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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: Twitter: Dereklowe

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November 14, 2008

Sticking It to Proteins

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Posted by Derek

So, you’re making an enzyme inhibitor drug, some compound that’s going to go into the protein’s active site and gum up the works. You usually want these things to be potent, so you can be sure that you’ve knocked down the enzyme, so you can give people a tiny, convenient pill, and so you don’t have to make heaps of the compound to sell. How potent is potent? And how potent can you get?

Well, we’d like nanomolar. For the non-chemists in the crowd, that’s a concentration measure based on the molecular weight of the compound. If the molecular weight of the drug is 400, which is more typical than perhaps it should be, then 400 grams of the stuff is one mole. And 400 grams dissolved in a liter of solvent to make a liter of solution would then give you a one molar (1 M) solution. (The original version of this post didn't make that important distinction, which I'll chalk up to my not being completely awake on the train ride first thing in the morning. The final volume you get on taking large amounts of things up in a given amount of solvent can vary quite a bit, but concentration is based, naturally, on what you end up with. And it’s a pretty flippin’ unusual drug substance than can be dissolved in water to that concentration, let me tell you right up front). So, four grams in a liter would be 0.01 M, or 10 millimolar, and foru hundred milligrams per liter would be a 1 millimolar solution. A one micromolar solution would be 400 micrograms (0.0004 grams) per liter, and a one nanomolar solution would be 400 nanograms (400 billionths of a gram) per liter. And that’s the concentration that we’d like to get to show good enzyme inhibition. Pretty potent, eh?

But you can do better – if you want to, which is a real question. Taking it all the way, your drug can go in and attach itself to the active site of its target by a real chemical bond. Some of those bond-forming reactions are reversible, and some of them aren’t. Even the reversible ones are a lot tighter than your usual run of inhibitor.

You can often recognize them by their time-dependent inhibition. With a normal drug, it doesn’t take all that long for things to equilibrate. If you leave the compound on for ten, twenty, thirty minutes, it usually doesn’t make a huge difference in the binding constant, because it’s already done what it can do and reached the balance it’s going to reach. But a covalent inhibitor, that’ll appear to get more and more potent the longer it stays in there, since more and more of the binding sites are being wiped out. (One test for reversibility after seeing that behavior is to let the protein equilibrate with fresh blank buffer solution for a while, to see if its activity ever comes back). You can get into hair-splitting arguments if your compound binds so tightly that it might as well be covalent; at some point they're functionally equivalent.

There are several drugs that do this kind of thing, but they’re an interesting lot. You have the penicillins and their kin – that’s what that weirdo four-membered lactam ring is doing, spring-loaded for trouble once it gets into the enzyme. The exact same trick is used in Alli (orlistat), the pancreatic lipase inhibitor. And there are some oncology drugs that covalently attach to their targets (and, in some cases, to everything else they hit, too). But you’ll notice that there’s a bias toward compounds that hit bacterial enzymes (instead of circulating human ones), don’t get out of the gut, or are toxic and used as a last resort.

Those classes don’t cover all the covalent drugs, but there’s enough of that sort of thing to make people nervous. If your compound has some sort of red-hot functional group on it, like some of those nasty older cancer compounds, you’re surely going to mess up a lot of other proteins that you would rather have left alone. And what happens to the target protein after you’ve stapled your drug to it, anyway? One fear has been that it might present enough of a different appearance to set off an immune response, and you don’t want that, either.

But covalent inhibition is actually a part of normal biochemistry. If you had a compound with a not-so-lively group, one that only reacted with the protein when it got right into the right spot – well, that might be selective, and worth a look. The Cravatt lab at Scripps has been looking into what kinds of functional groups react with various proteins, and as we get a better handle on this sort of thing, covalency could make a comeback. Some people maintain that it never left!

Comments (22) + TrackBacks (0) | Category: Drug Assays | Toxicology


1. JWB on November 14, 2008 10:31 AM writes...

"And 400 grams dissolved in a liter of solvent would then be a one molar (1 M) solution."

