New ligands for everyone's favorite protein

A landmark event in structural biology and pharmacology occurred in 2007 when the structure of the ß2-adrenergic receptor was solved using xray crystallography by Brian Kobilka's and Raymond Stevens's groups at Stanford and Scripps respectively. The structure was co-crystallized with the inverse agonist carazolol. Until then the only GPCR structure available was that of rhodopsin and all homology models of GPCR were based on this structure. The availability of this new high resolution structure opened new avenues for structure-based GPCR ligand discovery.

The ß2 binding pocket is especially suited for drug design since it is tight, narrow and lined with mostly hydrophobic residues with polar residues well-separated. Two crucial residues, an Asp and a Ser bind to the ubiquitous charged amino nitrogen present in most catecholamines and the aromatic section of the molecule docks deep into the hydrophobic pocket. These particular features also make computational docking more facile; a mix of polar and non-polar features with bridging waters can make docking and scoring more challenging.

Since the ß2 structure has been published, attempts are being made to use it as a template to build homology models of other GPCRs. A couple of months back I described an interesting proof-of-principle paper by Stefano Costanzi that sought to investigate how well a homology model based on the ß2 would perform. In that study carazolol itself was used as a ligand for docking into the homology model. Comparison with the original crystal structure revealed that while the ligand docked more or less satisfactorily, an important deviation in its orientation could be explained by a counterintuitive orientation of a Phe residue in the binding site. The study indicated that the devil is in the details when one is considering homology models.

However, finding ligands for the ß2 itself is also an important and interesting endeavor. Virtual screening could help in such studies. To this end Brian Shoichet, Brian Kobilka and their group have used the DOCK program to virtually screen one million lead-like ligands from their ZINC database against the ß2. Out of the 1 million ranked poses, they chose and clustered the top 500 compounds (0.05% of the database) into 25 unique chemotypes, a choice also guided by visual inspection of the protein-ligand interactions and commercial availability. They then tested these 25 compounds against the ß2 and found 6 compounds with IC50s better than 4 µM. One of these compounds with an IC50 of 9 nM is perhaps the most potent inverse agonist of the ß2 known. The binding poses revealed substantial overlap of similar functional groups with the carazolol structure. Two compounds turned out to have novel chemotypes and bore very little similarity with known ß2 ligands. A negative test was also run where a known predicted binder was chemical modified so that it would not bind.

Interestingly all the compounds found were inverse agonists. The ZINC library is somewhat biased against aminergic ligands as is most of chemical space. The catecholamine scaffold is one of the favourite scaffolds in medicinal chemistry. However, subtle difference in protein structure can sometimes turn an inverse agonist into an agonist. In this case, small changes in the orientation of the crucial Ser residue near the mouth of the binding pocket. In a past study for instance, slightly changing the rotameric features of the Ser residue thus resulting in a different orientation of the hydroxyl was sufficient to retrieve agonists.

The study thus shows the value of virtual screening in the discovery of new ß2 ligands and indicates the effect of library bias and protein structure on such ligand discovery. Many factors can contribute to the success or failure of such a search; nature is a multi-armed demon.

Reference:
Kolb, P., Rosenbaum, D., Irwin, J., Fung, J., Kobilka, B., & Shoichet, B. (2009). Structure-based discovery of ß2-adrenergic receptor ligands Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0812657106

Post-docking as a post-doc, and some fragment docking

I am now ready to post-doc. I am also now ready to post-dock, that is, engage in activities beyond docking. Sorry, I could not resist cracking that terrible joke. It's been a long journey and I have enjoyed every most moments of it. Thanks to everyone in the chemistry blogworld who regaled, informed, provoked and entertained on this blog. I am now ready to move on to the freakingly chilly Northeast. Location not disclosed for now, but maybe later.



Speaking of docking, here is a nice paper from the Shoichet group in which they use fragment docking to divine hits from a large library for a beta-lactamase. Fragment docking can often be tricky compared to "normal" docking since fragments being small usually demonstrate promiscuity, low-affinity and non-selectivity in binding. Fragment docking thus is not yet a completely validated technique.

