Enzymes as drug targets

"Enzymes catalyze multistep chemical reactions and achieve phenomenal rate accelerations by matching protein and substrate chemical groups in the transition state. Inhibitors that take advantage of these chemical interactions are among the most potent and effective drugs known. Recently, three new enzyme targets have been validated by FDA approval of new enzyme inhibitor drugs. These include mitogen-activated protein kinase, renin, and dipeptidyl peptidase IV. The drugs against these enzymes engage important enzyme functional groups, such as the active site serine in dipeptidyl peptidase IV."

There are more of them out there than we might imagine, and irreversible inhibitors are not always that bad.

"Some evidence now indicates that irreversible receptor protein-tyrosine kinase inhibitors might have advantages over reversible inhibitors in circumventing acquired resistance. The receptor protein-tyrosine kinases undergo ligand binding, homo-dimerization, hetero-dimerization, and internalization, whereas the intracellular nonspecific protein-tyrosine kinases lack these physiological processing events. Investigators speculate that reversible inhibitors may dissociate during receptor dimerization and internalization, and that significant signal trafficking can occur during the internalization.

By contrast, irreversibly inhibited receptor protein-tyrosine kinases would maintain inhibition throughout receptor recycling. Mutations that enhance receptor internalization would generate resistance to reversible inhibitors, but not to irreversible inhibitors. This is an interesting concept that remains to be explored.

Octagenarian worms on Prozac

"One goal of ageing research is to find drugs that would increase lifespan and vitality when given to an adult animal. To this end, we tested 88,000 chemicals for the ability to extend the lifespan of adult Caenorhabditis elegans nematodes. Here we report that a drug used as an antidepressant in humans increases C. elegans lifespan. In humans, this drug blocks neural signalling by the neurotransmitter serotonin. In C. elegans, the effect of the drug on lifespan is reduced or eradicated by mutations that affect serotonin synthesis, serotonin re-uptake at synapses, or either of two G-protein-coupled receptors: one that recognizes serotonin and the other that detects another neurotransmitterm octopamine. In vitro studies show that the drug acts as an antagonist at both receptors. Testing of the drug on dietary-restricted animals or animals with mutations that affect lifespan indicates that its effect on lifespan involves mechanisms associated with lifespan extension by dietary restriction. These studies indicate that lifespan can be extended by blocking certain types of neurotransmission implicated in food sensing in the adult animal, possibly leading to a state of perceived, although not real, starvation."

Nobel laureate Linda Buck's group finds out the ecstatic effects of human antidepressants on increasing worm lifespan. I am familiar with this "perceived state of starvation". I projected it during a Thanksgiving feast on Thursday leading to much generosity on the part of my hosts. Recall that caloric restriction is also possibly caused by wine molecule resveratrol.

Never a dull pharmacophore

The word "pharmacophore" was coined by Paul Ehrlich (a great word coiner; he also coined "magic bullet") as a reference to the essential chemical functionalities in a drug that makes it active. In the time since then, that word has been modified to mean the essential set of features in a drug, including their geometry and 3D orientation, that makes the drug active.

Pharmacophore modeling has become a stable of drug discovery and virtual screening, especially in the context of Computer Aided Drug Design (CADD). But even the average medicinal chemist is always interested in pharmacophores because he wants to know what's common between diverse structures that bind to a receptor that makes them active. Thus arises the goal of finding the "common pharmacophore" for different structures that bind to a site.

Sadly, the methods used for achieving this goal often fail because of some fundamental mistaken assumptions. The first assumption is that there is one, single pharmacophore. But ask anyone who uses a decent pharmacophore modeling program, and he or she will tell you that the program generates many pharmacophores, and it is not easy to decide which one among them is the "correct" one. The usual flaw in trying to come up with a common pharmacophore is to overlap compounds and look at common functionalities (hydrophobic, aromatic, charged etc.) in the same places. While this is a justifiable technique, the simple fact as noted above is that there are many ways to do this. One may overlay compounds so that say aromatic portions match with aromatic portions. But what about the other portions? How do we know that they don't constitute good binding features? Sometimes such trends or the lack thereof can be gleaned through traditional med chem and SAR, but more often than not they don't work.

