The same and not the same: more aggregates in HTS

High-throughput screening (HTS) is now a mainstay of drug discovery and usually the starting point for most drug discovery projects. Industry usually has a lot of resources invested in HTS and therefore needs to be aware of false positives and false negatives that could hamper useful results and lead one down an erroneous path.

Among the many factors responsible for false positives in HTS, one of the most startling and important factors recently unearthed is the non-specific and potent inhibition of enzymes by aggregates of molecules occurring under typical assay conditions. These aggregates are large enough to be observed under a microscope and to be detected by dynamic light scattering. The aggregates adsorb enzyme molecules on their surface, and one of the best tests for detecting their presence is to re-run the enzyme assay under high detergent concentration. High detergent concentrations usually break up the aggregates and lead to a loss of potent inhibition. The phenomenon of aggregation-based inhibition was accidentally discovered by Brian Shoichet's group at UCSF and has been comprehensively explored by him and his students in a series of papers throughout the last decade, although much is still to be known about the exact physical nature of these aggregates. The reason why this has become a big deal is because it has been observed in an unusual number of cases, which leads to the suspicion that much effort might have been already expended in drug discovery campaigns in pursuing such false leads.

In a recent paper, Shoichet and Craik's groups at UCSF accidentally discovered aggregate-based inhibition in discovering inhibitors for the enzyme cruzain which is a part of the metabolic machinery of the parasite responsible for Chagas disease. The authors had started with an initial hit from a virtual screening campaign and were engaged in the usual process of modifying the hit based on SAR. The initial tinkering led to a series of oxadiazole inhibitors which exhibited potent inhibition of cruzain.

However, many of these molecules failed to show activity in cell-based assays. Such a discrepancy between enzyme and cell-based assays can be traced back to many reasons including poor permeability. But in this particular case, kinetic measurements hinted at aggregates of the oxadiazoles that were inhibiting the enzyme. At this stage it was also discovered that unlike the initial hits series, the oxadiazole series had been accidentally assayed under low detergent conditions. The molecules also inhibited another intensely studied enzyme in the Shoichet group- AmpC beta-lactamase. The quintessential test for aggregate-based inhibition, namely increasing the concentration of detergent (Triton in this case), also proved positive confirming the phenomenon. Interestingly the initial set of hit molecules also seemed to exhibit this phenomenon but only in case of AmpC lactamase and not in case of cruzain. In case of cruzain, experiments with differing detergent concentrations proved that the initial set of molecules were equally potent under both conditions, while the oxadiazoles lost activity under high detergent conditions, indicating divergent modes of inhibition between the two sets of molecules.

Finally, note that the aggregation-based inhibition would likely have not been discovered if the oxadiazole series had been assayed under the same low detergent condition as the initial hit series. What seemed like similar molecules turned out to behave very differently under different assay conditions. Sometimes mistakes can reward you with unexpected treasures, and similarity needs to be pried out from the eye of the experimenter. Never underestimate the importance of going wrong (of course revealed only in retrospect).

As the authors narrate, the moral of such studies should not be lost on medicinal chemists, who usually interpret high and low potency of related molecules based on local structural features like hydrogen bonding, electrostatics and hydrophobicity. Aggregation-based enzyme inhibition proves that chemists have to look beyond single molecule structural features toward supramolecular features of several molecules that are interacting with each other. Chemists regularly engaged in HTS campaigns might well keep this valuable piece of advice in mind. Scientific enumeration, it seems, has to always be done at several different levels.

Note: Apologies to Prof. Roald Hoffmann for appropriating the title

Ferreira, R., Bryant, C., Ang, K., McKerrow, J., Shoichet, B., & Renslo, A. (2009). Divergent Modes of Enzyme Inhibition in a Homologous Structure−Activity Series Journal of Medicinal Chemistry DOI: 10.1021/jm9009229

Solid strands of knowledge

The Strand Bookstore in New York City on 12th street, close to NYU, is a book lover's paradise. Multiple copies of used and new books grace the shelves together (like they do in Powell's bookstore in Portland). Almost all of them are discounted. I love their collection described as "18 miles of books"; in my eyes the description evokes a never ending magic carpet full of novelties.