No, it does not.

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2. Mark M on November 14, 2008 10:36 AM writes...

Thanks JWB.

Yeah, I think more than a few of us cringed at that.

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3. Derek Lowe on November 14, 2008 10:48 AM writes...

Aargh, fixed that one. I added a line or two of explanation - people outside the field tend to find molarity confusing enough without me hosing it up for them even more.

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4. Still Scared of Dinosaurs on November 14, 2008 12:46 PM writes...

Well, Derek, I give you a lot of credit for acknowledging the error instead of just editing the post and pretending it never happened. Using the event to make the post more informative to non-chemits is all the better. It makes all the other stuff you post more trustworthy.

Speak Dude to Power.

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5. Sili on November 14, 2008 2:27 PM writes...

What's the name of that one success to come out of 'traditional' Chinese medicine? Artemicine or something to combat plasmodium?

That's certainly springloaded for effectivity.

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6. Derek Lowe on November 14, 2008 3:10 PM writes...

That's artemisinin, and yep, it's ready to go, all right.

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7. The Pharmacoepidemiologist on November 14, 2008 4:58 PM writes...

Anyone hearing about departures from BMS in light of below cost of living increases in salaries for next year? I'm hearing from friends at the company of lots of discontent, and recruiters I know have said the last couple of days they are being deluged with resumes from BMSers. One recruiter compared it to the dark days of 2002. Anyone know anything about this? Is BMS also going through the "reduce the headcount" blues?

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8. milkshake on November 14, 2008 5:58 PM writes...

One loaded functional group that can do covalent modifications on cytochrom oxidases is cyclopropyl amine. The same chemists that objected to us how ugly Sutent structure was (because it "contained a Michael acceptor", uh-huh) didn't think twice about that cyclopropyl in their molecule that miraculously solved the first pass metabolism problem.

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9. CRH on November 14, 2008 6:00 PM writes...

Not to be a pessimist, but to your point The Pharmacoepidemiologist, where would those discontented BMS individuals be going? Seems the entire industry is laying off by the dozens (or more) so getting a smaller than expected increase in salary is much better than getting a large decrease in salary (i.e., layed off). Below cost of living increases is, unfortunately, going to be the norm this year. Have you not heard there's a bit of a problem with the economy; and the pharma industry has had its problems for some time now. I'd love to talk with these "recruiters" and see where exactly they are going to try and place these individuals.

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10. Anonymous BMS Researcher on November 14, 2008 10:50 PM writes...

To The Pharmacoepidemiologist: we are waiting to learn who gets hit by impending layoffs, about which news is expected very soon. We have also been repeatedly told to cut every cost that can be cut. Signals indicate the current round of layoffs will NOT be the last by any means. Morale is the lowest I can recall since the Great Plavix Debacle a while back. Management uses the phrases "cost-disciplined science" and "productivity transformation" all the time (and "next-generation biopharma model" whatever that means).

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11. Cindy on November 14, 2008 11:08 PM writes...

Great article for you folks who support the Lowe, trade your way to the bottom of a hole argument.

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12. T on November 15, 2008 12:12 AM writes...

I have a friend in discovery chemistry at BMS. He told me they are in a hiring freeze - and that layoffs are on the horizon.

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13. The Pharmacoepidemiologist on November 15, 2008 12:12 PM writes...

That fits with what I'm hearing. It's not just the PRI either, it's in marketing too. Big cuts. Even a rumor of leaving 345 Park again. Just like in 2001. Princeton is "less expensive" and the exec offices are still in place at Captopril Castle. Whether Cornelius will do this or not isn't clear, as yet.

The situation in Groton isn't much better for Pfizerites. There are supposedly so many homes on the market, many are even listed but known to be for sale by word of mouth, and some locals refer to the place as "Merced on the Atlantic". Ouch!

