idea of the sheer variety of projects physicists work on. Physics books may still sell, but the physics they describe is something which most of the world's physicists don't do. I cannot see how this cannot be called a PR problem.

So who is responsible for this situation? Well, in one sense, nobody. The fact is that the public has always shown a taste for "big picture" topics like cosmology and quantum mechanics and physicists have been indulging this taste for quite a while now. And who can blame the public for being attracted to relativity with its time paradoxes or quantum mechanics with its cats and famous personal rivalries. Even in the 1920s, the popular physics literature sported the likes of Arthur Eddington and James Jeans who were pitching nuclear physics and relativity to packed audiences. The mantle was passed on in the postwar era to scientists like George Gamow and Isaac Asimov who spread the gospel with gusto. And the trend continues to the present day, with even a mind-numbingly well-trodden topic like the history of quantum theory finding eager expositors like Louisa Gilder, Manjit Kumar and David Lindley. All their books are highly engaging, but they are not doing a favor to other equally interesting branches of physics.

The popular physics literature has also started turning quasi-religious, and writers like Brian Greene and Michio Kaku are unfortunately responsible for this development. Greene in particular is a remarkably charismatic and clear writer and lecturer who has achieved almost rock-star status. Sadly, his popular expositions are seeming more like rock concerts rather than serious physics lectures. Part of the problem is his almost evangelical espousal of highly speculative, experimentally unverified (and perhaps even unverifiable) but deliciously tantalizing topics like string theory and multiple universe. Greene's books seem to indicate that the more speculative the topic, the more eagerly it will be assimilated by lay audiences. This cannot but be a disturbing trend, especially for those thousands of physicists whose research may sound pedestrian but which is also more solidly grounded in experiment and as interesting as perpetually splitting universes. One suspects that even the famous popular physics writers of lore like George Gamow would have been hesitant in pitching highly speculative topics merely for their "Wow" factor. If the biggest selling point of a popular physics book is its dependence on experimentally unverified ideas that sound more like science fiction, popular physics is in trouble indeed. 

In addition, whatever lacks the "Wow" factor seems to evidence the "Yawn" factor. By this I am referring to books constantly repackaging old wine in new bottles. A good example is Lisa Randall's latest book. It's an extremely well-written and spirited volume but it mostly treads the same tired ground of quantum mechanics, relativity and the Large Hadron Collider. The bottom line is that the popular physics literature seems to have reached a point of diminishing marginal returns. It's become very difficult to write anything on the subject that's either not well-trodden or highly speculative.

There is another unintentional effect of this literature which is more serious. Today's popular physics gives people the impression that the only questions worth addressing in physics are those that deal with unified theories or the birth and death of the cosmos. Everything else is either not worth doing or is at best done by second-rate minds or graduate students (take your pick). Not only does this paint a skewed picture of what's important and difficult in the field, it also inflates the importance and intellectual abilities of physicists working on fundamental problems at the expense of those working on more applied ones. This again does a great disservice to very many challenging problems in physics and the people addressing them. Building a room-temperature superconductor, understanding turbulence, designing new materials for capturing solar energy, keeping atoms stable at cold temperatures, kicking DNA around with lasers and of course, beating nuclear fusion at its own thermodynamic game are still long-unsolved problems that promise to engage the finest minds in the field. Yet the myth that the greatest problem in physics is finding the theory describing "everything" persists. This constant emphasis on "big" questions provides a biased view not just of physics but in fact of all of science, most of which involves solving interesting but modest problems. As MJ says in his post, most physicists he knows aren't really after 3 laws that describe 99% of the universe but would be content finding 99 laws that describe 3%. 

So what's the solution? As with other problems, the first step would be to acknowledge that there is indeed a problem. Sadly this would mean somewhat blunting the public's starry-eyed impression of cutting-edge physics, which the leading expositors of physics would perhaps be unwilling to do. At least some physicists might be basking in the public's mistaken grand impression that cosmology and quantum theory are all that physicists work on. If I were a soft condensed matter physicist and if I told someone at a cocktail party that I do physics, the images that response would evoke would most likely include long-haired professors, black holes, bosons and fermions, supernovae, nuclear weapons and time-travel. I may be excused for sounding hesitant to dispel this illusion and emphasize that I actually work on understanding the exact shape of coffee stains.

