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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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March 1, 2010

Calorimetry: What Say You?

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Posted by Derek

I've been involved in a mailing list discussion that I wanted to open up to a wider audience in drug discovery, so here goes. We spend our time (well, a lot of it, when we're not filling out forms) trying to get compound to bind well to our targets. And that binding is, of course, all about energy: the lower the overall energy of the system when your compound binds, relative to the starting state, the tighter the binding.

That energy change can be broken down (all can all chemical free energy changes) into an enthalpic part and an entropic part (that latter one depends on temperature, but we'll assume that everything's being done at a constant T and ignore that part). Roughly speaking, the enthalpic component is where you see effects of hydrogen bonds, pi-pi stacking, and other such "productive" interactions, and the entropic part is where you're pushing water molecules and side chains around - hydrophobic interactions and such.

That's a gross oversimplification, but it's a place to start. It's important to remember that these things are all tangled together in most cases. If you come in with a drug molecule and displace a water molecule that was well-attached to your binding pocket, you've broken some hydrogen bonds - for which you'll pay in enthalpy. But you may well have formed some, too, to your molecule - so you'll get some enthalpy term back. And by taking a bound water and setting it free, you'll pick up some good entropy change, too. But not all waters are so tightly bound - there are a few cases where they're actually at a lower entropy state in a protein pocket then they are out in solution, so displacing one of those actually hurts you in entropy. Hmm.

And as I mentioned here, you have the motion of your drug molecule to consider. If it goes from freely rotating to stuck when it binds (as it may well), then you're paying entropy costs. (That's one reason why tying down your structure into a ring can help so dramatically, when it helps at all). And don't forget the motion of the protein overall - if it's been flopping around until it folds over and clenches down on your molecule, there's another entropy penalty for you, which you'd better be able to make up in enthalpy. And so on.

There's been a proposal, spread most vigorously by Ernesto Freire of Johns Hopkins, that drug researchers should use calorimetry to pick compounds that have the biggest fraction of their binding from enthalpic interactions. (That used to be a terrible pain to do, but recent instruments have made it much more feasible). His contention is that the "best in class" drugs in long-lived therapeutic categories tend to move in that direction, and that we can use this earlier in our decision-making process. People doing fragment-based drug discovery are also urged to start with enthalpically-biased fragments, so that the drug candidate that grows out from them will have a better chance of ending up in the same category.

One possible reason for all this is that drugs that get most of their binding from sheer greasiness, fleeing the water to dive into a protein's sheltering cave, might not be so picky about which cave they pick. There's a persistent belief, which I think is correct, that very hydrophobic compounds tend to have tox problems, because they're often just not selective enough about where they bind. And then they tend to get metabolized and chewed up more, too, which adds to the problem.

And all that's fine. . .except for one thing: is anyone actually doing this? That's the question that came up recently, and (so far), for what it's worth, no one's willing to speak up and say that they are. Perhaps all this is a new enough consideration that all the work is still under wraps. But it will be interesting to see if it holds up or not. We need all the help we can get in drug discovery, so if this is real, then it's welcome. But we also don't need to run more assays that only confuse things, either, so it would be worth knowing if drug-candidate calorimetry falls into that roomy category, too. Opinions?

Comments (26) + TrackBacks (0) | Category: Analytical Chemistry | Drug Assays


COMMENTS

1. p on March 1, 2010 9:44 AM writes...

Not a med chemist, but I can see a rationale for doing it this way simply because enthalpy will be enthalpy while the entropic drivers in solution may not be the same in solution and in a cell. It seems like the innards of a cell are sufficiently different (and more complex) than solution that any entropy you measure by mixing drug and protein in buffer will/could be wildly different in the cell. But if it has a highly favorable enthalpy, that will translate well.

I realize that doesn't go to your question at all. But I will say that I first saw people using a calorimeter in the early 90s and it was a pain in the ass. I have a colleague now who uses it and it seems like simplicity itself. If you know what you're doing, of course. In academic labs, at any rate, I think it's probably an underutilized tool and I'd be surprised if the same wasn't true in industry.

Permalink to Comment

2. RTW on March 1, 2010 10:18 AM writes...

Actually yes - J Med Chem. 2008 Jul 10;51(13):3804-13. Epub 2008 Jun 10.

Thermodynamic and structure guided design of statin based inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase.
Sarver RW, Bills E, Bolton G, Bratton LD, Caspers NL, Dunbar JB, Harris MS, Hutchings RH, Kennedy RM, Larsen SD, Pavlovsky A, Pfefferkorn JA, Bainbridge G.

Pfizer Global Research & Development, Ann Arbor, Michigan 48105, USA. Ronald.w.sarver@pfizer.com

Enjoy.

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3. been there, done that on March 1, 2010 10:23 AM writes...

