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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 15, 2012

A Good Example of Phenotypic Screening

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Posted by Derek

I like to highlight phenotypic screening efforts here sometimes, because there's evidence that they can lead to drugs at a higher-than-usual rate. And who couldn't use some of that? Here's a new example from a team at the Broad Institute.

They're looking at the very popular idea of "cancer stem cells" (CSCs), a population of cells in some tumors that appear to be disproportionately resistant to current therapies (and disproportionately responsible for tumor relapse and regrowth). This screen uses a surrogate breast cell line, with E-cadherin knocked down, which seems to give the dedifferentiated phenotype you'd want to target. That's a bit risky, using an artificial system like that, but as the authors correctly point out, isolating a pure population of the real CSCs is difficult-to-impossible, and they're very poorly behaved in cell culture. So until those problems are solved, you have your choice - work on something that might translate over to the real system, or ditch the screening idea for now entirely. I think the first is worth a shot, as long as its limitations are kept in mind.

This paper does go on to do something very important, though - they use an isogenic cell line as a counterscreen, very close to the target cells. If you find compounds that hit the targets but not these controls, you have a lot more confidence that you're getting at some difference that's tied to the loss of E-cadherin. Using some other cell line as a control leaves too many doors open too wide; you could see "confirmed hits" that are taking advantage of totally irrelevant differences between the cell lines instead.

They ran a library of about 300,000 compounds (the MLSMR collection) past the CSC model cells, and about 3200 had the desired toxic effect on them. At this point, the team removed the compounds that were flagged in PubChem as toxic to normal mammalian cell lines, and also removed compounds that had hit in more than 10% of the assays they'd been through, both of which I'd say are prudent moves. Retesting the remaining 2200 compounds gave a weird result: at the highest concentration (20 micromolar), 97 per cent of them were active. I probably would have gotten nervous at that point, wondering if something had gone haywire with the assay, and I'll bet that a few folks at the Broad felt the same way.

But when used the isogenic cell line, things narrowed down rather quickly. Only 26 compounds showed reasonable potency on the target cells along with at least a 25-fold window for toxicity to the isogenic cells. (Without that screen, then, you'd have been chasing an awful lot of junk). Then they ordered up fresh samples of these, which is another step that believe me, you don't want to neglect. A number of compounds appear to have not been quite what they were supposed to be (not an uncommon problem in a big screening collection; you trust the labels unconditionally at your own peril).

In the end, two acylhydrazone compounds ended up retaining their selectivity after rechecking. So you can see how things narrow down in these situations: 300K to 2K to 26 to 2, and that's not such an unusual progression at all. The team made a series of analogs around the lead chemical matter, and then settled on the acylhydrazone compound shown (ML239) as the best in show. It's not a beauty. There seems to be some rule that more rigorous and unusual a phenotypic screen, the uglier the compounds that emerge from it. I'm only half kidding, or maybe a bit less - there are some issues to think about in there, and that topic is worth a post of its own.
More specifically, the obvious concern in that fulvene-looking pyrrole thingie on the right (I use "thingie" in its strict technical sense here). That's not a happy-looking (that is, particularly stable-looking) group. The acylhydrazine part might raise eyebrows with some people, but Rimonabant (among other compounds) shows that that functional group can be part of a drug. Admittedly, Rimonabant went down with all hands, but it wasn't because of the acylhydrazine. And the trichloroaryl group isn't anyone's favorite, either, but in this context, it's just sort of a dessert topping, in an inverse sense.

But the compound appears to be the real thing, as a pharmacological tool. It was also toxic to another type of breast cancer cell that had had its E-cadherin disrupted, and to a further nonengineered breast cancer cell line. Now comes the question: how does this happen? Gene expression profiling showed a variety of significant changes, with all sorts of cell death and free radical scavenging things altered. By contrast, when they did the same profiling on the isogenic controls, only five genes were altered to any significant extent, and none of those overlapped with the target cells. This is very strong evidence that something specific and important is being targeted here. A closer analysis of all the genes suggests the NF-kappaB system, and within that, perhaps a protein called TRIB3. Further experiments will have to be done to nail that down, but it's a good start. (And yes, in case you were wondering, TRIB3 does, in fact, stand for "tribble-3", and yes, that name did originate with the Drosophila research community, and how did you ever guess?)

So overall, I'd say that this is a very solid example of how phenotypic screening is supposed to work. I recommend it to people who are interested in the topic - and to people who aren't, either, because hey, you never know when it might come in handy. This is how a lot of new biology gets found, through identifying useful chemical matter, and we can never have too much of it.