Nonetheless, this harsh assessment of reality might be necessary to cut the public's umbilical cord to the Hawkings, Greenes and Randalls. But this would have to be done by someone else and not by Brian Greene. Now let me make it clear that as speculative as I might find some of his proclamations, I don't blame Greene at all for doing what he does. You cannot fault him for not reminding the public about the wonders of graphene since that's not his business. His business is string theory, that's what he is passionate about, and nobody can doubt that he is exceedingly good at practicing this trade. Personally I have enjoyed his books, and in an age where ignorance of science seems to reach new lows, Greene's books provide at least some solace. But other physicists would have to tread into territory that he does not venture into if they want to solve physicists' PR problem. 

Gratifyingly some physicists have already started staking their claims in this territory, although until now their efforts have sounded more like tiptoeing and less like confident leaps. Nevertheless, James Gleick proved in the 1990s with his "Chaos" that one can indeed grab the public's attention and introduce them to an entirely new branch of science very successfully. In recent years this tradition has been carried on with varying degrees of success by other scientists, and they provide very promising examples of how the PR problem could be addressed. Let me offer a few suggestions. Robert Laughlin has talked about emergence and condensed matter in his "A Different Universe". David Deutsch has laid out some very deep thoughts in his two books, most recently in "The Beginning of Infinity". Philip Anderson expounds on a variety of interesting topics in his recent collection of essays. And while not entirely about physics, Stuart Kauffman's books have done a great job at dismantling the strong reductionist ethic endemic in physics and suggesting new directions for inquiry. The common emphasis of these authors is on emergent, complex, adaptive systems, a paradigm of endless opportunities and questions which has been generally neglected by the popular physics literature. In addition there are excellent, courageous critiques of string theory from Peter Woit and Lee Smolin that deviate from the beaten track.

Sadly most of these books, while exceedingly interesting, are not as engagingly written as those by Greene or Randall. But the modest success they have enjoyed seems to indicate that the public does have a taste for other areas of physics as long as they are described with verve, passion and clarity. Maybe someday someone will do the same for turbulence, DNA dynamics, non-Newtonian liquids and single-molecule spectroscopy. Then physics will finally be complete, at least in a popular sense.

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Striking Alzheimer's before it strikes

Those following the news on trials of drugs against Alzheimer's disease must be familiar with the depressing outlook from the front lines. There was a string of failures reported in the last few years for therapies intended to disrupt the beta amyloid protein in AD. The failures have sent researchers back to the drawing board and the beta amyloid hypothesis itself has been strongly questioned. Amyloid is almost certainly involved in some big way with the disease, but its exact role as a causative agent has been under scrutiny for a while now.

Several factors could be responsible for the failure of these trials, but one factor in particular was bandied about as an obvious one; perhaps the intervention came too late to help the patients. We now know that diseases like AD and cancer often kick in quite early when they are beyond the detection limit of current diagnostic techniques. Perhaps, the thinking goes, we might stand a chance of beating the disease if we intervene early enough.

This thinking is completely sound, except for the problem that there is even now no definitive test to detect AD at very early stages. Fortunately for scientists - and quite certainly unfortunately for those unlucky enough to draw from this lottery - there are certain populations which are genetically predisposed to the disease. Members of these families typically get the disease in their 40s and by the 50s they are completely debilitated by it. The most prominent of these groups is an unfortunate family in Colombia, and the New York Times reported on them in 2010.

Now the Times reports on a drug trial designed to test the early intervention hypothesis in this clan. The drug in question is an antibody targeted against amyloid called crenezumab. The antibody was developed by Genentech and the preventative study is being jointly funded by the company, the NIH and a private foundation. Naturally it's going to be a long-drawn project; suspected patients are going to be started on the treatment when they are as young as 30, and their progress will be monitored meticulously over the next several years through both diagnostic mental tests and non-invasive techniques like PET scans.

This is a very hopeful and well thought-out experiment, and just like Derek who blogged about this today, I wish both the patients and the researchers the very best. Sadly, the history of the AD trials cited above does not fill me with too much hope. The amyloid hypothesis has constantly been under attack for the last decade or so. The most significant discovery in this regard was the finding that small oligomers of the protein rather than the full misfolded form might be the real culprit; but crenezumab seems to function by attacking the full form.