Yes, some groups, big & small, are using calorimetry to help guide SAR. It's attractive for several reasons as you've pointed out, but also that it is doable; for example, there is no way to measure changes in entropy, so this is what one can do today.

One has to remember, though, that this is only a tool and may not always give the whole story. In one group that I've dealt with over the years who offer this type of assay to the broader organization, they make claims that are simply not true. One situation of trying to decide if a compound was reversible or not (slow dissociation or irreversible), they proposed studies that would not distinguish between these molecular mechanisms & were set right by others in the organization. As with any technique or method, there is nothing that substitutes for thinking about results, understanding the underlying techiques & their limits, putting into context. In other words, thinking!

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4. cynical1 on March 1, 2010 10:43 AM writes...

Interestingly, one of two examples that Freire sites in your link is HIV protease inhibitors. They all violate the 'Rule of five'. So who's right, Lipinski or Freire?

In my humble opinion, there is an inordinate amount of effort (and attention) spent trying to simplify the drug discover process: combichem, rule of 5, molecular modeling, LogP, MW, PSA, solubility and now calorimetry. One can always find a successful drug class that violates these rules. And every drug discovery executive will buy into one of them hook, line and sinker which will inevitably fail in the long term (e.g. Pfizer and Ro5). All of these have their place in the process but each one will have very serious flaws.

Why don't we just accept that it's much more complex than we have the technology or wisdom to understand or predict and realize that a lot of what we come up with involves a lot of luck, serendipity and tons of hard work. If you want to win the lottery, you don't buy only one ticket. Good, cheap, fast – pick any two.

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5. leftscienceawhileago on March 1, 2010 10:55 AM writes...

There was a lot of noise around "enthalpy arrays" coming from Xerox PARC a while ago:

http://www.pnas.org/content/101/26/9517.full

I think the groups are still publishing stuff, but it clearly hasn't panned out as something that is actually useful...cool idea though.

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6. Alex G on March 1, 2010 11:20 AM writes...

I hope it's not too much trouble to go completely off-topic to: I'm giving a literature seminar this evening and wanted to do an informal poll of the organic community

Where do you obtain your Wilkinson's catalyst from and how do you store it?

If you don't mind posting those two tidbits along with whatever response you would otherwise post to the blog, it would help me out a lot.

Cheers

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7. Alex G on March 1, 2010 11:23 AM writes...

If forgot the word "ask" in there somewhere.

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8. bamh1d on March 1, 2010 12:01 PM writes...

I agree with #3 and #4. The industry's headlong rush to expand the scale and scope of drug discovery has given rise to an unfortunate reductionism, a tendency to over-simplify the behaviors of complex systems. We need to remember that we're working with tools that have inherent strengths and limitations, and you over-interpret those results at your peril. However, this is NOT what your average project leader or department VP wants to hear.

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9. non-pharma chemist on March 1, 2010 12:23 PM writes...

@ #6 - pressure chemical (presschem.com) is always one of the cheapest suppliers of PGM salts and catalysts. Not as much of a product range as Strem, but much more affordable. As for storing the Wilkinson's...I'm from an organometallics group and I can never remember treating that stuff with very much respect (i.e., glove box storage or such). But then, Rhodium wasn't really our metal either...

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10. rhodium on March 1, 2010 1:20 PM writes...

To #6, I always made it (but then I tend to have some RhCl3 trihydrate around). I usually stored it in the refig, but that was just to keep it out of public view. I've used five year old material stored in a screw top vial without problems.

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11. chris on March 1, 2010 1:34 PM writes...

I was involved with a group who tried to use this and to be honest they found it very difficult to get really reliable results. Whilst the instrumentation seems to have improved can it relied upon as the front line screen?

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12. Wagonwheel on March 1, 2010 1:34 PM writes...

Another recent Pfizer reference...

Thermodynamic Optimisation in Drug Discovery: A Case Study using Carbonic Anhydrase Inhibitors
Andrew D. Scott, Chris Phillips, Alexander Alex, Maria Flocco, Andrew Bent, Amy Randall, Ronan O'Brien, Luminita Damian, Lyn H. Jones.
ChemMedChem
Volume 4 Issue 12, Pages 1985 - 1989
Published Online: 30 Oct 2009

http://www3.interscience.wiley.com/cgi-bin/fulltext/122669278/HTMLSTART

Permalink to Comment

13. HelicalZz on March 1, 2010 2:37 PM writes...

Just thinking out load, I wonder is some form of in-silico tool that might measure a hypothetical 'maximum enthalpic' binding energy to an 'idealized' target could be considered based on a compounds structure (bond-donors, acceptors, hydrophobic segments). Thus an 'efficiency of binding' to a real target could give some idea of whether measured / observed biding enthalpy is expected to be general or specific.

Zz

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14. gillespie on March 1, 2010 5:01 PM writes...

