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Nematodes And Your Compounds

Here’s an update on the research in Ethan Perlstein’s lab, in which he raises an interesting question. When you screen against some sort of purified protein, you’re optimizing on potency, but not necessarily on many other properties (except perhaps, at some level, solubility in the buffer system). But a cell-based screen for an intracellular target is a higher bar. Now your compounds not only have to hit the target, enough to affect the readout, but they have to get into cells in the first place. And that’s not always easy. Outside of the “normal” polarity and molecular weight ranges, cell penetration can be pretty haphazard. There are a number of mechanisms to get across the cell membrane, but they each have their own preferences and idiosyncracies, which are not always clear.

Perlstein, though, is testing in nematodes, and those organisms have a famously impermeable cuticle. So you’re not going to diffuse into them; everything they get is an oral dose, if that’s the right word. Then a compound has to face the somewhat alien nemadotinal (nematoid?) digestive system. They don’t have a liver per se, so digestion and metabolic clearance are sort of blended together. And again assuming an intracellular target, any compound that’s active in such an assay will have had to survive the digestive enzymes, get absorbed, and then pass through cell membranes to reach its site of action. So the question is, will these compound then have even more of a leg up than the ones from a traditional cellular assay?

The compound shown in the post certainly performed well after an oral dose in mice, but Perlstein himself isn’t sure how general an effect this might be, and neither am I. I’m going to betray my lack of nematodal (nematodish?) experience, but has anyone generated transgenic ones with humanize protein targets? Does that even work? If so, it might turn out to be a good way to work through a large screening set, cutting right down to the compounds that have some built-in advantages.

19 comments on “Nematodes And Your Compounds”

  1. Erebus says:

    Seems to me like a potentially valuable approach, if only as an intermediary step — after a protein screen but before experimentation in mice.

    A 1995 PNAS paper reported transgenic c.elegans expressing human ß-amyloid. I don’t know how this research was followed-up, if at all. Furthermore, I’m not familiar with other transgenic nematodes — but it seems as though they can be produced fairly simply.

    If I post a link, the spam filter is likely to eat my comment, so I’ll only note that the paper is at PMID 7568134. Citation:
    – Link CD. Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans. Proc Natl Acad Sci USA. 1995;92(20):9368-72.

  2. Argon says:

    This is a similar problem the agricultural companies face. Starting with an enzyme target rarely helps them because of the difficulties of getting the compounds into the crops or pests. So they’ve miniaturized whole organism screens and test for killing directly, instead of using proxies.

    We’d do the same in pharmaceutical discovery if it was possible and ethical. That’s one of the reasons why 3D cell culture, artificial tissues and more complex systems are being pursued.

  3. Jen says:

    I don’t know about nematodes, but I read an article about using zebra fish to screen drugs. Do similar issues you pointed out in nematodes also apply to zebra fish, albeit via different but just as challenging modes of delivery into the zebra fish?

  4. Wile E. Coyote, Genius says:

    “Nematode” is both a noun and an adjective.

  5. SM says:

    The worm is perfect for this for many reasons. A big one is the fact that c. elegans can just eat DNA and it gets integrated into their cells. So RNAi and CRISPR can just be fed to them. Also the worm is clear so visualizing reporter signals is super easy. Invariant cell lineage, 3 day life cycle, thousands of worms can easily be maintained by undergraduate level skill, super cheap… man worms are great.

  6. Mark Thorson says:

    Zebrafish have closed circulartory systems, liver, brain, etc. so they’re a better model in that sense, but it would take a pretty big facility to keep a million of them. A million nematodes is no big deal, probably could do it in a bookcase against the wall behind your desk.

  7. Swarchal says:

    In the past I’ve carried out a screen with transgenic C.elegans expressing human beta-amyloid as a model of inclusion body myositis, the model was crap but it was a nice method. There are also a fair few drugs that penetrate the cuticle, and you can also use a bus-8 mutant crossed with your disease model that increases permeability through the cuticle by disrupting collagen crosslinking.

  8. A point of clarification: the Niemann-Pick C nematode model we used in the primary screen is a CRISPR-generated null allele, not a human transgene. PLab’s MO is to take advantage of disease gene orthologs in model organisms. For example, NPC1 is an ancient gene conserved in all animals, including yeast.

  9. Peter Kenny says:

    Couldn’t help wondering about suppositories as a means to circumvent first pass metabolism in nemadodes…

  10. NJBiologist says:

    “So you’re not going to diffuse into them; everything they get is an oral dose, if that’s the right word.”

