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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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In the Pipeline: Don't miss Derek Lowe's excellent commentary on drug discovery and the pharma industry in general at In the Pipeline

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June 19, 2012

Watch Your Cell Assays

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Posted by Derek

I've written here before about the (now) well-known problem of compound aggregation in screening assays. You can get false positives when a compound itself forms a colloidal mass that pulls the target protein into it. The readout looks as if the protein has been inactivated by the small molecule itself, just the way you were hoping for, but if you add a bit of detergent the activity mysteriously goes away.

The Shoichet lab has a paper out that warns people to look out for this in cellular assays as well. This time you'll get false negatives - the colloidal aggregates don't act right compared to the free molecules, as you could well imagine. Update: I see that Wavefunction has covered this same paper! Reformulating the assay restores the activity, but the trick is knowing that there was a problem to start with. Something to keep in mind when your cell assay numbers are wonky (as if there weren't enough reasons for that to happen already).

Comments (19) + TrackBacks (0) | Category: Drug Assays


COMMENTS

1. luigi on June 19, 2012 8:48 AM writes...

Interesting that the authors didn't bother to replicate the data they report - as reflected by a lack of estimates of variability in Table I. As we lament the inability to replicate data together with declining standards and transparency in science (we all read Begley and Ellis in Nature) - its surprising (not really) that the ACS journals continue to consider repletion of data as optional.

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2. Curious Wavefunction on June 19, 2012 8:57 AM writes...

As I mention in my post, one wonders how many drugs we may have missed because of this illusory lack of activity.

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3. Joe Q. on June 19, 2012 9:02 AM writes...

For a minute there, I wondered which Shoichet lab you were referring to... and then I realized that it was both (a sibling collaboration!)

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4. Rick Wobbe on June 19, 2012 9:14 AM writes...

I wonder how often this plays a role in the annoying phenomenon of screening hits whose titrations plateau well below 100% inhibition.

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5. anon on June 19, 2012 9:23 AM writes...

id love to hear solutions here (no pun intended)...first and foremost, Derek, to your question, how would one know to begin with?? are there reasonable steps to take to be sure you have inactivity versus insolubility?

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6. Pete on June 19, 2012 9:54 AM writes...

Plasma protein binding is measured routinely in Drug Discovery and this study has potential implications for how we interpret these measurements. I am not too familiar with the fine details of PPB assays but I seem to recall a sensitivity to lipid concentration in the assay media (perhaps somebody who knows about this could comment). Also people have looked at binding to Albumin using SPR which would provide insight into binding stoichiometry. In general, I would like to see aqueous solubility measured in protein free media for studies of aggregation.

On an unrelated note, I'm not sure if a cysteine protease such as Cruzain is an ideal enzyme with which to study aggregation phenomena since inhibition can result from oxdidation of the catalytic cysteine. I need to look art some of the earlier studies but, in the meantime, does anybody know whether researchers have looked much at the effect of these detergents on enzyme activity in the absence of inhibitors?

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7. Rick Wobbe on June 19, 2012 10:40 AM writes...

anon 5, It's not perfect, but one thing I and others have done is run compounds through a light scattering assay at a couple of concentrations (one in the 1-10 micromolar range and one in the 10-100 micromolar range) in a consensus aqueous buffer (I've used PBS). Some companies have made that part of their analytical process following synthesis or acquisition of compounds or libraries.

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8. Rick Wobbe on June 19, 2012 10:54 AM writes...

as an addendum to #7, my groups have used the DynaPro dynamic light scattering instrument to get throughput and it's worked well.

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9. NJBiologist on June 19, 2012 11:46 AM writes...

@7 Rick Wobbe: How did you choose those concentrations? Are they the highest concentrations you expect to deliver to your in vitro system?

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10. Rick Wobbe on June 19, 2012 12:49 PM writes...

NJBiologist 9, Essentially yes. They roughly bracket the concentrations I've most often used in primary screens and the highest concentration I'd use in hit titration, so it's better suited for library evaluation. For analoging and lead optimization, you'd want to change the concentrations.

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11. Alig on June 19, 2012 1:24 PM writes...

This paper has a similar error as the patent did in your post yesterday. A very unusual tautamer for the imidazole in Nilotinib.

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12. Biologist At Large on June 19, 2012 5:09 PM writes...

Anyone ever see an "aggregator" have an activating effect in either a biochemistry or cell-based assay? Normally they mess up your antagonist assays or enzyme inhibition assays, by taking-out the activity you're looking for so they appear as false positives. But can they ever have the opposite effect?

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13. NJBiologist on June 19, 2012 11:13 PM writes...

@10 Rick Wobbe: I'm glad to hear you say that. I had someone explain to me that solubility couldn't be an issue for their compound in vivo, based on solubility testing at 10 micromolar. He was surprised when we walked through the calculations and found that his high dose group had gotten something like 5 millimolar....

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14. MoMo on June 20, 2012 8:42 AM writes...

Dynamic Light Scattering! Exactly! Leave it to chemistry to help biologists understand things.

Now if you all were really on the ball you would study all of your screening compounds for cell membrane perturbation via flow cytometry.

Then half of all screening compounds would disappear and with it half of all biologists.

And it would be a good day.

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15. Biologist At Large on June 20, 2012 10:29 AM writes...

MoMo @14....it was the biologists at our digs that set up the Dynamic Light Scattering assay to help the whole team understand the compounds....just sayin'...

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16. Rick Wobbe on June 20, 2012 7:07 PM writes...

MoMo #14, Similar to Biologist At Large's account (#15), I, a biologist, was the instigator of doing DLS on compounds after we ran into compounds whose titration curves topped out at 80% inhibition. The chemists told us it couldn't be a solubility problem, that we must be screwing up the assay somehow (we were only biologists, after all) and the solubility testing was too hard anyway. We had a DynaPro in the screening lab, so we did it in the screening group. Turned out solubility was the problem.

However, you will be relieved to know that a few years later, I lost my job and, after 2 years of failing to find a job, tried teaching high school. I guess I'm one of that 50% of biologists who got what was coming to them, whose disappearance makes it a good day for you. Please excuse me for not sharing your exuberance.

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17. Biologist At Large on June 21, 2012 10:25 AM writes...

Rick Wobbe @16

..the number of times I've been told that "solubility isn't the problem"....one of these days {inset name}...*POW*...right in the kisser.

And sorry to hear about your job scenario. It's a bad time for all of us in pharma/biotech - chemists AND biologists.

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18. Rick Wobbe on June 21, 2012 3:00 PM writes...

Thanks B.A.L. (17). I hate wearing my heart out on my sleeve like that, but sometimes life, and science, are plenty hard enough without the attitude-copping. I needed to get that off my chest.

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19. A. Postdoc on November 28, 2012 2:02 PM writes...

@12, biologist at large,

Check out: http://pubs.acs.org/doi/abs/10.1021/ja208350u

Essentially, they found compounds that formed fibrils and turned the enzyme on. Not quite aggregators, but a whole other class of molecules.

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