The store is inhabited by hungry creatures with scruffy beards, colorful attire, thick glasses, exotic skirts and a generally zombie-sh look in their eyes. It is hard not to get infected. Last Sunday I could finally visit the place and acquired a handsome set of volumes. I had to force myself out but plan to visit again as soon as possible. My acquisitions include the following:

* A Question of Balance: Weighing the Options on Global Warming Policies- William Nordhaus (reviewed by Freeman Dyson)
* Six Impossible Things Before Breakfast: The Evolutionary Origins of Belief- Lewis Wolpert
* The Cambridge Quintet: A Work of Scientific Speculation- John L. Casti
* Six Questions of Socrates: A Modern-Day Journey of Discovery Through World Philosophy- Christopher Phillips
* The Way of the World: A Story of Truth and Hope in an Age of Extremism- Ron Susskind
* The Age of Wonder: How the Romantic Generation Discovered the Terror and Beauty of Science- Richard Holmes
* Deciphering the Cosmic Number: The Strange Friendship of Wolfgang Pauli and Carl Jung- Arthur I. Miller

The penultimate volume looks fascinating and has gathered a highly appreciative review in The Times. The last book especially was a revelation. I don't know how they managed to price a newly published book at less than half the price.

Ron Susskind's book is a riveting read that draws together the stories of disparate individuals; when I first checked out the hardback from the library I could not put it down for the entire night. As the synopsis says,

The Way of the World simultaneously follows an ensemble of characters in America and abroad who are turning fear and frustration into a desperate—and often daring—brand of human salvation. They include a striving, twenty-four-year-old Pakistani émigré, a fearless UN refugee commissioner, an Afghan teenager, a lawyer fighting for a Muslim man incarcerated in Guantanamo, a state department official desperately working to keep nuclear weapons out of terrorist hands, a Holocaust survivor’s son, and Benazir Bhutto, who discovers, days before her death, how she’s been abandoned by the United States at her moment of greatest need. They are all testing American values at a time of peril, and discovering solutions—human solutions—to so much that has gone wrong. For anyone hoping to exercise truly informed consent and begin the process of restoring the values and hope—along with the moral clarity and earned optimism—at the heart of the American tradition, The Way of the World is a must-read

I am planning to combine Wolpert's book with Robert Wright's recent "The Evolution of God" which talks about a similar theme. Christopher Phillips had entertained and informed many years back with "Socrates Cafe" and this work seems to further explore his travels and encounters. Nordhaus who is at Yale is one of the preeminent environmental economists in the world and considered a leading authority on economic solutions to climate change. The problem has now passed on from the hands of the scientists to those of the economists.

Enabling an antibody to jump the species barrier

Antibodies are invaluable tools not just in fighting disease but in exploring fundamental biological pathways and mechanisms. As is well known, they owe their useful properties to their very high specificities. Unfortunately these specificities can also hinder the application of an antibody from one species in the study of antigens in another species. For instance, an antibody designed for a human protein could bind much more unfavorably to a mouse ortholog of the same protein because of subtle differences in residue identity and placement.

Emerging computational design of protein-protein interfaces has the potential to make contributions in redesigning antibody surfaces to bind to orthologs. To this end, Craik, Jacobson and others from UCSF describe energetic analyses of combinations of mutations that allow an antibody for a human protein implicated in cancer to bind with high affinity to its mouse ortholog. The wild-type antibody binds about 300 times less strongly to the mouse ortholog. Simple biochemical and structural studies fail to pinpoint the reason for this reduced binding affinity, since only three residues differ between the human and mouse proteins and these don't seem to be directly involved in binding.

The authors decided to predict mutations at the antibody-protein interface that could possibly improve binding affinity for the mouse protein. They identified six protein residues in the antibody that were in proximity to the three differing residues. They then built a homology model of the mouse protein based on the crystal structure of the antibody-human protein complex and placed the mouse protein homology at the same location as the human protein in the complex.

