Yesterday, while having a project discussion, we got into asking how strong a salt-bridge is, and realised that we are trying to answer one of the perpetually alive and kicking questions of chemistry. I then realised that this question belongs into the class of some other PAQs (Perpetually Alive Questions)
1. How strong is a hydrogen bond?
2. Do "low-barrier, strong" hydrogen bonds exist?
3. How do enzymes exactly stabilize transition states and bring about such enormous stabilization? What forces contribute to this?
4. How do you distinguish between a 'weak' hydrogen bon and a Van der Waals contact?
5. Why do molecules adopt one crystal structure and not another equienergetic one?
6. What is the origin of the rotation barrier in ethane?
Some of these questions (such as 4.) depend as much on convenience and arbitrary definition as on having definite answers. There are also ones where one can make good general guesses and yet lack predictive ability (such as 5.). The protein folding problem also falls into this category.
Many of these questions concern my favourite topics, especially those related to hydrogen bonds. While hyperconjugation has been advanced as the source of the rotation barrier in ethane, proton sponges have been postulated as model systems for demonstrating "strong" hydrogen bonds. According to Dunitz, crystal structure prediction really boils down to choosing between equienergetic possibilities rather than asking why one of them exists. As for enzymes, Kendall Houk seems to think that efficiencies above a certain extent may imply covalent rather than non-covalent binding.
All questions make for exciting discussion and much fun, and the great thing is that even partial answers make for great intellectual debate and even scientific advancement. Roll on! Chemistry remains alive because of such questions. But PhD.s may get prolonged, an emphatic disadvantage.
More such questions?
Of course I can google it, but...
"If someone is interested in the details, I will be happy to talk to them later"...these words of mine in a group meeting presentation were met with amusement and subdued smirking. I was puzzled. I remember hearing these words often a few years ago in talks. They made me feel happy, because they seemed to indicate that the speaker was genuinely interested in explaining the fine points, and indeed even the general points of his talk later to those who were interested. So what had changed between then and now? Two factors I think among others: Google, and Powerpoint.
Powerpoint allows you to display a long list of references with the tacit assumption that the audience will scan and memorize them instantaneously. Surely all the references you need will be in there. So for details, just look into those.
Google made avoiding human communication even more easy. Some experience that I have supports this. Let's say someone was talking about a project that involved RNA interference (RNAi). I would cringe asking them "What is RNAi?", because more than once I have received the response, "O RNAi...that's...why don't you google it?" Well, of course I can google it, but it's not a crime to sometimes yearn for human communication. In 'older' times, the speaker knew that you would have to probably go to the library and browse through books to get such a question answered. To save you that trouble, he or she would take out a few minutes to answer your question. Even today, there are a few speakers who are gracious enough to be patient and try to answer even a general question by taking a few minutes. But the percentage is alarmingly dwindling, even those who are willing to talk to you in detail later. If you want to ask them about the direct details of their research, fine. If it's something general, you can always...
I understand of course, the enormous benefits of having Google and the internet at your fingertips, which in fact allow you to instantly access such information. Interestingly, it works both ways; today in a presentation, a colleague highlighted a drug for tuberculosis, a well-known antibiotic. I was tempted to ask her what protein target in the tuberculosis bacterium it targets. But I was stricken with the 'information at your fingertips syndrome'; why should I ask her that if I could get the information right away from Al Gore's information superhighway? (This syndrome has also led more people googling in presentations than paying attention to the talk)
Naturally, Google is God. But I wonder if human communication in presentations has been stifled because of the tacit assumption on the part of both speaker and audience, that they can always google it. As for me, I still love to say "If someone is interested in the details, I will be happy to talk to them later" as a catch-all phrase, and I think I am going to continue doing so. For the sake of good old fashioned banter, if not anything else.
Powerpoint allows you to display a long list of references with the tacit assumption that the audience will scan and memorize them instantaneously. Surely all the references you need will be in there. So for details, just look into those.