In pharmacophore based virtual screening (VS), the technique is to come up with a pharmacophore and then compare it to a 3D library of possibly hundreds of thousands of compounds to find a lead that matches the pharmacophore in its 3D conformation. Since the pharmacophore essentially includes 3D information, the compounds in the databases have many conformers. In most cases, conformational searching for these compounds cannot be very exhaustive, but in most cases drug-like compounds have few rotatable bonds and so exhaustive conformation generation is not required.

Such simplistic techniques are seen in some early pharmacophore models. Say you have a peptide with a positive and negative end (NH2 and COOH) and you want to develop a good small molecule analog. You look at the minimum energy conformation of that peptide, or even the bioactive conformation if you know it. Then you note that the distance between the positive amino and negative carboxylate ends is 10 A. So with this pharmacophore, just an amino-carboxylate distance of 10 A, what you do is simply run this feature against a large 3D database of molecules hoping that you will come up with something that will also have the same distance between such two groups and therefore be active. This is admittedly the most simplistic pharmacophore ever, but Merck made it work for some fibrinogen agonists in 1992, which was an early example of pharmacophore-based virtual screening. The problem inherent in this protocol is that you are obviously neglecting other parts of the molecules which will dictate binding. Binding is a multipronged phenomenon and does not always depend on just two or three strong interactions.

This problem with investigating pharmacophores points to a larger truth; that ligands which bind to a common site in a protein may not bind the same way in many cases. For example, several people tried to come up with a "common pharmacophore" hypothesis for compounds binding to tubulin, such as taxol, epothilone and discodermolide. But a 2004 paper indicated that the binding site was promiscuous, that each ligand exploited the site in a unique way, and so trying to overlap them and come up with a common pharmacophore hypothesis was largely a futile effort.

If you have ever built a pharmacophore, you will also realise that it depends crucially on user input. Chemical intuition and knowledge of binding as well as known SAR data is very important for making decisions along the way. Pharmacophore modeling even more than other CADD protocols is not a black box.

In the future, pharmacophore models will continue to be used. Again, we must be realistic about their utility, which is seldom to come up with an ultra-potent lead, but to help in lead discovery. Some people think that because of this multiple-solution scenario, pharmacophore development is inherently flawed. I am more optimistic because first of all it is now possible to generate many pharmacophores and assess them against multiconformer libraries of a million compounds if necessary in a relatively short period of time. Secondly, model generation combined with careful analysis and astute chemical intuition has been known to pay dividends in the past, and there is no reason why it should not pay dividends in the future. At some point in the future, I am planning a post on some more details of pharmacophore modeling.

Of interest: A pharmacophore for kinase frequent hitters

Man-sized scorpion's claw found

It is morbidly wondrous to imagine such creatures paddling the oceans and swarming the earth in the past. No time machine for me...brrr.

Skin cells to stem cells

"As news of the success by two research teams spread by e-mail, scientists seemed almost giddy at the likelihood that their field, which for its entire life has been at the center of so much debate, may suddenly become like other areas of biomedical science: appreciated, eligible for federal funding and wide open for new waves of discovery."

Wow. I have said this many times before; no matter how much egghead Presidents and religious bigots suppress scientific research, science moves on. It may trudge or even falter sometime, but it will progress. Of that one thing we can be sure.

Now, researchers have come up with a method to use 4 genes to induce skin cells to transform into stem cells through a retroviral vector. I am yet to read the two reports in Science and Cell in detail (here and here) but the news for now sounds like a real breakthroughs; even the White House dispensed their insipid bleatings about it. But to hell with them for now. I always knew there could be a way around this, and now here it seems to be. Made the front page of the New York Times. More details later.

Links via email from Patrix

Mountains of malicious mutations

"Human cancer is caused by the accumulation of mutations in oncogenes and tumor suppressor genes. To catalog the genetic changes that occur during tumorigenesis, we isolated DNA from 11 breast and 11 colorectal tumors and determined the sequences of the genes in the Reference Sequence database in these samples. Based on analysis of exons representing 20,857 transcripts from 18,191 genes, we conclude that the genomic landscapes of breast and colorectal cancers are composed of a handful of commonly mutated gene "mountains" and a much larger number of gene "hills" that are mutated at low frequency. We describe statistical and bioinformatic tools that may help identify mutations with a role in tumorigenesis. These results have implications for understanding the nature and heterogeneity of human cancers and for using personal genomics for tumor diagnosis and therapy."

