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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

In the Pipeline

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November 20, 2012

What's A Phenotypic Screen, And What Isn't?

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Posted by Derek

The recent entry here on a phenotypic screen got some discussion going in the comments, and I thought I'd bring that out here for more. Some readers objected to the paper being characterized as a phenotypic screen at all, saying that it was just a cell-based screen. That got me to thinking about how I use the term, and to judge from the comments, there are at least two schools of thought on this.

The first says "Phenotypic" to mean something like "Screening for some desired effect in a living system, independent of any defined target". That's where I come from as well, since I've spent so much of my career doing target-based drug discovery. In a target-based program, you have cell assays, too - but they're downstream of the biochemical/pharmacological assay, and are there to answer two key questions: (1) does hitting the desired target do the right things to the cells, and (2) do the compounds break out into new SAR categories in cells that aren't apparent from their activity against the target? That last part can mean that some of the compounds are cytotoxic (while others aren't), or some of them seem to get into cells a lot better than others, and so on. But they're all subordinated to the original target idea, which drives the whole project.

The other definition of phenotypic screen would be something more like: "Screening simultaneously for a broad range of effects in a living system, independent of any defined target". I would call that, personally, a "high-content" screen (or more precisely, a high-content phenotypic screen, but (as mentioned) opinions vary on this. To the people who think this way, that Broad Institute paper I blogged on was merely a cell assay that looked at the most boring endpoint of all (cell death), and hardly lifted its head beyond that. But to a target-based person, everything that involves throwing compounds onto cells, with no defined target in mind, just to see what happens. . .well, that sure isn't target-based drug discovery, so it must be a phenotypic screen. And death is a phenotype, too, you know.

I like both kinds of screening, just for the record. But they're done for different purposes. High-content screening is a great way to harvest a lot of data and generate a lot of hypotheses, but for drug discovery, it can be a bit too much like a firehose water fountain. A more narrowed-down approach (such as "We want to find some compounds that make these kinds of cells perform Action X") is closer to actionable drug discovery efforts.

At any rate, a reader sent along some good high-content-screening work, and I'll blog about that separately. More comparisons will come up then.

Comments (9) + TrackBacks (0) | Category: Drug Assays


COMMENTS

1. Cytirps on November 20, 2012 11:56 AM writes...

It is necessary to represent the phenotype in a true phenotypic assay. Screening proton pump inhibitor with parietal cells does satisfy this criterion. The biggest problem in anti-cancer research is that most cancers have a very heterogeneous composition and their compositions do not just vary from one patient to another but also vary over time. An artificial cell line cannot be qualified as a phenotype.

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2. lynn on November 20, 2012 1:37 PM writes...

I have always used "phenotypic screen" to indicate that the screen has a readout that reflects inhibition of (or an effect on) a specific pathway or target. This is in contrast to "empirical screening" - which is not target based and, generally, relies on a death endpoint. As my field is antibacterial discovery, I'll note that almost all our antibacterial classes were found initially via empirical, kill-the-bug, screening. But for many years, we have tried to be more rational - employing whole cell screens with bacterial strains engineered to give a specific signal when some pathway or enzyme of choice is inhibited - such as turn on of a reporter gene when a specific protein is inhibited. Sometimes a phenotypic screen is set up so that the actual readout is death - but the screen entails detecting differential sensitivity of two strains; for example: a strain underexpressing a target enzyme of interest should be more sensitive to killing than an isogenic strain expressing the target at wild type or overexpressed levels.

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3. Olivier Boss on November 20, 2012 1:39 PM writes...

I fully agree with you, Derek: I'm going with the first definition of "Phenotypic" (screen, assay).
Congrats on your very clearly defined characteristics of both types of screens (phenotypic/functional and target-based/target-specific).
In fact, at Energesis Pharmaceuticals we are using a phenotypic screen, and the readouts can be high-content (imaging, fluo immunohistochemistry) or not (morphology, molecular markers mRNA).
We look for agents that induce the differentiation of our human brown adipocyte stem/progenitor cells (muscle-derived) into brown adipocytes
(improved from "A reservoir of brown adipocyte progenitors in human skeletal muscle. Crisan et al. Stem Cells 2008 Sep;26(9):2425-33").