Google made avoiding human communication even more easy. Some experience that I have supports this. Let's say someone was talking about a project that involved RNA interference (RNAi). I would cringe asking them "What is RNAi?", because more than once I have received the response, "O RNAi...that's...why don't you google it?" Well, of course I can google it, but it's not a crime to sometimes yearn for human communication. In 'older' times, the speaker knew that you would have to probably go to the library and browse through books to get such a question answered. To save you that trouble, he or she would take out a few minutes to answer your question. Even today, there are a few speakers who are gracious enough to be patient and try to answer even a general question by taking a few minutes. But the percentage is alarmingly dwindling, even those who are willing to talk to you in detail later. If you want to ask them about the direct details of their research, fine. If it's something general, you can always...
I understand of course, the enormous benefits of having Google and the internet at your fingertips, which in fact allow you to instantly access such information. Interestingly, it works both ways; today in a presentation, a colleague highlighted a drug for tuberculosis, a well-known antibiotic. I was tempted to ask her what protein target in the tuberculosis bacterium it targets. But I was stricken with the 'information at your fingertips syndrome'; why should I ask her that if I could get the information right away from Al Gore's information superhighway? (This syndrome has also led more people googling in presentations than paying attention to the talk)
Naturally, Google is God. But I wonder if human communication in presentations has been stifled because of the tacit assumption on the part of both speaker and audience, that they can always google it. As for me, I still love to say "If someone is interested in the details, I will be happy to talk to them later" as a catch-all phrase, and I think I am going to continue doing so. For the sake of good old fashioned banter, if not anything else.
Features of selective kinase inhibitors
It was quite recently that I came across this fantastic review of kinase inhibitors from 2005 by Kevan Shokat. The reason why I missed it is because it was published in a journal that is usually not in people's top ten list- Chemistry and Biology. So I am putting it into mine from now onwards.
In any case, I think this review should be read by anyone who is concerned with either the experimental or computational design and testing of selective kinase inhibitors. Even now, the holy grail of kinase inhibitor development is selectivity, and Shokat gives a succint account of what we know about designing such molecules until now. I thought there were a few points especially crucial to keep in mind.
1. IC50 is not equal to Ki...usually:
This is a central if simple fact that should always guide computational as well as experimental scientists in their evaluation. The IC50 and Ki values are generally related by the so-called Cheng Prusoff equation:
Here, Km and [S] are the Km and substrate concentrations for the natural substrate of the protein, ATP in this case, which is usually competitively displaced by inhibitors.
What does this mean on a practical basis? Let me take my own example in which this principle helped a lot. We are trying to design a selective kinase inhibitor, and found out that a compound which we had, showed some selectivity for one kinase versus the other. To investigate the source of this selectivity, we started looking at the interactions of the inhibitor with the two kinase pockets; presumably, better the interaction, more it would contribute to the smaller IC50. Or would it? No! Better the interactions, the more it would contribute to the smaller Ki. The point is, only the Ki has to do with how effectively the inhibitor interacts with the active site. But the IC50 is an experimental number which as the above equation indicates, also has to do with how well the natural substrate, in this case ATP, binds to the protein. So if the Km of the protein for ATP is really small, that means ATP binds very well, and even a compound with a low Ki will have a relatively large IC50 and will be a poor inhibitor. So just looking at the active site interactions does not help to rationalize anything about the IC50; what must be known is how well the competitor ATP binds to the site. The bottom line is, in kinase assays, one can only compare Ki's and IC50's if the ratio [S]/Km is kept constant. Otherwise it's not a controlled experiment.
2. There is a minimum threshold of potency below which an inhibitor cannot be selective, irrespective of the in vitro data:
Another important point. If the inhibitor is extremely lousy in the first place, then the dosage needed to achieve selectivity is going to be much higher. On a practical basis, as Shokat says, "more potent compounds are more selective because they can be used at a lower dose". What I take this to mean is that if your compound is extremely potent, then you can essentially use it at such a low concentration, that it binds to only one protein, and is denied to the others. What could be the 'threshold' for a kinase inhibitor? Well, it depends also on what kind of a clinical target you are targeting, but I would think that anything above maybe a micromolar Ki would be enough to raise serious doubts about selectivity.