Calorimetry is making it's way into diagnostics too- we are developing protocols to diagnose CNS tumors based on differential calorimetry profiles of CSF. Seems to be working quite well.

Permalink to Comment

15. JK on March 1, 2010 5:14 PM writes...

I think an emphasis on protein flexibility and dynamics has been a major trend in basic protein science the last few years. We still only have a sketchy understanding of how enzyme catalysis really works, if that. But it's looking like flexibility is more important than might have been thought some time ago. That would suggest entropy is important in enzyme inhibition, though I suppose the entropy of the protein and the inhibitor are different questions. And calculating protein entropy, even if we have a structure, is a whole other problem...

Permalink to Comment

16. Malcolm on March 1, 2010 8:19 PM writes...

#1 I'm not aware of any evidence that the thermodynamics of binding differs between in vivo and in vitro situations (of course, in the absence of conformational change in the receptor, presence of cofactors etc.)

A buffer solution of the appropriate pH and ionic strength should do just nicely.

Permalink to Comment

17. Rock on March 1, 2010 10:10 PM writes...

#4 You are right that there are no 'magic bullets'. But the idea is to put probability on your side. There will always be exceptions to any of the drug-like property rules. The larger question is, what percentage of compounds in that particular physicochemical space make it through development? I would prefer to work in space that has a higher probability. I believe starting from a hit with higher enthalpic binding can only be a good thing.

#13 What you are referring to is called the Andrews binding energy.

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18. Morten G on March 2, 2010 3:30 AM writes...

The nice thing about ITC is that it's probeless and possible under standard buffer conditions so you can just take your ligand saturated protein out of the cell and use it directly in x-ray crystallography. Or you could try to recover your protein through gel filtration, osmosis, or desalting columns but really...

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19. Anonymous on March 2, 2010 4:15 AM writes...

There is a fundamental problem with interpretating ITC results, since what is measured is the sum of all degrees of freedom in the system, not directly say hydrogen bonding between the ligand and receptor. For instance introducing a hydrogen bond between a ligand and receptor might mainly give an entropic contribution to the whole system by reducing the flexibilty of the protein.

Also according to our in-house enzyme experts entropy and enthalpy is much more sensitive to the buffer conditions than the free energy.

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20. mehere on March 2, 2010 8:19 AM writes...

#13 just a bit late, Andrews got there first. On this note - does anyone really use ABE much? It was flavour of the month for about a month at GSK a couple of years ago, then disappeared

Permalink to Comment

21. cynical1 on March 2, 2010 10:36 AM writes...

@Rock: "The larger question is, what percentage of compounds in that particular physicochemical space make it through development?"

Since there does not seem to be any compounds making it through development, I would argue that question is moot.

"I believe starting from a hit with higher enthalpic binding can only be a good thing."

Do you think these rules apply for allosteric inhibitors the same way they do for competitive inhibitors? Would you throw them out of a screen simply because of an ITC result? I suspect that there is not enough data to answer this question with any certainty. If I had to fathom a guess, I would say no but I could be proved wrong. (It’s happened before.)

I would tell Professor Friere from personal experience that enthalpic interactions are just one very small aspect of turning an HIV protease inhibitor into a drug and it was NOT the rate-limiting one. There is no direct correlation between enzyme inhibition and cellular antiviral activity (and I have published data to prove that).

Personally, I would rather let academics/NIH refine these new tools until their liabilities are well-characterized on multiple target classes before applying them in an industrial setting. We can not afford those liabilities at this juncture and people like me do not have the luxury of tenure.

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22. smurf on March 2, 2010 2:02 PM writes...

Useless technology for membrane bound targets.

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23. anon on March 3, 2010 1:02 PM writes...

Seems like a useful approach but see Int. J. Mol. Sci. 2009, 10, 2752-2762; doi:10.3390/ijms10062752

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24. kemi on March 3, 2010 2:06 PM writes...

#22 Agree as you won´t get the required protein densities within a native membrane that allows to reliably detect a specific interaction. But there are ways for tackling this. Have been using this myself with detergent-solubilized membrane proteins that have been enriched to a level that allowed to conduct ligand-binding studies with ITC. Works beautyfully even when you reconstitute the membrane protein into lipid vesicles. So it is all about reaching a sufficient concentration in your sample cell.

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25. David Formerly Known as a Chemist on March 4, 2010 12:06 PM writes...

We looked at this technology back when I worked in the industry. It provides valuable data, but it's expensive and inefficient. One big downside is the relatively large amount of protein required to get a good measurement.

Wasn't Ernesto Freire part of a startup focused on use of calorimetry-driven lead ID/optimization, Fulcrum Pharmaceuticals? What ever happened to that company?

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26. Brian on March 5, 2010 11:01 PM writes...

#25 - The latest generation of calorimeters use far lower amounts of protein. You can get good data now with 100 ug.

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