The calculations were done with an implicit solvent model and involved optimizing side chain conformations and then minimizing the energy of the complex using molecular mechanics. Admittedly this is a relatively crude, approximate process and the authors admit that factors like changes in protein conformational entropy which are very difficult to calculate have been neglected. In order to gauge the effect of mutations, each one of the six residues was mutated to every one of the 18 other amino acid residues. Many mutations failed to show an improvement in binding affinity. However eight mutations indicated possibly improved binding to the mouse protein.

To validate these calculations, antibodies with the predicted mutations were engineered using recombinant techniques and their binding affinity for the mouse protein were measured. One mutation from a proline to a histidine was predicted to lead to a positive change in affinity. Experiments however indicated decreased binding. This was an indication of increased flexibility (and probably some entropy loss) imparted by the more flexible histidine, a factor that was not included in the calculations. However, another calculated increase in affinity was validated by experiment; this involved a threonine to arginine mutation. The arginine was predicted to form a hydrogen bond with a backbone carbonyl and a glutamate side chain, contributing about 1.6 kcal/mol of binding energy. The experiments demonstrated a 30 fold increase in binding affinity thus confirming the prediction. The structural prediction also explained why the human ortholog did not bind as well with the mutant; the glutamate there was replaced with an aspartate, whose side chain was not long enough to interact with the arginine.

The real value of any model is in prediction. This is a goal that climate modelers, financial modelers and computational chemists struggle with. The various factors that are at play are usually too complex to model and approximations are essential. It's nice when, once in a while, one of these approximations actually works. Such results provide hope for future design of protein-protein interfaces, one of the most challenging and potentially immensely useful aspects of biomedical research.

Farady, C., Sellers, B., Jacobson, M., & Craik, C. (2009). Improving the species cross-reactivity of an antibody using computational design Bioorganic & Medicinal Chemistry Letters, 19 (14), 3744-3747 DOI: 10.1016/j.bmcl.2009.05.005

Can a religious person head the National Institutes of Health?

Francis Collins is an unusual scientist. A physical chemist and doctor who rose to prominence as the leader of the Human Genome Project, he has recently been appointed by President Barack Obama as head of the NIH, the largest biomedical funding and research organization in the country. Collins is unusual because along with this undoubtedly distinguished scientific credentials he brings another kind of background to the job; that of a pious, church-going Christian. A few years ago Collins published a book that argued for a scientific basis for belief in God, and not just a theological one. Needless to say, his views have caused concern among a number of atheist scientists and secular scientists in general.

It is one thing to be a deist, namely someone who believes in the kind of abstract God embodied in the laws of nature who could have set the universe in motion and then let it run its course without interfering, but quite another to be a proper theist, a person who believes in the kind of material God that most people who believe in God invoke, one who helps out or doles out punishment in daily life and personal matters. Collins seems to be more of a theist. And therefore his appointment to the NIH again raises a question which has fomented reams of arguments, and sometimes almost violent argument, on blogs and in books. The central question is; can a scientist truly be religious? Since this question immediately gets you into a morass of conflicting views and definitions, I will simply state my one line answer to the question in this specific context; from an empirical standpoint of course you can be religious and a scientist, as demonstrated by the existence of many religious scientists like Collins. But from a philosophical standpoint, you then have to accept that there is some very strange compartmentalization in your mind that allows you to essentially sustain two opposite and clearly conflicting paradigms simultaneously, one paradigm in which faith without evidence is positively eschewed and another in which faith without evidence is positively extolled. As an aside, it is a fascinating scientific question how such compartmentalization can occur.

Collins has come under fire from, among others, one of my favorite writers Sam Harris, whose controversial book The End of Faith made a compelling case against religious faith. In a piece in the New York Times, Harris criticizes Collin's views, best enumerated in his book, which essentially proclaim that God must exist since some things are beyond the scope of science. Both Harris and me find this kind of reasoning remarkably simple-minded. How does someone know that things that are beyond the scope of science today would always be so? For instance Collins quips that God must have adjusted the values of the so-called fundamental constants of nature (such as Planck's constant, the speed of light etc.), since life seems to depend on an incredibly precise fine-tuning of their values. But how do we know that science is never going to provide an answer for their origin? If "God" is simply a place card for "we don't know" then it sounds fine, but Collins seems to actually imply some kind of interventionist God here.