3. Common features of kinase inhibitors:
This could be educated observation and guesswork at best, but Shokat says many inhibitors show dramatic SAR relationships. The hydrogen bonds between the adenine ring nitrogens and a crucial backbone residue are duplicated by many inhibitors for example. I can vouch for the ubiquity of this particular interaction, as it has shown up even in docking poses. This is what can be called a 'correlated' pair of hydrogen bonds, one which is strong and conserved. The other point about kinase inhibitors being usually relatively rigid and entropically constrained is also interesting. One thing is for sure; kinase inhibitors seem to promise yet another bounty for heterocyclic chemists (We who criticize 'flatland' should quietly slink away now...)
And of course, this is only for ATP competitive inhibitors. Allosteric inhibition will be quite another unexplored terrain. Overall, a highly informative and practically useful review. It helped me ask our biologists questions which they wouldn't have expected from a modeler. The search for selective inhibitors is surely one of the most vigorously explored areas of med chem. The dozens of publications literally every week on Src, PKC, p38 Map, CDK, and Bcr kinases represent only a fraction of the research that is being currently done in pharma as well as academia.
In any case, I think this review should be read by anyone who is concerned with either the experimental or computational design and testing of selective kinase inhibitors. Even now, the holy grail of kinase inhibitor development is selectivity, and Shokat gives a succint account of what we know about designing such molecules until now. I thought there were a few points especially crucial to keep in mind.
1. IC50 is not equal to Ki...usually:
This is a central if simple fact that should always guide computational as well as experimental scientists in their evaluation. The IC50 and Ki values are generally related by the so-called Cheng Prusoff equation:
Here, Km and [S] are the Km and substrate concentrations for the natural substrate of the protein, ATP in this case, which is usually competitively displaced by inhibitors.
What does this mean on a practical basis? Let me take my own example in which this principle helped a lot. We are trying to design a selective kinase inhibitor, and found out that a compound which we had, showed some selectivity for one kinase versus the other. To investigate the source of this selectivity, we started looking at the interactions of the inhibitor with the two kinase pockets; presumably, better the interaction, more it would contribute to the smaller IC50. Or would it? No! Better the interactions, the more it would contribute to the smaller Ki. The point is, only the Ki has to do with how effectively the inhibitor interacts with the active site. But the IC50 is an experimental number which as the above equation indicates, also has to do with how well the natural substrate, in this case ATP, binds to the protein. So if the Km of the protein for ATP is really small, that means ATP binds very well, and even a compound with a low Ki will have a relatively large IC50 and will be a poor inhibitor. So just looking at the active site interactions does not help to rationalize anything about the IC50; what must be known is how well the competitor ATP binds to the site. The bottom line is, in kinase assays, one can only compare Ki's and IC50's if the ratio [S]/Km is kept constant. Otherwise it's not a controlled experiment.
2. There is a minimum threshold of potency below which an inhibitor cannot be selective, irrespective of the in vitro data:
Another important point. If the inhibitor is extremely lousy in the first place, then the dosage needed to achieve selectivity is going to be much higher. On a practical basis, as Shokat says, "more potent compounds are more selective because they can be used at a lower dose". What I take this to mean is that if your compound is extremely potent, then you can essentially use it at such a low concentration, that it binds to only one protein, and is denied to the others. What could be the 'threshold' for a kinase inhibitor? Well, it depends also on what kind of a clinical target you are targeting, but I would think that anything above maybe a micromolar Ki would be enough to raise serious doubts about selectivity.
3. Common features of kinase inhibitors:
This could be educated observation and guesswork at best, but Shokat says many inhibitors show dramatic SAR relationships. The hydrogen bonds between the adenine ring nitrogens and a crucial backbone residue are duplicated by many inhibitors for example. I can vouch for the ubiquity of this particular interaction, as it has shown up even in docking poses. This is what can be called a 'correlated' pair of hydrogen bonds, one which is strong and conserved. The other point about kinase inhibitors being usually relatively rigid and entropically constrained is also interesting. One thing is for sure; kinase inhibitors seem to promise yet another bounty for heterocyclic chemists (We who criticize 'flatland' should quietly slink away now...)