More intriguing and disturbing are the potential consequences for the normal health of the patients. Amyloid is definitely part of the normal functioning of the human body but nobody knows its exact function yet. However, it seems clear that the misfolded and normal soluble form of the protein are in some kind of equilibrium with each other. More interestingly, recent reports have implicated amyloid as an antibacterial agent. There's also some longstanding studies that suggest that amyloid forms free radicals which are usually toxic, but which may also help kill bacteria. I myself had speculated on amyloid's possible evolutionary role as a defense mechanism.

All this makes me skeptical about disturbing the normal vs misfolded amyloid equilibrium as a long-term strategy; the process may well be a crucial one, and killing the messenger might kill the message. Everyone wants this trial to succeed but we probably shouldn't be surprised if something disappointing shows up. One thing's for sure; this stuff will generate a lot of data, and that's what science is about.
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John LaMattina on the new NIH drug discovery center

There's a post by ex-Pfizer research chief John LaMattina about the new NIH drug discovery center, and predictably he does not seem too happy about it. While the initial center was supposed to be all about translational research, the latest idea is to use the NIH's resources for "repurposing", or discovering new indications for old drugs.

LaMattina echoes some of the dissatisfaction that a few of us have earlier expressed about this idea. The main point here is that the NIH should not be in the business of discovering new drugs; it should be in the business of doing the basic biological research that may enable such potential discoveries. In fact one might argue that the biggest challenge facing drug discovery today is an incomplete understanding of the complexities of the biology underlying major diseases. Just think of the conflicting data and the complications that have emerged from attacking beta amyloid in Alzheimer's disease for instance. There's hardly any doubt that better treatments can only result from a proper evaluation of the basic biology of disease. And it's also clear that this understanding is not going to come from industry. Only the NIH and academic labs can accomplish this, and spending money on therapies when it could more fruitfully be spent on such fundamental studies seems to be folly. So ironically, funding drug discovery may hinder an understanding of the very foundations that may truly enable it.

Nor is what the NIH doing truly novel. As LaMattina points out, repurposing is an obvious route and an attractive one at that, since finding a novel indication for an old drug means that the drug has already run the gauntlet of FDA approval. So we can bet that industry would have worked on repurposing if they could possibly do it. Now granted, there's always going to be compounds that were dropped for financial or project-related reasons which may be potentially valuable agents for all kinds of conditions. And we can also assume that these numbers might have grown during the last few years when projects have been axed and personnel laid off in increasing numbers. But what are the chances that hidden among those dusty vials on the shelf is the next cure for pancreatic cancer? Of course one may never find out if one does not look, but the NIH's announcements make it sound like there's pure gold among those neglected compounds, waiting to be discovered. The fact is that examples of truly repurposed drugs are quite few; as LaMattina points out, even the two repurposed drugs cited by NIH director Francis Collins are drugs for which the "other" indications were rather obvious based on their mechanism of action. Repurposing by itself is not entirely misguided, but repurposing at the cost of basic biomedical research draws resources away from more worthy endeavors.

Thus, by and large LaMattina's arguments seem to be cogent. Unfortunately the indignation on the other side of the equation is not as justified as it sounds. LaMattina refers to a statement by legendary Merck ex-CEO Roy Vagelos along the lines that if there was real benefit to something that the NIH wants to do, pharma would already be doing it. Sadly this is increasingly not the case. In the last few years pharma has defined "benefit" based on whether something's going to affect the next quarter's profits. Working on Alzheimer's disease and other CNS disorders where the rewards are long-term but undoubtedly stellar is no longer considered a beneficial strategy. So we have a situation here where industry is rightly advising the NIH to work on basic research rather than drug development, but not committing itself to its part of the deal. As well-intended as it may be, the impact of your advice gets blunted a little if you stop looking in the mirror.

A history of metallocenes: Bringing on the hashish

Following on the heels of the comprehensive article on metal-catalyzed reactions noted by Derek, here's another one by Helmut Werner specifically about the history of ferrocene and other metallocenes. It's got lots of interesting trivia about priorities, personalities and chemical developments. The article traces early priority disputes in the discovery of ferrocene followed by an account of the rush to explore other metal-organic systems.

