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Diabetes and Obesity

Zebrafish, Finally Taking Over as Foretold

The idea of phenotypic screening is not new, but once you bring up the topic, you find that different people have different ideas about what a phenotypic screen really is. As has been discussed around here before, not everyone buys into the concept of a cell-based screen as “phenotypic”, but I’m willing to believe in the category. But cells aren’t animals. And “animal” itself is a concept that needs some clarification as well. Fruit flies and nematodes are noble creatures, in their way, and have provided many an insight into living systems, but for human drug discovery you need to move a bit closer to humans.

But doing such a screen in a whole vertebrate animal is not easy to realize. You’re going to need thousands of them, many thousands, in the same way that a screen against a cloned protein target uses many thousands of wells. So that rules out anything larger than. . .what? Xenopus frogs and zebrafish? Doing those on scale is pretty nontrivial, too. I remember back at the Wonder Drug Factory, when I was part of a task force (a good ten or twelve years ago now) trying to think of new ways to generate projects and leads, and one of the ideas we kicked around was the feasibility of just that sort of screening. “So which building are we going to turn into an aquarium?” was one response.

I wondered about this just a year or so ago, now a new paper has come along detailing the largest zebrafish screen I have yet heard of. The authors are looking at diabetes pathways, and have generated transgenic fish embryos with fluorescent reporter genes tied to various pancreatic cells – one wavelength specifically for beta-cell growth, and another for general pancreatic cell differentiation (as measured by delta-cells). The appearance and timing of these tissues in zebrafish development is pretty well worked out, so compounds that enhance or disrupt aspects of it will be apparent on inspection.

On inspection . . .there’s the rub. How do you “inspect” the thousands and thousands of zebrafish embryos that you’re going to need. This paper details an automated system that seems to do a pretty good job (but I’m mindful that people have been working on automated imaging tools for such things for many years now – it’s not easy, and you run a significant risk of generating, at significant cost, a terabyte of so of noise). These authors, actually, had previously tried a less-automated pancreatic growth assay in zebrafish, and seem to have been spending some time trying to avoid doing it that way again. The result is what they’re calling “the first truly high-throughput whole-organism drug screen in a vertebrate model”, and they may well be right about that, although there are a lot of press releases over the years that would appear to disagree.

So, how many compound are we talking about here? Probably not as many as you’re thinking. Their collection was 3348 (mostly approved) drugs, but you need 16 zebrafish embryos per concentration (six levels) across the dose-response, which added up, in the whole screen, to over 500,000 fish embryos. That gives you one drug per 96-well plate. This data-point buildup happens, naturally, because the data are noisy in whole animals, and if you don’t work on this scale you run the risk of that terabytes-of-noise effect, especially in what is, in the end, a (sorry) fishing expedition. (And if this makes you wonder if many rodent studies are underpowered, you may be onto something there, although keep in mind that many of these are narrowed down to very specific readouts).

225 compounds showed signs of activity at first, but 48 of these were false positives (fluorescent interference or general effects on embryo viability). 23 of the remaining compounds really looked as if they were really affecting islet cell formation, and 23 more might have been, so these compounds were taken into orthogonal secondary assays. 11 of the compounds (“Lead I”) from the best set also performed in an endocrine reporter assay, but they also went back to look for compounds that might have been increasing the beta-cell mass without such effects, and 15 of these  (“Lead II”) were identified by further screening.

One possible commonality in the Lead I set was an affect on NF-kB function, and in the Lead II set, serotinergic activity looked like a possible common trait. Serotinergic compounds (like fluoxetine or serotonin itself) that weren’t in the original screening set also showed the desired effects on beta-cells, which leads some weight to the idea. Another recent (although smaller and less automated) zebrafish screen looking for beta-cell effects also identified serotonin as a hit, interestingly, and the two screens seem to generally overlap in activity classes, which must be reassuring.