More importantly, does such a belief in God mean that we should automatically stay away from certain things and not apply critical questioning to them because they have been declared by religions to be supernatural by definition? Even Collins will admit that pushing any question under the rug by default by declaring that it is beyond the purview of science is completely antithetical to rational inquiry.

Harris is particularly concerned about Collins's enunciations about neuroscientific research. The last few decades have provided spectacular and remarkable insights into attributes that were for many years considered almost mystical, things like human emotions, love and the nature of faith itself. But in recent years, science is gradually opening a window into these attributes, answering questions like; What happens when someone is praying? How does the brain look when it is experiencing intense emotion? Studies like functional magnetic resonance imaging (fMRI) are making significant headway into answering such questions and detecting common patterns of neuronal activity that are at play. Collins essentially says that there is something special about faith and human emotions and that God injected these qualities into human beings at some point in our evolution. Would Collins then have us not explore the scientific basis of such human attributes by assuming that they are beyond scientific inquiry? These questions worry Harris and some others like evolutionary biologist Jerry Coyne of the University of Chicago, and they worry me. Some of Collins's statements from a set of slides that are cited by Harris and Coyne really sound preposterous.

Is it dangerous to have a bona fide Christian heading the most important biomedical research agency in the country, and one of the most significant of its kind in the world? In spite of the above concerns I would say not per se. I would say that Collins should be given the benefit of doubt. After all, there does not seem to be a shred of evidence that his religious leanings have affected his capacity for objective scientific judgement. The NIH is a scientific organization and Collins's leadership of it should be judged purely based on his handling of the science. We should not care as much about what he says as about what he does. In the absence of evidence that his religious views are affecting his ability to allocate funding for specific kinds of research, he should be closely watched but not condemned.

At the same time, I share Harris, Coyne and others' sense of unease for a totally different reason; Collins's appointment might unfortunately have the disastrous side effect of enabling creationists and proponents of intelligent design to clamor and push their agenda for turning science and religion into convenient bedfellows. Creationists might make Collins's appointment a case for asserting that not only is religion compatible with science but it's even necessary, as purportedly exemplified in the highest echelons of science policy and research. Collin's appointment may sadly make it easier for the creationists to weasel their way into sensible scientific debate. For this reason the appointment may cause problems, and we will have to make sure that Collins is constantly watched and criticized wherever appropriate. The price of scientific freedom, it seems, is indeed eternal vigilance.

The leap forward

In 5 to 10 years, I could walk into a doctor's office and for less than 1000$ have my genome sequenced. This could tell me how likely I am to get Alzheimer's disease, cancer or heart disease. Such possibilities open up enormous technical, legal and moral challenges.

That this would be possible in 5 to 10 years is what Craig Venter says. Coming from a lesser man this would seem like wishful speculation, but Venter is the man who single-handedly raced the government to "shotgun" sequencing the human genome and he has a knack of turning dreams into reality. If there's one word to describe Venter it's "big". Even after the human genome the man has not rested on his laurels. Over the last few years he has trawled the seven seas in his boat, gathering marine water samples everywhere and analyzing them for interesting bacteria and unusual DNA sequences. Genetically engineering these bugs could give us organisms that could mop up CO2, that would produce biofuels. Recently Venter has teamed up with Exxon (who could imagine!) for investigating genetically engineered algae that would produce hydrocarbons for transportation fuel.

Venter aims to have a unique genomic sequencing facility where he can sequence the genome of almost any organism one can think of. The computational and sequencing power in this facility is incredible and it seems to get bigger and more efficient every day. Here he shows the facility and has a conversation about it with Richard Dawkins. Venter says that mammalian genes are no longer very interesting because they don't show that much variation compared to some other genomes such as insect and bacterial genomes. Right now Venter has a room full of sequencers and behind a secret curtain he already has a large sequencer that would replace this entire room. The rate at which the technology is growing is breathtaking. This is a one of a kind tour and the entire video is worth watching. When Dawkins asks Venter if he is trying to "play God", Venter quips that he "does not play mythological characters"...