The future, ladies and gentlemen (or at least a small part of it). Commentary here.

Ethanol: saviour of the unfortunate...and the stupid

Ethanol, the much reviled yet irresistible satanic temptation that has plagued the human world for centuries, can actually be a giver of life in some situations...

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Increasing the bioavailability of Curcumin

"To improve the bioavailability of curcumin, numerous approaches have been undertaken. These approaches involve, first, the use of adjuvant like piperine that interferes with glucuronidation; second, the use of liposomal curcumin; third, curcumin nanoparticles; fourth, the use of curcumin phospholipid complex; and fifth, the use of structural analogues of curcumin"

The article is by Bharat Aggarwal, who first demonstrated that curcumin inhibits NfkB. The strategy can likely be used for other compounds too.

Give the kid S-15535, said Dad

"The researchers also found that they could alter the aggressive behaviour of their rats by manipulating the serotonin system. They gave the rats S-15535, a compound that binds exclusively to a neuron 'autoreceptor' that acts to dampen the serotonin system. This autoreceptor is called 5-HT1a. Binding to it seems to bring serotonin levels in the rats back to normal.

When even very low doses of S-15535 were used to bind to the receptors, de Boer found that both the serotonin and the violence of the pathologically aggressive rats returned to normal levels. The much more limited violence of naturally low-aggression rats could also be brought back to normal by the compound — but only by using higher doses. The compound seems to work by ‘fixing’ the serotonin breaking system, which becomes dysfunctional in the super-aggressive rats, says de Boer.

Hmm...drugs calming down violent rats...now who is going to convince Ann Coulter to enroll in the trial?

A simple substitution to possibly avoid phenol elimination

Several discussions on KP and other sites have focused on the unfeasibility of phenols as drugs because of their tendency to get glucuronidated, become more polar and get rapidly eliminated through the kidneys. So it seems helpful to know what one can do to a phenol in order to retain its electronic properties/steric properties but to prevent glucuronidation and elimination.

Just by chance I came across the following simple substitution: a phenol--->indole change, where the NH of the indole functions like the phenolic OH in hydrogen bonding situations but avoids the unfavourable glucuronidation. Certainly not a general solution, but a useful one that may work in some cases



Reference:
5-, 6-, 7- and 8-amino-2-(N,N-di-n-propylamino)-1,2,3,4-tetrahydronaphthalenes: centrally acting DA and 5-HT1A agonists
European Journal of Medicinal Chemistry, Volume 28, Issue 9, 1993, Pages 693-701
P Stjernlöf, T Elebring, B Andersson, A Svensson, K Svensson, A Ekman, A Carlsson and H Wikström

Paraquat, paraquat...

We had a Fraser Stoddart protege over for a talk today, and I have to say I can now really understand the appeal of Sir Fraser's chemistry. The molecules are really pretty and simple, the synthesis is really simple, the physical methods used are elegant and simple, the effects are simple to understand...everything just looks so simple! The appeal clearly lies in the simplicity and the potential applications. Science should be fun, but slogging through a 50 step total synthesis can tend to at least temporarily sap the fun out of it. In this kind of science, it seems the fun is much easier to retain. No wonder it's creating a buzz.

Why I have scant patience with postmodernists

This echoes my sentiments perfectly:

"There are lots of things I don't understand -- say, the latest debates over whether neutrinos have mass or the way that Fermat's last theorem was (apparently) proven recently. But from 50 years in this game, I have learned two things: (1) I can ask friends who work in these areas to explain it to me at a level that I can understand, and they can do so, without particular difficulty; (2) if I'm interested, I can proceed to learn more so that I will come to understand it. Now Derrida, Lacan, Lyotard, Kristeva, etc. --- even Foucault, whom I knew and liked, and who was somewhat different from the rest --- write things that I also don't understand, but (1) and (2) don't hold: no one who says they do understand can explain it to me and I haven't a clue as to how to proceed to overcome my failures. That leaves one of two possibilities: (a) some new advance in intellectual life has been made, perhaps some sudden genetic mutation, which has created a form of "theory" that is beyond quantum theory, topology, etc., in depth and profundity; or (b) ... I won't spell it out."- Noam Chomsky

Postmodernists positively irk me (like they did a few days ago) when they contend that science is "just another way of knowing the world" and that other ways of "knowing" the world have equal validity. For them scientific truth holds no overwhelming importance, because it is as much a product of culture as anything else.

