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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

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October 21, 2010

Laser Nematode Surgery!

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Posted by Derek

There's a headline I've never written before, for sure. A new paper in PNAS describes an assay in nematodes to look for compounds that have an effect on nerve regeneration. That means that you have to damage neurons first, naturally, and doing that on something as small (and as active) as a nematode is not trivial.

The authors (a team from MIT) used microfluidic chips to direct single nematodes into a small chamber where they're held down briefly by a membrane. Then an operator picks out one of its neurons on an imagining screen, whereupon a laser beam cuts it. The nematode is then released into a culture well, where it's exposed to some small molecule to see what effect that has on the neuron's regrowth. It takes about 20 seconds to process a single C. elegans, in case you're wondering, and I can imagine that after a while you'd wish that they weren't streaming along quite so relentlessly.

The group tried about 100 bioactive molecules, targeting a range of known pathways, to see what might speed up or slow down nerve regeneration. As it happens, the highest hit rates were among the kinase inhibitors and compounds targeting cytoskeletal processes. (By contrast, nothing affecting vesicle trafficking or histone deacetylase activity showed any effect). The most significant hit was an old friend to kinase researchers, staurosporine. Interestingly, this effect was only seen on particular subtypes of neurons, suggesting that they weren't picking up some sort of broad-spectrum regeneration pathway.

The paper acknowledges that staurosporine has a number of different activities, but treats it largely as a PKC inhibitor. I'm not sure that that's a good idea, personally - I'd be suspicious of pinning any specific activity to that compound without an awful lot of follow-up, because it's a real Claymore mine when it comes to kinases. The MIT group did check to see if caspases (and apoptotic pathways in general) were involved, since those are well-known effects of staurosporine treatment, and they seem to have ruled those out. And they also followed up with some other PKC inhibitors, chelerythrine and Gö 6983, and these showed similar effects.

So they may be right about this being a PKC pathway, but that's a tough one to nail down. (And even if you do, there are plenty of PKC isoforms doing different things, but there aren't enough selective ligands known to unravel all those yet). Chelerythrine inhibits alanine aminotransferase, has had some doubts expressed about it before in PKC work, and also binds to DNA, which may be responsible for some of its activity in cells. Gö 6983 seems to be a better tool, but it's is in the same broad chemical class as staurosporine itself, so as a medicinal chemist I still find myself giving it the fishy eye.

This is very interesting work, nonetheless, and it's the sort of thing that no one's been able to do before. I'm a big fan of using the most complex systems you can to assay compounds, and living nematodes are a good spot to be in. I'd be quite interested in a broader screen of small molecules, but 20 seconds per nematode surgery is still too slow for the sort of thing a medicinal chemist like me would like to run - a diversity set of, say, ten or twenty thousand compounds, for starters. And there's always the problem we were talking about here the other day, about how easy it is to get compounds into nematodes at all. I wonder if there were some false negatives in this screen just because the critters had no exposure?

Comments (16) + TrackBacks (0) | Category: Biological News | Drug Assays | The Central Nervous System


COMMENTS

1. Bang on October 21, 2010 9:49 AM writes...

Off topic, but fun:

Nitoglycerine detonation film:

http://www.youtube.com/watch?v=r17czTWHFmU&feature=player_embedded

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2. barry on October 21, 2010 9:53 AM writes...

getting any meaningful hits out of a screening set of "about a hundred" is pretty remarkable. The authors improved their odds by including a compound like staurosporine, precisely because it is potent against a large number of targets. It now remains to disambiguate that result by screening a collection of (more) specific protein kinase inhibitors. To leap from a staurosporine hit to the conclusion that the effect is mediated by PKC is bold, to say the least.

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3. cynical1 on October 21, 2010 10:02 AM writes...

"The most significant hit was an old friend to kinase researchers, staurosporine. Interestingly, this effect was only seen on particular subtypes of neurons, suggesting that they weren't picking up some sort of broad-spectrum regeneration pathway."

Unless I read the article wrong, staurosporine inhibited regrowth of neurons not stimulated regeneration. IMO, a multi-kinase inhibitor that inhibits neuronal regrowth at greater than 5 uM and shows toxicity at 10 uM isn't all that interesting to me.........even if I was a worm.

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4. ronathan richardson on October 21, 2010 10:16 AM writes...

Along these lines, I was hoping for a comment on Steve McKnight's ridiculously labor-intensive small molecule neuroprotection screen published in Cell this year: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-50GM1DG-9&_user=501045&_coverDate=07%2F09%2F2010&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000022659&_version=1&_urlVersion=0&_userid=501045&md5=cabfead53f2eb556c892dbdc026a7e14&searchtype=a

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5. GC on October 21, 2010 10:30 AM writes...

