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.