Fine, I say. Next time you get a bacterial infection, don't take antibiotics because they constitute just one particular approach of looking at the world advocated by one particular school of thought. After all, the fact that antibiotics kill bacteria does not constitute "truth", right? Instead, just pray, or visit shamans, or rub lotion all over your body.

See you later if you stay alive.

GHB in Aqua Dots

So there it is...they are ominously out to poison everybody, especially through a sinister delayed action transformation of a precursor into a deadly chemical. I could not be more thankful that I did not play with Aqua Dots as a kid...

In any case, does anyone know what it was in the dots that transformed into GHB (Dr. Sanjay Gupta probably does not)? Wikipedia lists two possible prodrugs, the lactone which is ring opened and 1,4 butanediol which is oxidised to GHB by alcohol dehydrogenase. Or maybe it's just proprietary ("Better killing through proprietary means")

As bad as it gets: a debate with two surprisingly creationist gentlemen

How a debate with two friends about creationism turned into a defense of science itself...

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You've lost that gut feeling

I came across a nifty Pfizer review published in JMC a couple of years ago about property-based optimization of drug candidates. There are many words of wisdom in it about various pharmacokinetic parameters and processes and related property optimization. One of the most interesting analyses I found was of gut absorption. Apparently:

1. Gut absorption is of two types; passive diffusion through lipid bilayers and passage through tight gap junctions. For drugs that undertake the latter route, size is obviously important. It turns out that dog GI gap junction pores are larger than in humans, so that a drug that passes readily through dogs won't pass readily through humans, highlighting the drawbacks of using dog models for studying human absorption. Also, Caco-2 cells seems to be bad models for drugs that undergo passage through this route. Fortunately, most drugs seem to pass via the first route, that is diffusion through lipid bilayers.

2. P-glycoprotein (PGP) and CYP3A4 (cytochrome P450 isozyme) are important in the gut, even if CYP3A4 expression is not as much as in the liver. The most interesting fact I read was that in case of PGP, the expression increases from top to bottom in the human digestive tract, with the least in the duodenum and the most in the colon. But for CYP the trend is opposite. Clearly this will influence structural considerations based on where the drug is going to be absorbed as well as drug delivery tactics.

3. CYP and PGP often work in concert in the gut, with PGP recycling substrates and offering them multiple times to CYP.

SAR for PGP and CYP definitely seems to be scarce, not surprisingly given that their substrate scope is quite large. Certain molecules such as the well-known immunosuppresants (eg. cyclosporin) have been identified to be rapidly metabolised by these enzymes. On the other hand, hyperforin from St. John's Wort can induce CYP expression and women on birth control pills might suddenly get a nasty surprise and find themselves pregnant; the hyperforin causes excessive metabolism of birth control molecules and reduces their blood levels to non-efficacious doses. Lipophilic drugs with electron rich groups can be choice fodder for CYP. Given the recent review highlighted by KinasePro which concluded that reducing lipophilicty should be a goal for avoiding poor absorption and promiscuous binding, this seems to be another good reason for losing the grease.

What doth a methylene?

One common strategy used by medicinal chemists for enhancing potency is to add a methylene carbon in a ring that's fitting into a lipophilic pocket. A methylene group usually contributes about 4 kJ/mol of affinity....or maybe not.

Gerhard Klebe's group at Marburg are studying inhibitors of thrombin in which they replace a cyclopentyl group by a cyclohexyl group. To their surprise they observe (DOI: 10.1002/anie.200701169) no change in binding affinity. Crystallography indicates no electron density for the part occupied by the cyclohexyl group but robust density for the cyclopentyl. The authors conclude that while the cyclohexyl binding is enthalpically unfavourable, entropically the six-membered ring can flip and twist and dance, which is favourable, and also does not provide much crystal density because of its flexibility. So unfavourable enthalpy is balanced by favourable entropy. MD simulations support this contention.