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4. Boghog on November 20, 2012 4:30 PM writes...

It is not necessary to monitor more than one phenotype in a phenotypic screen, but it is often useful monitor several phenotypes in order to eliminate compounds that work through mechanisms that you are not interested in. High content screens with several readouts also allow you to build in controls into your assay.

Finally it should be obvious that the phenotypic screen should be a natural read out and not rely on a genetically engineered reporter system.

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5. rx90087 on November 20, 2012 7:59 PM writes...

Derek I agree with your definition of a phenotypic assay. I would add that a PDD assay should capture as much of the complex biology as practical. Depending on the biology this might be a cell line, primary cells, co-cultures of progenitor cells, or entire organisms. We have had similar discussions on the Phenotypic Drug Discovery SIG blog http://www.linkedin.com/groups?home=&gid=4443471&trk=anet_ug_hm&goback=%2Egmr_4443471

I have copied an entry that I posted about this recent "In the Pipeline" stream.

One of the “Pipeline” bloggers was critical of the Carmody et al manuscript. The basis of the criticisms “this is actually not a phenotypic screen - just a plain low content surrogate assay.... it is unclear what these cells have to do with stem cells ....” are related to topics discussed in our forum.

Broadly speaking, the issues seem to relate to what constitutes a phenotypic assay and how does it translate/relate to “real” biology?

Yes a recombinant model of “cancer stem cells” was utilized; but realistically are there better alternatives? What is a cancer stem cell..do you have a bucket of them for a screen? The authors utilized two methods to induce epithelial to mesenchymal transition (EMT), a cellular process thought to be important for the metatasis, proliferation, and drug resistance of some tumor models. In deed some aspects of cancer cell “stem-ness” seem to correspond to traits of tumor cells following EMT.

Was this a perfect model of “cancer stem cells”? Probably not.... but it is a practical alternative and provides an opportunity to make scientific advancements NOW. Was the assay endpoint a plain low content surrogate? Granted a cell survival assay is not “high content” but it is relevant to the experimental question/biological system at hand and is gain....practical.

The Carmody manuscript highlights the difficulty in developing a phenotypic assay which broadly translates. The authors identify compounds which are cytotoxic to “M cells” induced by two methods over a near isogenic control cell line without M induction. Although compounds are identified that are, to varying degrees, M-selective, the translation of activity from the isogenic EMT model does not necessarily translate to non-isogenic models (MDA-231 which I believe has an M-like transcript profile and the low adhesion SUM159 cancer stem cell model).

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6. Cellbio on November 21, 2012 12:47 AM writes...

Agree with boghog. One phenotype suffices. More than one is a complimentary phenotypic screen that greatly facilitates true hit identification from general poisons. High content is, imo, many measures in one system.

Also agree native, not engineered, and would add that immortal cell lines mostly useless.

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7. Morten G on November 21, 2012 5:08 AM writes...

Weird, I've always thought of phenotypic screens as Paul Erhlich with his 1000 compound library and the syphilitic rabbits, i.e. a whole organism screen. Of course you could do single cell work but that would require that you were looking at a single cell organism.

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8. Boghog on November 21, 2012 3:37 PM writes...

Cellbio. Also agree on immortalized cells being strongly suspect. But how do you feel about redifferentiated iPSCs?

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9. Cellbio on November 22, 2012 7:35 PM writes...

I think the iPSCs are likely good. The problem I have with immortal cell lines is that I have taken hit lists from a screen of them and did follow-up on primary cells and got zero concordance. While I have not done the same with iPSCs, I would bet the wiring is not totally unpredictable.

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