In their study, the present authors screen their ZINC library for fragments binding to the ß lactamase CTX-M by docking using the program DOCK. They also screen a lead-like library for larger molecules. The top hits from the fragment docking results were assayed and showed micromolar inhibition against the lactamase. These included tetrazole scaffolds not seen before. Importantly, five of these hits could be crystallized and the high-res crystal structures validated the docking modes.

What was interesting was that the same tetrazole scaffolds in the larger lead-like library were ranked very low (>900) and would not have ever been selected had their tetrazole fragments not showed up at the top in the fragment docking results. These compounds, when assayed showed sub-milimolar to micromolar activity against the lactamase. Thus, the protocol essentially demonstrated that fragment docking can reveal hits that can be missed by docking larger lead-like molecules. One of the reasons DOCK succeeds in this capacity is because of its use of a physics-based scoring function that has no bias against fragments. It also helps that the active site of CTM-X is relatively rigid with little protein motion.

The fragments were also assayed against another lactamase for Amp C. Usually, hits for CTM-X and Amp C are mutually exclusive. What was seen was that the higher the potency of the fragments for CTX-M, the higher the specificity for CTX-M, not surprising considering that increased potency translates to a much better complementary fit of the fragments for CTX-M.

Fragment docking can be messy since fragments can bind non-selectively and haphazardly to many different parts of many different proteins. But this study indicates that fragment docking is not an uninteresting strategy to possibly find hits from other lead-like libraries that may be otherwise concealed.

The potencies of the compounds found may look pretty weak, but because there are extremely few molecules inhibiting these medicinally important lactamases, such advances are welcome. Lactamases are of course an important target for overcoming resistance in antibiotic treatment.

Reference:
Chen, Y., & Shoichet, B. (2009). Molecular docking and ligand specificity in fragment-based inhibitor discovery Nature Chemical Biology DOI: 10.1038/nchembio.155

It's been a good ride...

...and I am (hopefully) on its last leg. In a very short time from now I will finally have to summarize everything that I have learnt in five years in 45 mins.

45 mins could possibly be enough for everything I have done but it could never be enough for everything I have learnt, an exercise that goes far beyond the classroom, the library and the laboratory. In any case, this is one of those instances where the journey is so much more important than the destination. So it's been enlightening, and I thank everyone concerned for making it so.

"As you set out for Ithaka
hope your road is a long one,
full of adventure, full of discovery.
Laistrygonians, Cyclops,
angry Poseidon-don't be afraid of them:
you'll never find things like that on your way
as long as you keep your thoughts raised high,
as long as a rare excitement
stirs your spirit and your body.
Laistrygonians, Cyclops,
wild Poseidon-you won't encounter them
unless you bring them along inside your soul,
unless your soul sets them up in front of you.


Hope your road is a long one.
May there be many summer mornings when,
with what pleasure, what joy,
you enter harbors you're seeing for the first time;
may you stop at Phoenician trading stations
to buy fine things,
mother of pearl and coral, amber and ebony,
sensual perfume of every kind-
as many sensual perfumes as you can;
and may you visit many Egyptian cities
to learn and go on learning from their scholars.


Keep Ithaka always in your mind.
Arriving there is what you're destined for.
But don't hurry the journey at all.
Better if it lasts for years,
so you're old by the time you reach the island,
wealthy with all you've gained on the way,
not expecting Ithaka to make you rich.
Ithaka gave you the marvelous journey.
Without her you wouldn't have set out.
She has nothing left to give you now.


And if you find her poor, Ithaka won't have fooled you.
Wise as you will have become, so full of experience,
you'll have understood by then what these Ithakas mean"...

Constantine P. Cavafy

An honest heretic

I am deeply buried in my dissertation but I had to take some time out for this.

"He is a short, sinewy man with strawlike filaments of excitable gray hair that make him resemble an upside-down broom. Every day he dresses with the same frowzy Oxbridge formality in L. L. Bean khaki trousers (his daughter Mia is a minister in Maine), a tweed sport coat, a necktie (most often one made for him, he says, by another daughter, Emily, many years ago “in the age of primary colors”) and wool sweater-vests. On cold days he wears a second vest, one right over the other, and the effect is like a window with two sets of curtains. His smile is the real window, a delighted beam that appears to float free from his face, strangely dynamic with its electric ears and quantum nose, and his laugh is so hearty it shakes him. The smile and laughter have the effect of softening Dyson’s formality, transforming him into a sage and friendly elf, and also reminding those he talks with that he has spent a lifetime immersed in efforts to find what he considers humane solutions to dire problems, whose controversial gloss never seems to agitate him. His eyes are murky gray, and whatever he’s thinking beyond what he says, the eyes never betray."

