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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 13, 2009

Lumpy Assay Results

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Posted by Derek

When we screen zillions of compounds from our files against a new drug target, what can we expect? How many hits will we get, and what percentage of those are actually worth looking at in more detail?

These are long-running questions, but over the last twenty years some lessons have been learned. A new paper in J. Med. Chem. emphasizes one of the biggest ones: if at all possible, run your assays with some sort of detergent in them.

Why would you do a thing like that? Compound aggregation. The last few years have seen a rapidly growing appreciation of this problem. Many small molecules will, under some conditions, clump together in solution and make a new species that has little or nothing to do with their individual members. These new aggregates can bind to protein surfaces, mess up fluorescent readouts, cause the target protein to stick to their surfaces instead, and cause all kinds of trouble. Adding detergent to the assay system cuts this down a great deal, and any compound that's a hit without detergent but loses activity with it should be viewed with strong suspicion.

The authors of this paper (from the NIH's Chemical Genomics Center and Brian Shoichet's lab at UCSF) were screening against the cysteine protease cruzain, a target for Chagas disease. They ran their whole library of compounds through under both detergent-free and detergent conditions and compared the results. In an earlier screening effort of this sort against beta-lactamase, nearly 95% of the hits (many of them rather weak) turned out to be aggregator compounds. This campaign showed similar numbers.

There were 15 times as many apparent hits in the detergent-free assay, for one thing. Some of these were apparently activating the enzyme, which is always a bit of an odd thing to explain, since inhibiting enzyme activity is a lot more likely. These activators almost completely disappeared under the detergent conditions, though. And even looking just at the inhibitors, 90% of the hit set in the detergent-free assay went away when detergent was added. (I should note that control cruzain inhibitors performed fine under both sets of assays, so it's not like the detergent itself was messing with the enzyme to any significant degree).

They point out another benefit to the detergent assay - it seems to improve the data by keeping the enzyme from sticking to the walls of the plastic tubes. That's a real problem which can kick your data around all over the place - I've encountered it myself, and heard a few horror stories over the years. But it's not something that's well appreciated outside of the people who set up assays for a living (and not always even among some of them).

So, let's get rid of those nasty aggegators, right? Not so fast. It turns out that some of the compounds that showed this problem during the earlier beta-lactamase work didn't cause a problem here, and vice versa. Even using different assays designed to detect aggregation alone gave varying results among sets of compounds. It appears that aggregation is quite sensitive to the specific assay conditions you're using, so trying to assemble a blacklist of aggregators is probably not going to work. You have to check things every time.

One other interesting point from this paper (and the previous one): curators of large screening collections spend a lot of time weeding out reactive compounds. They don't want things that will come in and react nonspecifically with labile groups on the target proteins, and that seems like a reasonable thing to do. But in these screens, the compounds with "hot" functional groups didn't have a particularly high hit rate. You'd expect a cysteine protease to be especially sensitive to this sort of thing, with that reactive thiol right in the active site, but not so. This ties in with the work from Benjamin Cravatt's group at Scripps, suggesting that even fairly reactive groups have a lot of constraints on them - they have to line up just right to form a covalent bond, and that just doesn't happen that often.

So perhaps we've all been worrying too much about reactive compounds, and not enough about the innocent-looking ones that clump up while we're not looking. Detergent is your friend!

Comments (11) + TrackBacks (0) | Category: Drug Assays | Life in the Drug Labs


1. Curious Wavefunction on November 13, 2009 10:39 AM writes...

Shoichet has been writing about this for many years. Many HTS screens now use his tests as standard tests for determining aggregation.

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2. Practical Fragments on November 13, 2009 10:44 AM writes...

Thanks for publicizing this. It's a real problem, particularly as more academic groups move into screening, and can even trip up people who are aware of the problem. Brian Shoichet and Adam Renslo published a related paper a few months ago in which they spent some time optimizing a series of cruzain inhibitors only to discover that they had 10-fold less detergent in their assay than they thought. The compounds even had interpretable SAR, but it turned out they had been optimized for aggregation, not binding to cruzain (see summary at Fortunately they discovered the error and their analysis makes a nice lesson, but many other papers likely report artifacts, not inhibitors.

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3. Fries with That? on November 13, 2009 5:11 PM writes...

How many different ways can a lab milk the same concept over and over again? Maybe the next paper will characterize what type of hits they get when they accidently add ten times greater detergent than they thought they had. Maybe they can try Tide, or Woolite.

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4. Fries with That? on November 13, 2009 5:11 PM writes...

How many different ways can a lab milk the same concept over and over again? Maybe the next paper will characterize what type of hits they get when they accidently add ten times greater detergent than they thought they had. Maybe they can try Tide, or Woolite.

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5. qetzal on November 13, 2009 7:17 PM writes...

For those of us poor, deprived individuals without online journal access:

What detergent(s) and concentration(s) did they use? Any indication that different 'aggregators' require different detergents (or concentrations)?

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6. Spectrochimico on November 14, 2009 2:00 AM writes...


They used 0.01% Triton X-100 for the detergent-present runs and 0.00005% Triton X-100 for the "detergent-free" runs.

They didn't report results for any other concentrations or surfactants, and didn't really give much of a rationale for the choice of the ones they did use, as far as I could see.

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7. qetzal on November 14, 2009 12:00 PM writes...

Thanks, Spectrochimico.

I'm not surprised they used Triton X-100. I figured it would be either that or Tween 20. Odd that 'detergent-free' = 0.00005% Triton, though.

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8. Ty on November 14, 2009 1:18 PM writes...

Long been wondering.. what about cellular assays? including antibacterial assays. Do aggregators do anything ? anybody know?

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9. drughuntcrapshoot on November 15, 2009 7:48 AM writes...

in the past, shoichet & colleagues have followed up such work with a docking paper so there will probably be another paper coming out that deals with the non-problematic actives' docking performance.

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10. Rico on November 17, 2009 9:05 PM writes...

Cells Baby! do it in cells! none of these issues come up from cell-based assays. of course, other challenges (opportunities?) do arise nonetheless the false positive rate is nil.

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11. drug_hunter on November 18, 2009 5:46 PM writes...

Instead of adding detergents, you can always, you know, screen actual drug-like molecules instead of brightly-colored coal tar derivatives... we have uniformly very low false positive rates in our screens because we actually think about what to screen beforehand. Most people do this, yes? I'm not claiming any unique insights here.

And, absolutely, do more cell assays!

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