    Could you call the dosing “per stoma”?

  11. Dave says:

    Would using DMSO as a solvent help the drugs penetrate the barrier?

    Dave

  12. worm pharmacogeneticist says:

    Many human orthologs functionally rescue worm phenotypes when expressed as transgenes under the appropriate worm promoter. Making worm transgenics is (relatively) trivial. In the time it takes to make 1 transgenic mouse you can make hundreds of transgenic worm lines– the only real limitation is the throughput of micro-injecting C. elegans.

    That said, for screening while there are certainly going to be pharmacological differences between C. elegans targets and their human orthologs, given the limited level one is sampling chemical space in a primary screen, I would suggest these differences may be smaller than we think.

    I have always thought that one of the features of C. elegans for compound discovery is this oral availability requirement for compound penetration. While Pearlstein writes that it should not be surprising that oral availability in invertebrates is conserved in mice, In my own personal experience arguing for this type of work I find the consensus opinion strongly against this from known differences between mouse/rat/dog/human. Congratulations to the Pearlstein lab for getting data that at least proves the exception to the consensus.

    From both a target pharmacology perspective as well as a conservation of oral availability perspective I think invertebrates such as C. elegans get one at least in the ballpark at the stage of the primary screen. Albeit perhaps a different part of the ballpark than is accessed in a mammalian cell based screen– this is not necessarily a bad thing.

  13. Davide Marini says:

    This work by the Ausubel lab is particularly compelling in my opinion, as it illustrates the power of whole-organism screens:

    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2745594/

    Excerpt: “We identified 28 compounds and extracts that were not previously reported to have antimicrobial properties, including 6 structural classes that cure infected C. elegans animals but do not affect the growth of the pathogen in vitro, thus acting by a mechanism of action distinct from antibiotics currently in clinical use.”

  14. gshamilton says:

    Nematodes are amenable to droplet-based microfluidic platforms (Chemistry & Biology, 2008, 15, 427-437) for possible screening.

  15. HTSguy says:

    One issue that comes up when testing compounds against yeast (S. cerevisiae) is that they have lots of MDR-style pumps to keep compounds out. Does the GI tract of C. elegans have a similar repertoire of transporters?

    1. Erich Schwarz says:

      Yes, C. elegans has MDR transporters that do contribute to drug resistance. See (for instance) PubMed ID 18708066.

  16. milkshaken says:

    the best deadline-related excuse on a group meeting: “The nematodes ate my compounds”

  17. Andre says:

    I am happy for hear that the Ethan Perlstein’s lab has been able to identify a drug candidate for the treatment of Type C Niemann-Pick disease (NPC) on the basis of a phenotypic drug screen using a C. elegans mutant. It seems also that they were lucky in that their drug candidate PERL 101 has good oral bioavailability and in vivo PK characteristics. From the data presented in the Perlstein blog, it is however unclear whether the drug candidate has any therapeutic effect in mammalian models of NPC. This will be the real challenge. Do they know the target of their compound? In general, I do not believe that one can generalize from one promising example that in vivo screens in C. elegans select in general for drug candidates with promising PK and ADME characteristics. As pointed out by others here, C. elegans is for most human diseases not an ideal model as it is not a vertebrate to start. It lacks a many organs of the human body, such as a liver, a lung, a cardiovascular system, an immune system, and excretory organs with compelling resemblance to human kidneys. Furthermore, the evolutionary distance to man is with about 630 million years enormous (for example, see Fig. 6 in dos Reis et al. 2015 Curr. Biol. 25: 2939-2950; http://www.cell.com/current-biology/abstract/S0960-9822(15)01177-X). Therefore, phenotypic drug screening using vertebrate models, such as zebrafish (430 million years) and Xenopus (340 million years) are more likely to yield drug candidates with efficacy in human patients. This last points remains however to be proven. To date, no drug candidate has made it from zebrafish (or Xenopus) to FDA approval. To be fair, the drug screening community using zebrafish or Xenopus is tiny in comparison to the manpower and resources available in the pharmaceutical industry.

    A real verdict would require several hundred millions of investment (as was done for siRNA in the past) to prove or disprove the utility of fish and amphibians in drug discovery and development. At present, the industry prefers to invest large sums into other unproven technologies, such as mRNA-based therapeutics (Morderna!) and organs-on-a-chip.

  18. Brendan says:

    Congrats to the Perlstein Lab!

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