And of course, this is only for ATP competitive inhibitors. Allosteric inhibition will be quite another unexplored terrain. Overall, a highly informative and practically useful review. It helped me ask our biologists questions which they wouldn't have expected from a modeler. The search for selective inhibitors is surely one of the most vigorously explored areas of med chem. The dozens of publications literally every week on Src, PKC, p38 Map, CDK, and Bcr kinases represent only a fraction of the research that is being currently done in pharma as well as academia.
Discodermolide unraveled?
Drugs affecting microtubule dynamics are familiar chemical players in med chem by now. First came Taxol, then the epothilones, then discodermolide, and the list continues with peluroside, eleutherobin, and dictyostatin to name a few of the better known entities.
Like it is for other drugs, one of the major questions asked about these molecules is how they bind to their target. Taxol and epothilone have been subjected to immense SAR and analog preparation by some of the hard hitters in the synthetic arena. Their binding conformations have been postulated with reasonable confidence. The common pharmacophore hypothesis, tempting but misleading and not true in this case, has been convincingly questioned. But for discodermolide, the binding conformation is not yet known. Now, groups from Spain and the UK have applied the "INPHARMA" NMR methodology to probe the interaction of disco with tubulin.
Admittedly, INPHARMA is a nifty technique- here is the original reference. It relies on magnetization transfer to a protein proton from a proton of a molecule that binds in the active site. This magnetization is then again transfered from the protein proton to a proton of another molecule that binds to the same site. For this to happen, the rate constants for binding have to be much smaller than the relaxation times for the protons.
Thus, the magnetization transfer sequence for two ligands A and B that bind to the same active site is
Naturally, this happens if both H(A) and H(B) are close to the same protein proton. Thus you see cross peaks between two protons A and B of two different ligands, mediated by a protein proton. Information from many such cross peaks allows us to map the protein protons to the ligand protons that are near them. In the end, not only does a picture emerge of the binding conformation of both ligands separately, but this information also allows us to suggest a common pharmacophore for the two ligands. And Paterson has now used this technique for disco and epothilone.
I am sure the technique has to be done carefully and that it was, and I also don't doubt the postulated conformation of disco. Most of the paper is really interesting and it's a neat study. But what concerns me is the fact that the end result, the binding conformation of disco can be mapped onto the x-ray conformation of disco proposed earlier, as well as the solution conformation of dictyostatin. Where my mind snags is in accepting this conclusion, because a single or even one dominant conformation for a flexible molecule derived in solution is unrealistic. It's what is called a 'virtual' solution. It's virtual simply because it's an average conformation. And since the average is a juxtaposition of all possible individual conformations, it simply does not exist in solution by itself. It's like saying that the contiguous structure of fan blades seen when a fan is moving very fast actually exists. It does not, because it is an average, and the resolution time of our eye is not short enough to capture individual positions of the fan blades.
So I wonder how the binding conformation of disco could be mapped onto the conformations of one x-ray conformation and one single dominant conformation in solution. Now I am sure there is more to this story, and I am still exploring the paper, but for commonsense reasons, a little red light in my brain always turns on (or at least should turn on) when a single or dominant conformation for a highly flexible molecule in solution is postulated.
More cogitations to come soon.
Protein-protein interactions and academic bounties
Whistling's post on the neat article published by researchers at Sunesis Pharma on tethering as a strategy to discover caspase inhibitors reminded me of Jim Wells, who had come to give a talk at Emory a couple of months ago. Wells moved from being president of Sunesis to UCSF. At UCSF, he commands a formidable repertoire of resources, including NMR, X-Ray and High Res Mass, as well as synthesis and molecular biology facilities. Who in academia can compete with such an immense wall of capability? I am sure Wells must have been offered great incentives including these facilities at UCSF to facilitate his transition from industry to academia. His move is symbolic of the power that academia has now started to command. Part of this power no doubt comes from it being allowed to have patents on drugs, from which they can get considerable finances through royalties. My own advisor, Prof. Dennis Liotta, got Emory 500 million $ in royalties, as one of the co-discoverers of the anti-HIV drug Emtricitabine (Emtriva®). That is a good sign, because researchers would gladly move back into the intellectually more stimulating environment of academics, if they were also provided good incentives and facilities.