Hm. Being MIT, I'd think they'd get with their Comp Sci department and have 'em rig up a vision system that can find neurons and target them for the laser. That'd speed things up, or at least get the man out of the loop.

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6. Sili on October 21, 2010 10:32 AM writes...

Nematodes with friggin' laserbeam on in their heads.

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7. Anon on October 21, 2010 11:40 AM writes...

I wonder what PETA thinks of this.

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8. Ed on October 21, 2010 11:58 AM writes...

Sili - I await the follow up paper, with giant mutated sea bass.

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9. milkshake on October 21, 2010 12:27 PM writes...

@4 In your article, these are Dimebone-like Serono carbazole compounds working through stabilization of mitochondria. They are not kinase compounds. It is not a neuron-specific anti-apoptosis effect. I am unsure how generally useful for neurogenesis they will be, outside this engineered mouse model.

Staurosporin analogs: I think they are not very good probes because they carpet-bomb the entire kinome. We and others have had some luck with Sutent-related compounds for neuroprotection (especially if one cleans up their rather broad profile)

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10. Maks on October 21, 2010 3:29 PM writes...

Talking about stupid/labour intensive screens:
dx.doi.org/10.1016/j.neuron.2010.09.001

300 compounds manually screened by electrophysiology. Papers like that should be rejected just because of poor screening design . Should be a topic by itself, stupid screens in academy which either produce obvious hits or useless hits, or are extremely labour intensive....

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11. ronathan richardson on October 21, 2010 4:30 PM writes...

I was more interested in having the debate about what level of "labor intensity" is optimal in screening. If it isn't obvious, there's a bit of a tradeoff between applicability to 1536, penny-per-well assays and relevance in biology--as we see the many phase II failures of compounds from target-based binding screens. My inclination is much more towards the "complex" (i.e. in vivo) assays, even better if they are in a real tissue and not just a cell line, and just put in the money and effort up-front and only screen 10-20k compounds of a diversity set--I think it's false that screening 500,000 compounds is much more likely to give you a drug than 10,000 (as evidence, see how low throughput chemical screening gave us most of the great drugs on the market right now.

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12. Esteban on October 21, 2010 7:24 PM writes...

Hmmm...perhaps an early candidate for next year's Ignoble's...

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13. Lacerta Bio on October 22, 2010 7:28 AM writes...

Nematodes? Really? Wow.

Derek, you make an interesting comment on the desire to assay compounds in the most complex systems you can. I agree with you for some areas, like neurology, or perhaps immunology. But for other areas, perhaps not so much? Would cardiovascular drugs like beta blockers be better suited in a simpler model)? Also, does anybody know how relevant this model is to mammals, let alone humans?

Great find, Derek.

Permalink to Comment

14. partial agonist on October 22, 2010 10:51 AM writes...

We often read about screening with nematodes, or zebrafish, or fruit flies, or other easily bred/easily handled critters.

It always sounds pretty interesting, but I'm curious if anything that worked well in any of these types of systems actually translated to in vivo activity in mammals, including man.

Anybody know?

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15. Marc on October 26, 2010 3:31 PM writes...

@14:

As a fruit-fly biologist who thinks a lot about drugs, we have a lot of examples now that _could_ have been discovered in flies but weren't - there are lots of compounds that cross-react and seem to have similar specificity. I suspect strongly that if someone comes up with the right assay the advantages of flies/worms may contribute a lot to screening for specific classes of compounds. This is what exelixis was supposed to do, back in the day.

That said, the screen is critical, and so is knowing what to look for. And once you get your compound, PK and tox are going to be just as bad coming from worms or flies as from cell culture—these things have totally different repertoires of cytochromes p450 from mammals. So you lose a lot of the apparent virtues that come from being in vivo.

All this, plus the relatively low levels of effort being put into this by pharma (as far as I know), suggests to me that we'll be waiting a long time yet until the first compound comes from flies into humans - but I would love to be proven wrong.

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16. Anonymous on October 29, 2010 12:47 PM writes...

These guys are experts on the instrumentation - the microfluidics, laser surgery, speed of screening... I wouldn't focus too much on their feeble conclusions on the medicinal chemistry. They are going to stretch to find a good looking nail for their golden hammer. They are spinning out a company using similar methdology with zebrafish as a screening service. The initial interest from a number of discovery groups would imply that it is something to watch. The business case is being sold on similar logic as expressed by Marc in #15 - that there supposedly are a large number of much later-stage failures that are subsequently shown to have been predictable in zebrafish. Is that true?

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