"What lesson can be learned from this example? Usually ligands are optimized in congeneric series. The addition of functional groups is expected to enhance binding; for example, the change from a five- to a six-membered ring should augment binding affinity by approximately 3–4 kJ/mol
. However, even very similar ligands can exhibit very different binding properties that destroy a simple structure–activity relationship"

Also, the gain in binding affinity from a methylene depends on the system. Model systems based on octanol-water partitioning offer a value of about 0.7 kcal/mol. But of course octanol unlike a protein does not have a problem reorienting itself around the substrate. Thus, the value can be less for a protein. On the other hand, in cases where an extra methylene or methyl just about fills the remaining space in a pocket and packs tightly can provide 5 times as much binding affinity. This is true for example in case of tRNA synthatases (I got this from Alan Fersht's excellent book) As usual, exceptions abound to the generalisations, and Klebe's study provides another interesting example.

The Traffic Light

Part of the reason I made the trip to London in September was a single goal; to stand at a particular traffic light near the British Museum and take a photo of myself standing there. Later when I told people about the reason, most thought it was silly, and perhaps it was. But all of us have a romanticised impression of certain people, places and events in our mind which other people could find silly. In this particular case, the person, place and event involved were profound even if little known to the general public. In fact this light was so important for me that I had made up my mind to visit London once in my lifetime for the sole purpose of standing at the light, if not for anything else. What was so special about this traffic light?

It was 1933. Adolf Hitler had come to power in January, The Depression was raging and the future looked bleak to many. On the morning of September 12, 1933, on a miserable, wet, quintessentially English autumn day, at the intersection where Russell Square meets Southampton Row, Leó Szilárd waited irritably at a traffic light waiting for it to change from red to green. He had just attended a lecture by the great English physicist Ernest Rutherford. Rutherford, known to many as the father of nuclear physics, was discussing the newly prophesied release of energy from atoms, most notably by science-fiction pioneer H G Wells in his book The World Set Free. In his baritone voice, Rutherford, acknowledged master of the atomic domain, dismissed this fanciful idea as nonsense. Any thought of releasing the energy locked in atoms, he said, was "moonshine".

Szilárd was irritated by this flippant repudiation. Accomplished as he was, how could even the great Lord Rutherford know what the future held in store? Szilárd, peripatetic Hungarian genius, imperious habitué of hotel lobbies, soothsayer without peer among scientists, had himself thought deeply about nuclear matters before, most often during his extended morning bathtub ablutions. Now waiting for the light to change, Szilárd pondered Rutherford's words...I will let the acclaimed nuclear historian Richard Rhodes do the talking here. It was the riveting description of this event in Rhodes's magnificent book that engraved it in my mind like nothing else:

"In London, where Southampton Row passes Russell Square, across from the British Museum in Bloomsbury, Leo Szilard waited irritably one gray Depression morning for the stoplight to change. A trace of rain had fallen during the night; Tuesday, September 12, 1933, dawned cool, humid and dull. Drizzling rain would begin again in early afternoon. When Szilard told the story later he never mentioned his destination that morning. He may have had none; he often walked to think. In any case another destination intervened. The stoplight changed to green. Szilard stepped off the curb. As he crossed the street time cracked open before him and he saw a way to the future, death into the world and all our woes, the shape of things to come"

Time cracked open indeed. What Szilard realised as he stepped off that curb, was that if we found an element that when bombarded by one neutron would release two neutrons, it could lead to a chain reaction that could possibly release vast amounts of energy. Leo Szilárd had discovered the nuclear chain reaction long before anyone else, six years before the discovery of nuclear fission and any inkling that anyone could have had about the release of atomic energy, let alone the woeful apocalyptic future that would await the world because of its release.

I first read Rhodes's book in 2000. The book begins with this story. The description is so riveting, the tale so profound and evocative, the person so singular and the implications so prophetic, that I resolved to visit Szilárd's traffic light, my traffic light, even if I had to once make a trip to London for just that. Since then, the event has been etched in my mind like words in red hot steel. Seven years later I got a chance.