Comments (23) + TrackBacks (0) | Category: Cancer | Chemical Biology | Drug Assays


1. Hap on November 15, 2012 9:21 AM writes...

The fulvene's really just a tautomer of the 2-pyrrolecarboxaldehyde acylhydrazone - it's functionally equivalent (addition to the fulvene with a nucleophile would occur at the aldehyde carbon to regenerate the pyrrole core and generate an acylhydrazine hemiaminal derivative, which would be the reaction you might expect from the acylhydrazone).

Considering how electron-deficient the fulvene tautomer is (the enamine and electron-deficient fulvene segregated in a sort of charge separation) while the acylhydrazone should be relative electron-neutral I wouldn't figure it to be a significant species. Since acylhydrazones were used as stable aldehyde characterizing derivatives, I would not figure that addition to the hydrazone would be facile, although I'm not an enzyme.

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2. barry on November 15, 2012 9:44 AM writes...

regardless of tautomer, that fulvene or pyrrole is probably trouble. One of Lipitor's biggest surprises is the pyrrole. They're rare in drugs because they're oxidatively/metabolically labile. Lipitor seems to get away with it by huge steric encumbrance.
But how do you move this series forward? If you don't proceed to demonstrate productive binding to TRIB3 (which I don't find in the PDB) you have a lot of modifying to do in the dark to get to a drug-like molecule. You may not be able to get an x-ray co-crystal structure, but you surely should try for one (or several).

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3. Cytirps on November 15, 2012 9:54 AM writes...

It seems to be a cell based assay instead of a phenotypic screening.

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4. B on November 15, 2012 10:05 AM writes...

@3: Death is my phenotype of choice.

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5. Morten G on November 15, 2012 10:17 AM writes...

It's quite small. In some libraries that would have been characterised as a fragment (21 non-hydrogen atoms).
Anyone want to bet that the activity is due to it being a "dirty" molecule?

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6. Hap on November 15, 2012 10:48 AM writes...

I don't know if lots were in the original libraries tested, but couldn't you make a whole bunch of five-membered heterocyclic aldehyde acylhydrazones? If pyrroles are a problem, various thiazole- or imidazole carboxaldehydes might work better.

It seems like you could make a fishing expedition relatively easily with this kind of molecule - lots of heterocyclyl aldehydes, and lots of aryloxyacetyl hydrazides, and the same sets of assays. I don't know if it would work, but with a lead, and good assays, it's not blind chance.

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7. anonymous on November 15, 2012 11:47 AM writes...

@4 B: Death is not a phenotype! (tongue-in-cheek), obviously you never TA'ed for a geneticist

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8. David Borhani on November 15, 2012 1:44 PM writes...

Looks a lot like nitrofurazone as well. I think the analogy with Rimonabant is a bit less relevant, because tautomers of the acylhydrazide arn't possible in Rimonabant.

The trichlorophenol has a pKa ~6.2, i.e. the position alpha to the carbonyl may be mildly activated for displacement.

This compound may be binding to and/or modifying a specific target in the NF-kB pathway, like TRIB-3, but I'm putting my money on it being junk. Too many chemically questionable features, and I suspect the observed induction of free radical & apoptotic pathways in non-isogenic control cell lines (read: most other cells in your body) spells trouble.

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9. J on November 15, 2012 1:50 PM writes...

$100 that this compound is a prodrug of some sort. activated best in the most sensitive cells and least activated in the controls. Not sure how to demonstrate this but it is the Occam's razor explanation of the observation.

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10. Pete on November 15, 2012 2:44 PM writes...

The other tautomer can be observed in the crystal structures of a number of analogues (check the CSD). In this tautomer the hydrazone will exert an electron-withdrawing effect on the pyrrole which might be hypothesised to stabilise the pyrrole with respect to oxidation. If you're interested in tautomeric issues in this context, you might want to take a look at: doi:10.1038/nchembio.995

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11. MoMo on November 15, 2012 2:51 PM writes...

Ye Gods! More of these Evil compounds from the MLSMR! I would have thought the craniums in Broad would have been wary of such beasts, but you go where the action is!

The molecule reminds me of a poem:

Tautomers! Tautomers! cried the Queen
If I had 2, I'd be King!

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12. Chris on November 15, 2012 3:46 PM writes...