It's hard for us today to imagine the shock that was felt on witnessing the existence of the first sandwich compound, a complex of iron sandwiched between two cyclopentadienyl rings. Before ferrocene the division of chemistry into inorganic (especially metallic) and organic compounds was assumed to be virtually set in stone, and this was one of those classic developments that shatters the mirror between two realms. The world of transition metal-mediated chemistry that the discovery inaugurated completely transformed the academic and industrial practice of chemistry, led to several Nobel Prizes and turned out to be one of the most beneficial scientific developments of the latter half of the twentieth century. 

The novelty of the new compound is best captured by what must surely be the most memorable reply sent by a journal editor to a submitting author, this one being from Marshall Gates (the editor of JACS) to R. B. Woodward:

"We have dispatched your communication to the printer but I cannot help feeling that you have been at the hashish again. 'Remarkable' seems a pallid word with which to describe this substance"

Perhaps the most extraordinary part of the story is the candid and rather dramatic note from Woodward to the Nobel committee lamenting his exclusion from the 1973 Nobel Prize awarded to Geoffrey Wilkinson and Ernst Fischer.

"The notice in The Times of London (October 24, p. 5) of the award of this year's Nobel Prize in Chemistry leaves me no choice but to let you know, most respectfully, that you have - inadvertently, I am sure - committed a grave injustice"

Woodward went on to rather pointedly emphasize his individual contributions to the discovery, making it sound like he had done Wilkinson at least a minor favor by putting his own name last on the manuscript. 

"The problem is that there were two seminal ideas in this field-first the proposal of the unusual and hitherto unknown sandwich structure, and second, the prediction that such structures would display unusual, "aromatic" characteristics. Both of these concepts were simply, completely, and entirely mine, and mine alone. Indeed, when I, as a gesture to a friend and junior colleague interested in organo-metallic  chemistry, invited Professor Wilkinson to join me and my colleagues in the simple experiments which verified my structure proposal, his initial reaction to my views was close to derision . . . . But in the event, he had second thoughts about his initial scoffing view of my structural proposal and its consequences, and all together we published the initial seminal communication that was written by me. The decision to place my name last in the roster of authors was made, by me alone, again as a courtesy to a junior staff colleague of independent status".

Interestingly, his recollection almost completely differs from that of Wilkinson's who stated in a 1975 review that he thought of the structure right away while Woodward immediately started thinking about its reactions. It's intriguing - and probably futile - to psychoanalyze the reasons for this very public expression of disappointment, especially coming from one who was not exactly known for publicly airing his personal feelings (for instance, his Cope Award lecture is the only time Woodward really provided personal biographical details). By 1973 Woodward had already won the Nobel Prize, and while he was always known to be extraordinarily ambitious, he must have known that his place in chemical history had already been secured; at that point he had even published the landmark papers on the Woodward-Hoffmann rules. Perhaps he sincerely felt that he deserved a share of the prize; nevertheless, it's a little curious that such a towering figure in the field made it a point to convey his disappointment at not winning a prize so publicly and strongly. Whatever the reason, Woodward's note makes it clear that scientists - both famous ones and otherwise - are keen to stake their priority. They are after all human.

To be fair to the prize committee, the award was given for the more general field of organometallic chemistry that the discovery of ferrocene launched rather than for the structure of ferrocene itself. Even at the beginning Wilkinson had been more interested in the new structural class of metallocenes while Woodward had been more interested in the kind of reactions the novel compounds would undergo. After the initial finding, while Wilkinson immersed himself in investigating the interactions of other metals with similar organic systems, Woodward went back to his life's love; the chemistry of natural products. Thus, it seems sensible in retrospect to have the prize given to Wilkinson and Fischer if the purpose had been to honor a new field of chemistry. Woodward died in 1979, and I am not familiar with his later thoughts on the subject if he had any. But of course, his place in the annals of science had long been assured, and ferrocene has turned into little more than an interesting historical footnote in his list of superlative achievements.

Note: The quotes by Woodward come from an article by Thomas Zydowsky from the Northeastern Section of the ACS that I had noted in the mailing list ORGLIST...in 2001. Time flies.