But coming back to the scientific side, Wells is one of the pioneers in developing small molecule inhibitors for disrupting protein-protein interactions, a notoriously tricky endeavor. Proteins can interact with other proteins in as many ways as small molecules can interact with them. Finding a protein-protein interaction is not simply a matter of finding a good complementary fit, but is much more complicated, because the protein essentially interacts with another protein through flexible maneuvering. Not only can it simply slide into a hydrophobic complementary site, but it can also catch hold of loops, causing immense conformational changes in them, and then only be in a comfortable position to dock with the other protein. Needless to say, programs which depend on rigid body protein docking often fail miserably, like ClusPro, which gave me horrendous results on my system. Also, proteins may not always dock in a theoretically optimum manner in real systems, but only in an orientation that is optimum to cause further action.
Protein-protein docking will remain a holy grail for both experimentalists and computational scientists, more so with the huge number of protein-protein interactions impliccated in diseases now. This whole discussion reminded me of two excellent reviews on protein-protein interactions, which give a succint view of the field.
1. Small-molecule inhibitors of protein-protein interactions: progressing towards the dream- Michelle R. Arkin, James A. Wells, Nature Reviews Drug Discovery 3, 301 - 317 (01 Apr 2004)
2. Strategies for Targeting Protein–Protein Interactions With Synthetic Agents- Hang Yin and Andrew D. Hamilton, Angewandte Chemie International Edition Volume 44, Issue 27, Date: July 4, 2005, Pages: 4130-4163
But coming back to the scientific side, Wells is one of the pioneers in developing small molecule inhibitors for disrupting protein-protein interactions, a notoriously tricky endeavor. Proteins can interact with other proteins in as many ways as small molecules can interact with them. Finding a protein-protein interaction is not simply a matter of finding a good complementary fit, but is much more complicated, because the protein essentially interacts with another protein through flexible maneuvering. Not only can it simply slide into a hydrophobic complementary site, but it can also catch hold of loops, causing immense conformational changes in them, and then only be in a comfortable position to dock with the other protein. Needless to say, programs which depend on rigid body protein docking often fail miserably, like ClusPro, which gave me horrendous results on my system. Also, proteins may not always dock in a theoretically optimum manner in real systems, but only in an orientation that is optimum to cause further action.
Protein-protein docking will remain a holy grail for both experimentalists and computational scientists, more so with the huge number of protein-protein interactions impliccated in diseases now. This whole discussion reminded me of two excellent reviews on protein-protein interactions, which give a succint view of the field.
1. Small-molecule inhibitors of protein-protein interactions: progressing towards the dream- Michelle R. Arkin, James A. Wells, Nature Reviews Drug Discovery 3, 301 - 317 (01 Apr 2004)
2. Strategies for Targeting Protein–Protein Interactions With Synthetic Agents- Hang Yin and Andrew D. Hamilton, Angewandte Chemie International Edition Volume 44, Issue 27, Date: July 4, 2005, Pages: 4130-4163
Low is still better
One of the pieces of news (NYT link) making waves is the finding that resveratrol, a substance in red wine, can offset the effects of a high-calorie diet and prolong longevity...in mice at least. But the graph is revealing, and is also relieving, because it emphatically shows that wine enthusiasts gleefully running out to buy (and justify) large stores of Chianti should pause for thought. The graph clearly shows that a standard low calorie diet still is better than a high-calorie one fortified with red wine, at least in the long term. So think again before you douse yourself with wine and cheese.
Copyright: NPG
Original Nature article
Now, let me get back to my creme-filled donut
Copyright: NPG
Original Nature article
Now, let me get back to my creme-filled donut
Fishy no more
I better fry that fish for dinner today instead of waiting for the weekend. How many times do you see a front page news headline on BBC saying "Only 50 years left for sea fish"? But that's what it says, and this is one of the scarier changes that's going to take place as we irreversibly modify our planet. No need to have Halloween as a special celebration anymore.