The traffic light itself is completely non-descript, standing among dozens of other non-descript lights. We almost missed it; as I mused aloud about my great disappointment to my friend in a cafe and wished I had a map, a Spanish tourist sitting at the next table saved my life and procured one. The intersection was there. We had missed it by a block. Back we went and indeed there it was, with not an indication that a famous and prophetic physicist had seen into the future at that light some 75 years ago.

As it turned out at the time, Szilárd's choice for the element he was thinking about turned out to be wrong. Nuclear fission would be discovered only six years later in Germany after a series of close misses in Italy and France. But Leo Szilárd went down in history as the man who saw death before anyone else, a glimpse into mankind's Faustian pact with fate, the shape of things to come.

Ironically, when the first atomic bomb test was conducted in the New Mexico desert in the deathly stillness of the morning, in the midst of war and hope, the flash was so bright that it would have been seen reflected off the moon. It was, literally, "moonshine". The rest was history.

But I lived one of my dreams that day at that traffic light in London. Szilárd's traffic light. My traffic light.

Nuclear terrorism's unheeded assumptions?

Nuclear terrorism forms an important part of the armamentarium of one of the Bush administration's favourite pastimes- threat inflation. While it is true that the potential damage that terrorists could cause with even a 1 kT nuclear weapon is tremendous (Times Square NYC, noon on a weekday), there are many very realistic obstacles they need to overcome before they can acquire, process, build, transport and use any kind of a nuclear weapon.

The more realistic fear that governments and the public have is about dirty bombs, explosives packaged together with low-tech dispersive radioactive material that would largely circumvent the need to achieve the myriad steps needed to be in charge of a bonafide atomic device.

Writing in the Bulletin of the Atomic Scientists, Sonia Ben Ouagrham-Gormley challenges two assumptions made by proponents of a nuclear terrorist attack scenario: access to knowledge and the existence of a nuclear black market (exemplified by black market czar Pakistani scientist A. Q. Khan). Gormley correctly tackles the myth of easy access to nuclear material and knowledge and identifies the slip between the cup and the lip- from knowledge to working product.

She also questions the ease of facilitation of trade in the nuclear black market and doubts the existence of a dedicated clientele, an essential feature of any black market. The clientele should also have the understanding and sophistication to purchase and process nuclear material (In the early days of Al Qaeda, Bin Laden was had when someone sold him mercuric oxide as yellowcake).

Lastly, she questions the nature of materials that have been implicated in nuclear smuggling until now, most of which included depleted uranium and isotopes like Osmium 167, too ineffectual in a dirty bomb, let alone a weapon.

But I think she is missing out on three other important isotopes which are widespread products of research reactors, large scale reactors as well as medical research reactors- Iodine 131, Cesium 137 and Strontium 90. Out of these, Iodine 131 can be absorbed by the thyroid gland and leads to thyroid cancer, but its effects can be thwarted rather easily by ingesting tablets made of normal non-radioactive iodine, provided such tablets are easily available (the slow dissemination of these tablets was partly responsible for the large number of deaths from Chernobyl). Cs 137 and Sr 90 pose more serious problems, and I would think that more than anything else they would be choice materials for a dirty bomb. Both isotopes seem to strike the golden mean for radioactive lethality, possessing half-lives of 30 years and 28 years respectively; long enough to compare to a human life span, and short enough to be intensely radioactive. Moreoever, both elements chemically resemble two key elements in the human body. Cs 137 behaves somewhat like potassium and distributes throughout body fluids and compartments, whereas Sr 90 resembles calcium and deposits in bones, greatly increasing the risk of bone cancer. Both elements if ingested in reasonable amounts will pose almost irreparable risk and cause permanent damage.

I certainly don't think one should be immediately paranoid about these isotopes, but it is clear that if they wanted to, terrorists could steal them from multiple sources. I would think that any perceived scenario involving terrorists and dirty bombs should include discussion of these three isotopes, which because of their ease of access and purity are in some ways much more lethal than uranium or plutonium.

A chat with Nobel laureate Peter Agre

It’s not everyday that you get to have a relaxed, inspirational and informal almost one-on-one chat for an hour with a Nobel Prize winner. Yet that was what it was today morning...

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