Overall, I have to say that I’m impressed by efforts like these. There are still plenty of chances to go off the rails, but this screen seems to have been designed to take as many of these into account as possible. The next question is how many other screens of this sort are achievable – that is, how many different types of tissue can be usefully labeled? And how many non-embryonic-development endpoints can it be extended to? That zebrafish aquarium building may have merely been ahead of its time. . .

14 comments on “Zebrafish, Finally Taking Over as Foretold”

  1. Kent Brockman says:

    I for one welcome our piscine overlords!

  2. bhip says:

    A few (rhetorical) questions & a comment:
    1- What is the connection btwn affecting organ/cell development vs. altering organ/cell function in fully developed entity i.e. a patient?
    2- We have many, many examples of animal models in drug development where mice ≠ rats ≠ dogs ≠ people- why would we think that a positive effect in zebrafish would translate predictably to patients?
    3- A compound that generally impairs (fish) embryonic development (examples?) would be disregarded/rejected- this could be a lead compound in an adult animal/patient?

    A general comment- – A (smallish) industrial collection of 1,000,000 compounds equates to ~ 15 million embryos based on the screening paradigm described by Derek. Never raised or phenotyped zebrafish but that sounds like a lot of fish….

    1. hypnos says:

      Well, I’m not sure how many of those 1M compounds would actually dissolve in an aquarium or enter the fish without being injected. If you want to do in vivo screening, you probably have to apply some pretty strict filters around PhysChem- and ADME-properties. (Which might be one reason why they focused on drugs.)

      One positive aspect of our typical in vitro assays is that you can get away and answer isolated questions with some pretty lousy compounds (considering their overall profile).

  3. Anon says:

    “But doing such a screen in a whole vertebrate animal is not easy to realize. You’re going to need thousands of them…”

    How big is the US prison population?

  4. anonymous says:

    The title “Zebrafish, Finally Taking Over as Foretold” sounds a bit too hyped up to my ear.

  5. Derek Lowe says:

    Anonymous, it’s meant to be slightly joking, actually. . .

  6. Mildweasel says:

    That title made me cackle. Thats the best one since “Sand wont save you…”

  7. Agent Smith says:

    This is what the Matrix is for.

  8. milkshaken says:

    I would propose to apply a gentle overpressure, to pump out the fluorescently labeled zebra fish through a narrow transparent tubing – to perform flow ichthyometry. (And since we are using species that is naturally striped, we could modify a simple supermarket barcode reader to serve as a detector.)

  9. Gene says:

    I initially read “Lead I” as Pb and thought to myself, “Yes, that would probably impact pancreatic development.”

  10. Anonymous BMS Researcher says:

    “Flow ichthyometry,” I laughed out loud at that!

  11. Andre says:

    The study by Wang et al. 2015 is surely a tour de force. I do not want to imaging the costs required for the screen. I am however not sure whether is was necessary to screen six concentrations (4 µM to 125 mM) of each compound tested. The other study mentioned by Derek, Tsuji et al. 2014, screened at 10 µM and recovered hits implicating glucocorticoids, retinoic acid and serotonin signaling. They also pointed out that compounds causing lethality at 10 µM were rescreened at lower concentrations. Seems to me like a wise strategy to reduce the screening of compounds to a reasonable level. EC50 values can later be established for the bioactive hits. Returning back to the list hits reported by Wang et al., the top two hits inducing endocrine different differentiation are N-acetylmuramic acid and methylthiouracil. The first is an amino sugar and constitutes a building block of the peptidoglycan polymer of Gram-positive bacterial cell walls. The other methylthioruracil is an antithyroid preparation. It is unclear to me how these compounds act to promote endocrine differentiation in zebrafish. Any suggestions out there?

  12. Smoki says:

    This reminded me of a recent cool paper describing high throughput robotic handling of fruit flies:
    http://www.ncbi.nlm.nih.gov/pubmed/26005812

    A popular science look at the study:
    http://nerdist.com/robots-can-now-perform-brain-surgery-on-fruit-flies/

  13. This reminded me of a recent cool paper describing high throughput robotic handling of fruit flies

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