I would very strongly recommend Venter's autobiography. His journey from a careless, unfocused, fun-loving teenager who, shaped by harrowing experiences as a medic in Vietnam brought pin point focus to his career is amazing. Venter has been called reckless, audacious, arrogant, egoistic, curt and apathetic, and to be honest all these qualities shine forth on many pages of his book (however he appears pretty modest and engaging in most of his interviews including the one above). He has managed to alienate many people who he worked with (as someone quipped, he will never win a rightly deserved Nobel prize because of his personality traits). But it doesn't matter; he is a born entrepreneur and he is nothing but brilliant and ambitious, an intrepid risk taker. He is one of those people who will bulldoze his way through problems and not care what other people think. Nobody has accomplished what he has.

Just when I was going to order the t-shirt...

Does anyone else see the problem here?.
Nothing gets an organic chemist's goat like wrong structures. Except maybe for structures that don't exist.

The importance of being patient

The determination of the ß-adrenergic receptor GPCR structure in 2007 was a breakthrough in structural biology. Combined with the earlier structure of rhodopsin, this provided a template for structure-based design for GPCRs. However, there was a lurking mystery in the structure, a mystery which was not always discussed but which has started to come to light recently.

Th mystery is exemplified by a recent paper in which authors from D E Shaw Research in New York use extremely long molecular dynamics simulations to uncover a peculiar conformational characteristic of the ß2 AR. The original structure was crystallized bound to an inverse agonist named carazolol. The receptor as crystallized was thought to be in an inactive state. In this state, two helices of the receptor were at some distance from each other. However, this observation did not square with biochemical experiments that indicated proximity of the two helices mediated by a crucial ionic lock, a salt bridge between a glutamate and arginine. This lock however was absent in the crystal structure, raising questions about the exact role of the lock in activating the receptor and the nature of the inactive state.

In the present study, the authors used extremely long, microsecond MD simulations on the crystal structure. They used the DESMOND program recently introduced by Schrodinger and D E Shaw to perform simulations of the GPCR in a lipid bilayer.

All they really had to do was wait.

The first 150 ns were not very interesting from the perspective of the salt bridge. However, the salt bridge spontaneously formed after 150 ns and then stayed put like a fly on fly paper. Notice the N-O distance (blue) and how it stabilizes after 150 ns.




The bridge also involved local movement of the helices and some important residues. The authors also did the simulation in the presence and absence of the ligand and found that this lock forms irrespective of the presence of the ligand. They follow up with some mutagenesis experiments that reconcile the conformational changes with experimental observations. Interestingly, they mutate an aspartate that is also proximal to the arginine in the salt bridge. Mutation of this aspartate would be expected to "free up" the arginine and further encourage its interaction with the glutamate. However, the opposite seemed to happen, indicating an interesting role for the aspartate as a something of lock itself in holding the aspartate fixed.

The overall conclusion is that there are probably two inactive states, one in which the salt bridge is formed (the dominant one) and one in which it's broken (not highly populated) and the receptor recycles between the two. The less populated conformation is nonetheless the one that is crystallized which is interesting. This kind of observation is clearly important for further structure-based design since it implies that one could encourage GPCR activation if the "right" conformation of the receptor could be preferentially stabilized.

The thing to note here is the time. Nothing interesting would have been observed had the simulation been run for less than 150 ns. Researchers who ran the simulation for less than 150 ns may not have had something worth reporting. 150 ns is a reasonably long amount of time for any MD program or simulation. The fact that such simulations can be run for microseconds attests to the rapid development of hardware and software exemplified by D E Shaw's program DESMOND and their processor named ANTON.

Sometimes simply waiting long enough can lead to productive results. Echoing an unpleasant man's ominous pronouncement, "Quantity has a quality of its own".

