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Drug Assays

Watch Your Cell Assays

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).

19 comments on “Watch Your Cell Assays”

  1. luigi says:

    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.

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

  3. Joe Q. says:

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

  4. Rick Wobbe says:

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

  5. anon says:

    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?

  6. Pete says:

    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?

  7. Rick Wobbe says:

    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.

  8. Rick Wobbe says:

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

  9. NJBiologist says:

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

  10. Rick Wobbe says:

    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.

  11. Alig says:

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

  12. Biologist At Large says:

    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?

  13. NJBiologist says:

    @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….

  14. MoMo says:

    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.

  15. Biologist At Large says:

    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’…

  16. Rick Wobbe says:

    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.

  17. Biologist At Large says:

    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.

  18. Rick Wobbe says:

    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.

  19. A. Postdoc says:

    @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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