I do agree that phenotypic screening is the way to go. Unfortunately I don’t think you picked a “good” example. As other readers have commented, this is actually not a phenotypic screen - just a plain low content surrogate assay for cell death - and it is unclear what these cells have to do with stem cells in the first place. I would also argue that nothing was elucidated at all, in absence of validation in actual stem cells. The cells used are not truly an isogenic pair, and there are many red flags in the results (you mentioned one of them).

This is more of a good example of overhyped and misleading science article and I wish you had picked another one for this post.

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13. Derek Lowe on November 15, 2012 3:50 PM writes...

#12 Chris - suggestions welcome! If you try to put a bunch of links here in the comments, the spam filters will probably grab it and I may not be able to rescue it in time. But plaintext references will do fine - or send me some by e-mail. Thanks!

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14. David Borhani on November 15, 2012 3:57 PM writes...

@10 (Pete), @11 (MoMo):
In the NatChemBio paper, is Bam7 covalently modifying Bax? Reaction of Lys21 with hydrazone?

The SAR is odd (suppl. fig. 9), i.e. there is essentially no SAR. Changing the Bam7 EtO to (Me, Cl, or OH) decreases binding by at least 10-fold. Looking at Fig 1c (IC50 curves), Bam7 better than above analogs by at least 50-fold. Making any of a variety of other small changes (add halogens, etc., here or there) again makes no difference...only Bam7 is active. Possible, I suppose, but a bit suspicious, especially given the rather open binding site (i.e., what's so special about the EtO interactions?).

I think MoMo is right --- more Evil compounds from the MLSMR!

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15. barry on November 16, 2012 1:26 AM writes...

the isogenic control cells are a nice touch. But does it pass the Shoichet detergent test? I've seen too many molecules like this in too many seemingly disparate screens.

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16. LeeH on November 16, 2012 8:38 AM writes...

This was the compound that the Klingons used to poison the quadrotriticale.

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17. L2B2 on November 16, 2012 12:00 PM writes...

#3. Cytirps #12. Chris: I totally agree with you guys that this is just an engineered cell screen. The article's title is a bit self-propaganda kinda. When a Chemist hear phenotype, we think disease modification, right?

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18. MoMo on November 16, 2012 1:17 PM writes...

This MLSMR compound appears to be a socialist plot to derail our Great American Drug Discovery Research machine. Q1: How many man hours are wasted, yearly, on such compounds from them I wonder?

Q2: Has anyone out there, still employed, ever pulled a gem out of this library?

Q3: Why would Broad, home to the "DOS Universe" study such a compound with the Trillions they have in-house?

SOB! I am so confused!

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19. Anonymous on November 16, 2012 5:14 PM writes...

#18 MoMo: Relax. This is just a J.Biomol.Scr., paper. Nobody would have noticed it but because of this discussion. Give guys a break - they did the work, published the result in a very honest and straightforward way.
There were no press-released, no BS about curing the cancer, etc.

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20. SP on November 17, 2012 8:18 PM writes...

There seems to be some disagreement about what is meant by phenotypic and some people here are conflating it with high-content screening. As I understand it, phenotypic just means you're probing a complex system for particular behavior- whether it's changes in gene expression, changes in morphology, or, yes, death of the cells. The usual comparison is to target-based screening which is a system of one or more purified components you're looking to directly modulate. So the complaints that this isn't phenotypic because it's just a simple readout of cell viability seem misguided- @12 seems to be saying that the opposite of phenotypic is "low content", implying that phenotypic must be "high content" aka imaging or expression arrays.

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21. Anonymous on November 20, 2012 10:24 AM writes...

#20: SP: technically this might be correct, but "phenotypic screen" has become synonym of high content screen nowadays, and it is broadly used to refer to that type of screen. In that sense, I believe that the title is misleading.

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22. Anonymous on November 20, 2012 5:55 PM writes...

Death is a phenotype but because there are so many different ways of dying, it is not a very informative phenotype.

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23. Angler on December 5, 2012 8:30 AM writes...

I'd say death is a phenotype, just not a pretty one. I followed this paper up because my former boss was on it. To me the major difference between a phenotypic screen and a targeted screen is whether your screening targets are predefined or biased. A good analogy is fly fishing vs traditional fishing. If you know what's (target)in the pond, you might as well use the targeted approach. But if you have little clue what's out there (as we encounter all the time in a complex biological system), you may want to try fly fishing instead. The beauty of phenotypic screenings is that you never know what you are going to get and many times you might have a pleasant surprise. The problem of targeted approach sometimes is that you thought you know your target well enough, but it turns out what you know is not nearly enough! Knowing the limitation of both screening approaches then it becomes a choice.

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