Dror, R., Arlow, D., Borhani, D., Jensen, M., Piana, S., & Shaw, D. (2009). Identification of two distinct inactive conformations of the ß2-adrenergic receptor reconciles structural and biochemical observations Proceedings of the National Academy of Sciences, 106 (12), 4689-4694 DOI: 10.1073/pnas.0811065106

Lindau: The teachings of the savants

Marie Curie once said that "Science is about things, and not people". While this statement is true and profound, the fruits of science are unmistakably linked to their human origins, postmodernist relativism notwithstanding. The scientists who make discoveries are human beings, and they shoulder their share of foibles and successes, petty rivalries and forthcoming generosity, despair and triumph. Their life displays cycles that any young researcher will go through in his or her future career...

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Lindau: Who is the joke going to be on?

When the controversial and talented physicist Edward Teller was doing a PhD. with the great Werner Heisenberg at the University of Leipzig, the question asked at the end of every group meeting that focused on a complex sequence of problems was "Wo ist der Witz?", supposed to be translated as "What is the point"? but more correctly translated as "What is the joke?". The joke part of it consisted of turning a wry eye at the world, donning the hat of the court jester who laughs even as the fire that he predicted would engulf the world rages on. The question about global warming that we ask is also "Wo ist der Witz"? and we only hope that the joke is not upon us and we can actually still get the last laugh. Whether we might was the topic of discussion of a panel on global warming on the final day of the 59th Meeting of Nobel Laureates at Lindau...

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Lindau: From fullerenes to global education

When I visit my favourite restaurant for lunch or dinner, I usually order a legitimate food item from the main course. But once in a while, just to indulge, I order a sample platter of appetizers. The appetizers don't always provide the deep satisfaction that I get from eating a proper, expensive food item. But they provide me with a different kind of unique satisfaction; they give me a glimpse of what's new, what's possible. They provide a view of the diversity that can emerge in a plate of bite-sized chunks. And through their frequent novelty, they give me hope that there are new possibilities on the horizon. These appetizers constitute occasional but necessary fodder. Sir Harold Kroto's talk was one of the most satisfying platter of appetizers I have sampled, and I had not even ordered it...
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Lindau: The way dinner should be

When you first meet Aaron Ciechanover, he appears to have the distracted air of a man who feels slightly inconvenienced to be in whatever situation has been apparently imposed on him. But this preoccupied demeanor belies a mind which is ready to hold forth on a disparate variety of topics with infinite verve and enthusiasm and which is not reluctant to be politically incorrect, provocative and utterly honest. And it hides a broad smile which is very readily revealed at the mention of a favourite incident or fact.

If there is one word to describe the Israeli doctor, biochemist and Nobel Laureate it's passion, and this passion is pronounced no matter what the topic of discussion; from protein degradation to languages and traveling, from politics to history. Whether we were talking about protein structure or Israel-Palestine relations, Ciechanover's thoughts were always opinionated, honest, cogent, provocative and without a dull shade in them. This is the kind of stimulating person that you always want as a dinner companion...

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Lindau: the glowing joy of discovery

Last year's chemistry Nobel Prize was one of the most softball predictions ever made for the Nobel Prize. The Green Fluorescent Protein (GFP) has become so widely used in chemistry, biology and medicine that it is easy to forget that someone had to discover it and develop the technology. Every year Roger Tsien's name used to be on everybody's favorite candidate list along with Martin Chalfie's and Osamu Shimomura's. Then last year, he along with Shimomura and Chalfie finally put the tortuous process and spilling of ink to rest.

A post about GFP is a writer's dream for indulging in pretty pictures. I will restrict myself to two. GFP has become a poster boy for the science of biotechnology. Its barrel shaped ß-sheet structure shown above has become iconic in the scientific world. This is most emblematic in the odd and many varieties of glowing animals that now grace the covers of everything from scientific journals to websites and children's textbooks. If as some have predicted, we happen to "domesticate" biotechnology in the next few decades, it is very likely that one of the first things that our children would do would be to produce glowing pet rabbits, dogs, mice and cats. Along with a few other icons like DNA and the fruit fly, the image of glowing animals and fluorescent proteins is now deeply ensconced in our imagination as an example of what humans can do by manipulating biological systems. Perhaps one day our children can become friends with transgenic, green, glowing human beings, without the hulk-like physique and temper tantrums...

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