I still remember the first time I walked into the library and discovered Freeman Dyson's autobiography lying neglected in a corner of the college library, covered with years of fine dust. I dusted off the book and noticed that the cover was missing. In spite of its miserable condition, I was so entranced that I read the volume cover to cover that night. Ever since then it has been the single-best socioscientific memoir I have read. Briefly corresponding with Dyson by e-mail was one of the high points in my life.

I won't say much about Dyson since I have already written about him in detail before. He is considered an extraordinary scientist and humanist, one of the most highly respected of the last fifty years, having inhabited the lofty Institute for Advanced Study at Princeton with luminaries like Oppenheimer, EInstein, Witten and Gödel. Earlier Dyson had worked with Hans Bethe and Richard Feynman, both legends. But what I have found puzzling about him in recent times is his skeptical stance on global warming. Now Dyson is no Bjørn Lomborg, but some of his statements really bothered me. For instance, in spite of spending some of the most productive years of his life designing a safe nuclear reactor, Dyson still does not talk in favour of nuclear energy. At least some of his ideas make sense; he has espoused research into genetically modified plants that would soak up CO2 for instance, but I cannot see how any such measures could do no more than supplement solutions to climate change.

In any case, Nicholas Dawidoff has a well-written profile of Dyson in the New York Times magazine. The article is well-written and documents well Dyson's outstanding credentials as a scientist and humanist as well as his early years in war-torn England. Dawidoff documents Dyson's contrarian stance on climate change; indeed this seems to be the reason for the article. Dyson's fondness for coal is also jarring. But while you may strongly disagree with what he says (and there's at least some stuff in there which makes sense), of one thing you can be sure; Dyson's criticism is scientific and honest and he has no political axe to grind. The invective that he seems to have received in some emails is actually amusing.

Global warming stances aside, Dyson probably has the best command over both science and the English language of anyone that I have come across; as he himself says, he has two passions- "calculation and English prose". In his magnificent books Dyson liberally quotes from both the best scientists and the best poets and writers. Over the last fifty years his mind has ranged and soared high over topics as diverse as nuclear reactor engineering, space exploration, problems of population and poverty, poetry, solid-state physics, quantum electrodynamics, adaptive optics in telescopes, origins of life and genetic engineering. Still a sprightly 85, Dyson continues to inspire and awe. I hope to gather up enough mettle to try to ask him for an audience sometime.

Meta-substitution: challenging a classic textbook paradigm

With my graduate school circus hopefully about to fold up tent, I will leave you with the abstract for this recent interesting Science paper which challenges a classic sophomore organic chemistry notion; that electron donating groups on benzene direct para and ortho substitution in electrophilic aromatic substitution reactions. By using a clever copper catalyst the authors manage to coax an aryl group to neatly substitute meta to an amido substituent, thus effecting a valuable C-H bond arylation.

"For over a century, chemical transformations of benzene derivatives have been guided by the high selectivity for electrophilic attack at the ortho/para positions in electron-rich substrates and at the meta position in electron-deficient molecules. We have developed a copper-catalyzed arylation reaction that, in contrast, selectively substitutes phenyl electrophiles at the aromatic carbon–hydrogen sites meta to an amido substituent. This previously elusive class of transformation is applicable to a broad range of aromatic compounds."

I also want to state that I remember many in my sophomore organic class misunderstanding the facts about the effects of e-withdrawing and donating substituents. For some reason they used to think that electron donating groups activate ortho and para positions and electron withdrawing groups activate meta positions. But that's completely incorrect. The correct statement is one which I still remember from a then classic organic chemistry textbook (which sadly went out of print).