At lot of folks are thinking of taking a go at biotech, but a survey three months ago found that 75 percent of biotechs in the US with less than 200 employees have less than 12 months operating capital. That's right, they can survive for at most a year. Biotech isn't going to be a good hiding place once next summer hits.

Oh, and Merck's hitting a rough patch after it's failed efforts to market the HPV vaccine. At least in White House Station, houses are still selling, albeit slowly.

I suspect one implication of all of this downsizing is going to be a revisitation of previously looked at targets and previously evaluated compounds in the hope something useful was missed the first time round. Boring stuff, to be sure, but cheap to do from the company's perspective. Probably not a high yield process. Doubt there's even a Chantix or Vanlev present in the bunch. But happen it will.

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14. Nir. on November 15, 2008 12:41 PM writes...

I believe the answer for this problem would be to design protein inhibitors, It would be much easier to achieve specificity while obtaining better than nano-molar constants, albeit I agree we're not quite there yet, and that protein inhibitors have their down side as well.

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15. Anonymous BMS Researcher on November 15, 2008 4:00 PM writes...

To The Pharmacoepidemiologist:

What you are hearing certainly is consistent with what I'm hearing -- though the PRI isn't the PRI anymore, it has been redubbed R&D fairly recently. We have been told not to waste money tossing perfectly good stationery and the like, so I'm still using business cards that say "Bristol-Myers Squibb" Pharmaceutical Research Institute", and since I was promoted fairly recently I've got PLENTY of these cards. I do expect that some time in the next few weeks we're going to learn who among us will soon no longer be using our BMS business cards...

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16. Great Molecular Crapshoot on November 16, 2008 3:55 AM writes...

Nitriles can interact covalently and reversibly with cysteine and represent a perfectly acceptable approach to achieving inhibition of enzymes that exploit a cysteine thiol in the catalytic mechanism. The nitrogen of the thioamidate adduct can interact (non-covalently) with the protein which can result in selectivity with respect to other cysteines.

On an unrelated note, enzyme assays typically run with the inhibitor at a higher concentration than the protein. The IC50 values of very potent compounds may fall below the protein concentration used in the assay, resulting in tight binding and an apparent insensitivity of IC50 to structural modification (assuming that a number of analogs are tight binders).

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17. Jason on November 16, 2008 6:30 AM writes...

Tranylcipromine, a MAOI, also is irreversible.

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18. TNC on November 16, 2008 3:07 PM writes...

So why are covalent inhibitors associated with toxicity? This is something that seems to get assumed, but I don't know why. Is it that the molecule, while inhibiting the appropriate enzyme, is also inhibiting a lot of other enzymes?

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19. Cellbio on November 17, 2008 11:47 AM writes...

TNC, the argument that I have heard put forth is that covalent modifiers are reactive, to some degree, with non-target proteins. The ability to modify other proteins may be mostly without consequence, but in some patients at some time the modified proteins elicit an undesirable response,. A rare and unpredictable tox event is tough to manage, and a show stopper if the events include serious reactions. One potential serious reaction would be an immune response to the conjugate that would either create a hypersensitivity to further treatment, or cause epitope spreading and result in autoimmunity. With these potential outcomes, the risk of investing in a clinical program for a covalent modifier are deemed to high, for some, since no manner of clinical trial may show the potential risk for low probability, but "predictable" reactions that may become evident once the drug is on the market.

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20. Heisenberg on November 18, 2008 5:40 PM writes...

Aspirin is a covalent inhibitor. 40,000 tons produced annually can't be wrong.

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21. Cellbio on November 19, 2008 2:09 AM writes...

Interesting read on this topic.


Biochemical mechanisms of drug
action: what does it take for success?

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22. Anonymous BMS Researcher on November 19, 2008 7:05 AM writes...

Heisenberg on November 18, 2008 5:40 PM wrote...

> Aspirin is a covalent inhibitor.
> 40,000 tons produced annually can't be wrong.

At least once a week in some meeting I cite aspirin as the canonical example of an extremely useful drug that could NEVER make it very far in our pipeline today because it breaks so many of our rules.

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