This is not suprising. In an earlier post, I already commented about precipitous amphibian declines orchestrated by environmental damage. Now it's the fish, and in fact marine life in general. And no wonder; out of all systems, marine systems are probably the most delicate systems on earth. In fact, we haven't even understood the complex symphony involving fish, algae, other sea denizens and chemicals, that takes place below the water's surface. As one researcher said, marine biosystems are like a pack of cards, so intricably linked with each other, that disturb one, and you turn others topsy turvy. But doesn't the pack of cards go further in even more ways? After all, the oceans are the great equalizers of the planet, absorbing CO2 and being key for maintaining temperature. One of the scarier scenarios for global warming concerns the perturbation of the North Atlantic Circulation, which would throw Europe and the US into a new age of climate, possibly an ice age.
In the last two decades or so, we have been starting to see the effects of climate change on biodiversity in a very real way, with not only loss of habitats, but also the spread of disease vectors that thrive in warmer conditions. Finally, as I have already said, it's going to be the disruption of daily life that is going to be the final wake up call for people. The only critical question is whether it will be too late by then, and the answer increasingly seems to be yes. This is no longer a matter that needs to appeal to only morality and preserving the beauty of nature. This has to do with our modern way of life, and once we take a look, we realise that the matter of biodiversity destruction is linked to many others of our grotesque nemeses, including the oil crises, and religious and political conflict. The pack of cards packs deep indeed.
Critics of global warming who said that taking action against it would adversely affect economies need to open their eyes. The naysayers who don't wish to preserve the environment for its own sake could at least preserve it for their own sake. How many people's likelihood is related to seafood collection and processing? And again, how much of the world economic capital rests on providing seafood to populations? If this has nothing to do with economics, then I don't see what has. The fact that I may not get that stuffed pomfret on a lazy weekend will be the most trivial of all consequences.
I firmly believe that if humanity's end comes, it will not be because it lacked the technology and capability for solving problems, but because the problems were so intractably connected to each other and humans' way of life, that even solving one problem would make the entire system collapse. It would be the ultimate irony; the system's sheer complexity and overbearing influence precluding even the realistic solution of a problem, even when it is at hand.
This is not suprising. In an earlier post, I already commented about precipitous amphibian declines orchestrated by environmental damage. Now it's the fish, and in fact marine life in general. And no wonder; out of all systems, marine systems are probably the most delicate systems on earth. In fact, we haven't even understood the complex symphony involving fish, algae, other sea denizens and chemicals, that takes place below the water's surface. As one researcher said, marine biosystems are like a pack of cards, so intricably linked with each other, that disturb one, and you turn others topsy turvy. But doesn't the pack of cards go further in even more ways? After all, the oceans are the great equalizers of the planet, absorbing CO2 and being key for maintaining temperature. One of the scarier scenarios for global warming concerns the perturbation of the North Atlantic Circulation, which would throw Europe and the US into a new age of climate, possibly an ice age.
In the last two decades or so, we have been starting to see the effects of climate change on biodiversity in a very real way, with not only loss of habitats, but also the spread of disease vectors that thrive in warmer conditions. Finally, as I have already said, it's going to be the disruption of daily life that is going to be the final wake up call for people. The only critical question is whether it will be too late by then, and the answer increasingly seems to be yes. This is no longer a matter that needs to appeal to only morality and preserving the beauty of nature. This has to do with our modern way of life, and once we take a look, we realise that the matter of biodiversity destruction is linked to many others of our grotesque nemeses, including the oil crises, and religious and political conflict. The pack of cards packs deep indeed.
Critics of global warming who said that taking action against it would adversely affect economies need to open their eyes. The naysayers who don't wish to preserve the environment for its own sake could at least preserve it for their own sake. How many people's likelihood is related to seafood collection and processing? And again, how much of the world economic capital rests on providing seafood to populations? If this has nothing to do with economics, then I don't see what has. The fact that I may not get that stuffed pomfret on a lazy weekend will be the most trivial of all consequences.
I firmly believe that if humanity's end comes, it will not be because it lacked the technology and capability for solving problems, but because the problems were so intractably connected to each other and humans' way of life, that even solving one problem would make the entire system collapse. It would be the ultimate irony; the system's sheer complexity and overbearing influence precluding even the realistic solution of a problem, even when it is at hand.