Electron donating groups on benzene activate all positions; it's just that they activate ortho and para positions more than meta. Similarly, electron withdrawing groups on benzene deactivate all positions; it's just that they deactivate para and ortho more than meta. Thus the effect of any group, whether electron donating or electron withdrawing, is greatest at the ortho and para positions

Reference:
Phipps, R., & Gaunt, M. (2009). A Meta-Selective Copper-Catalyzed C-H Bond Arylation Science, 323 (5921), 1593-1597 DOI: 10.1126/science.1169975

Czechs halt missile shield progress

Finally, some promising development on this front. I have talked about the futility of missile defense several times before. In a nutshell, the only time an ICBM can truly fruitfully be intercepted is in midcourse, when it is descending to earth above the atmosphere. At this point it is being guided only by gravity, and it can release thousands of simple decoys from which it will be essentially indistinguishable for an incoming warhead. Several scientists over the last three decades have written articles arguing this point (read the excellent article in the Bulletin of Atomic Scientists), and yet missile defense stubbornly refuses to leave the minds of US presidents.

Plus, the underpinnings of missile defense totally miss the point and indicate vastly misplaced priorities. What in the name of Wotan is the possibility that N. Korea or Iran would attack the US with ICBMs and risk being reduced to dust? What on the other hand are the chances of someone slipping a small WMD through the incompletely guarded ports in the US? And what are the chances of alienating Russia by erecting such a shield a stone's throw away from Russian territory?

The former administration did not believe in the laws of physics, nor in the laws of human nature. Seems this one does.

So salt bridges are not stable in water? Shocking

Three salt bridges seen in this protein in the xtal structure were not observed by detailed NMR experiments in water. Here's the abstract:

NMR investigations have been carried out on the B1 domain of protein G. This protein has six lysine residues, of which three are consistently found to form surface-exposed salt bridges in crystal structures, while the other three are not. The Nζ and Hζ chemical shifts of all six lysines are similar and are not affected significantly by pH titration of the carboxylate groups in the protein, except for a relatively small titration of K39 Nζ. Deuterium isotope effects on nitrogen and proton are of the size expected for a simple hydrated amine (a result supported by density functional theory calculations), and also do not titrate with the carboxylates. The line shapes of the J-coupled 15N signals suggest rapid internal reorientation of all NH3+ groups. pKa values have been measured for all charged side chains except Glu50 and do not show the perturbations expected for salt bridge formation, except that E35 has a Hill coefficient of 0.84. The main differential effect seen is that the lysines that are involved in salt bridges in the crystal display faster exchange of the amine protons with the solvent, an effect attributed to general base catalysis by the carboxylates. This explanation is supported by varying buffer composition, which demonstrates reduced electrostatic shielding at low concentration. In conclusion, the study demonstrates that the six surface-exposed lysines in protein G are not involved in significant salt bridge interactions, even though such interactions are found consistently in crystal structures. However, the intrahelical E35−K39 (i,i+4) interaction is partially present.

The title was meant in half-jest of course and I don't mean to disparage such studies. But I think it just goes to show the kind of difficult, tedious and careful work that has to be often carried out in science even to reach "obvious" conclusions.

An an aside though, this conclusion was not at all obvious for a fair amount of time. There was a vigorous debate in the 90s kicked off by Bruce Tidor's paper arguing that salt bridges are not really that energetically important in protein stabilization, especially on surfaces. People who believed in the intense power of the holy electrostatic attraction did not really believe this. While the debate still continues, to my knowledge the general consensus is now on the side of the original Tidor proposition; salt bridges mostly provide only a marginal energetic gain (1-2 kcal/mol) to protein stability. This has been shown to be so primarily because of the loss in solvation and especially long-range solvation that formation of a salt-bridge incurs. Well, let the "obvious" research continue.

References:
1. Tomlinson, J., Ullah, S., Hansen, P., & Williamson, M. (2009). Characterization of Salt Bridges to Lysines in the Protein G B1 Domain Journal of the American Chemical Society DOI: 10.1021/ja808223p

2. Z.S. Hendsch and B. Tidor. Do salt bridges stabilize proteins? A continuum electrostatic analysis. Protein Sci. 3: 211-226 (1994)

A first-class mental workout

I am back from the CUP X OpenEye conference in Santa Fe. Of all the conferences I go to, none is more intellectually stimulating, and very few have the same quality of food for thought. I have also not been to another conference where people grapple with such fundamental scientific problems; shape, electrostatics, statistics, dipole moments, force fields, tautomers. It's a treat for the brain, and it's just the shot of intellectual energy that I need to re-energize myself for doing and discussing science. Plus, I get to visit Santa Fe whose mountains can inspire even the most muddle-headed scientific thinker to come up with at least a few inspired ideas. As Linus Pauling said, first you need to have lots of ideas, then throw the bad ones away.

The cast of characters this year was delicious since they had invited keynote speakers from all nine previous CUPs back. You therefore got to hear about a smorgasbord of topics from folks like Barry Honig, Vijay Pande, Tack Kuntz, Ajay Jain, Paul Labute, Anthony Nicholls, Chris Bayly, Yvonne Martin and many more. The topics ranged all over the place, the humor flowed abundantly, but the focus was always on the basic science. In computational chemistry (or finance or physics or biology for that matter...) we build so many models, yet how many of them reflect true understanding of the underlying physical basis? Simply adding parameters can make a model fit the data, yet how many of us would be nonchalant about using it for new prediction? As von Neumann said, with enough parameters we can indeed fit elephants on a curve. Yet who knows if we would be able to fit all those wondrous creatures that currently exist only in our imagination?

Scientists at CUP X grappled with these issues with infinite concern and zeal. They asked questions like; Can we say we can predict if we can't even get the dipole moment right? Would force fields ever reach the golden standard? Can we predict which tautomer of a molecule will bind to a protein? Can we make quantitative calculations of thermodynamic quantities that we can compare to accurate quantities obtained from ITC data? How can we predict solvation energies? What biases do we have in modeling? Can we get rid of them? And then, how can we ensure only the most rigorous standards for the experimental data itself? As someone indicated, a PDB or CSD structure of a molecule that you see on a screen is not the data, it is only a model of the data. And finally, an eternal question; can quantum mechanics get us to heaven?

If you are any kind of chemist concerned about and connected with building models of chemical and biochemical reality, I would strongly urge you to attend the OpenEye conference, held every year in March in the Land of Enchantment. Registration is free, a few meals are provided, alcohol splashes around with abandon during the poster sessions, and the conference is usually in a nice downtown Santa Fe hotel (the elegant and spacious El Dorado in this case), deals for which are usually cheap if done early. I am going to be here, if possible, every single year that I can. Being here reminds me of a reviewer's assessment of Douglas Hofstadter's magnificent "Gödel, Escher, Bach": It is like having a first-class mental workout in one of the finest intellectual gyms around

The real costs of generics

Details released by the agency last week reveal that, since at least 2006, Ranbaxy employees refrigerated samples that were supposed to be stored at room temperature to test their stability over time. In another instance, the plant reported stability test results for tablets of fluconazole, an antifungal drug, and ciprofloxacin, an antibiotic reserved for severe and life-threatening infections, as occurring at the required time intervals of months; but employees actually conducted all the tests on the same day or within a period of days. One audit of 15 applications approved for the US market found 1,676 errors, including errors in dates of analyses, packaging and stability test results. And during a 2008 inspection, plant records were found to contain the signatures or initials of Ranbaxy employees who were not present in the facility on the dates documented in the batch records

For some reason that does not sound surprising to me. There is a mad rush to plunge into the generics market once a lucrative drug patent expires, and companies may not engage in 100% quality control during this frenzy. Clearly, with great power comes great responsibility. Derek also has a post.

Why nature chose phosphates

I don't know if I have linked to this paper before, but if I haven't I definitely should. It's a seminal Science paper by Frank Westheimer in which he describes his thoughts on why nature chose phosphates as the most important signaling and building blocks in living organisms.

The entire paper is eminently readable and among other things, Westheimer compares the properties of phosphates with other possible groups such as arsenates and sulfates. Basically the bottom line is that phosphates possess the right value of pKa to be doubly ionized at physiological pH. The singly ionized form in the phosphodiester linkages of nucleic acids prevents the group from being easily hydrolyzed from nucleophilic attack by water, without making the group so stable that it won't undergo enzymatic hydrolysis.

As in other chemical schemes that Nature has developed, it's the right combination of stability and lability that makes phosphates ideal for being among the most fundamental chemical entities in life's inventory. And all essentially because of the right acid-base chemistry of phosphoric acid.