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What the Coronavirus Proteins Are Targeting

I wanted to mention this paper, which is one of the more comprehensive ones on the idea of repurposing existing drugs against the coronavirus. It’s a large multicenter team that clearly did a lot of very fast coordination to produce these results. What they’ve done is looked at the complete suite of proteins produced by the new coronavirus (some 27 to 29 of them, we think – viruses have relatively few moving parts). They were able to express almost all of them with a strep-tag in human cells (HEK293T), whereupon they used affinity-purification mass spectrometry (APMS) to identify human proteins that associate with them. With that technique, you lyse the cells and run that over beads containing a particular engineered form of streptavidin that the strep-tag binds do.. The strep-tagged proteins stick to the beads, along with whatever proteins are sticking to them, and then you clear these off by eluting with biotin (which binds to streptavidin like nothing on earth) and analyze them through mass spec proteomic techniques. It’s a classic chemical biology experiment, and works pretty well (with some known artifacts, naturally).

Looking at the interaction profile of 27 of the viral proteins, with suitable control experiments for nonspecific binding, etc., gives you 332 total interactions with the human proteome (well, the human proteome as expressed in HEK293T cells, anyway). To that point, the team went on to check the expression of these human proteins across 29 different tissue. Most interestingly, lung tissue (which is of course a lot more relevant to the infection!) had the highest expression of the overall suite of hits, which certainly argues for their functional importance in the viral mechanism. To go along with this, there’s recently been a report of protein expression changes during viral infection itself, and indeed, the interacting set also stands out in this group. In fact, a number of them also show up as hits in similar protein expression screens that had previously been conducted in West Nile and tuberculosis infection, both of which of course are also associated with lung pathways. So these pathogens may well have converged on some likely targets over time.

Running the human proteins through a Gene Ontology enrichment analysis to try to sort out functions showed some likely targets and pathways for several of the viral proteins – for example, the viral Nsp7 seems to be involved in nuclear transport processes. As you would expect, there are a lot of signs of the virus attacking the pathways of innate immunity and inflammation. You can even go down to the protein structural domain level and find enrichments – as an example, the viral Nsp1 protein’s human interactors are enriched in DNA polymerase domains, and the viral E protein’s interactors are enriched in BET and acetylated-histone binding domains.  In addition to these sorts of transcriptional targets, some of the processes targeted by the viral proteins include vesicle trafficking, lipoprotein pathways and lipid metabolism, and mitochondrial and cytoskeletal proteins. There are several potential targets in these that could also apply to other pathogens, which is good to know in the long term.

For the shorter term, though, the team tried to identify known compounds that interact with the human target proteins, in the hopes that some of these might go on to interfere with the viral protein-protein interactions that had been uncovered. There are about 25 approved drugs on the list, which are very likely of the most interest, since these can already be dispensed to patients. It’s an interesting list. Chloroquine shows up, but not by any of the mechanisms that anyone has proposed for it – it makes this list via its capacity as a Sigma1 binder, of all things. If it turns out that sigma receptor ligands are finally good for something I will be quite startled, since I’ve been hearing comments about ignoring sigma activity because everything hits it and no one knows what it does since, oh, about 1990 or so. Azithromycin didn’t make the list per se, but it is known to have off-target ribosomal effects, and compounds with more direct ribosome targeting were identified.

And the ACE inhibitors are on there, too, which brings up a key point. If you’re just looking at the level of “This compound interacts with this human protein, which apparently interacts with this viral protein”, which is all we can do for now, that doesn’t give you enough mechanistic detail to say if said compound is going to do anything beneficial, to turn out to have no effect, or indeed to possibly make things even worse. This is a very nice paper, but the authors themselves are careful to note that an interactome map like this can’t provide any mechanistic understanding by itself – we have to bring that, via brainpower and further experimentation. Some of these proteins and pathways are being hijacked by the virus or interfered with by it, while others may be involved in fighting it off, so you need to be sure what you’re messing with.

There are also a lot of compounds that are in clinical trials that show up on the list, which are one notch down compared to approved drugs, and a number of other compounds that are known in the literature as ligands, tools, etc. but have not been into humans yet. Those are way down in the bottom rank as far as I’m concerned. The big hurdle is getting drugs into patients, and having been through the preclinical studies needed for human trials (toxicity, dose formulation, stability, etc.) and especially having been into Phase I or II trials in actual human subjects counts for a great deal here. All the better if the compound proved safe enough (and efficacious enough in its intended role) for a regulatory agency to approve it. Those are actionable; pre-clinical compounds are far, far less so.

This paper points out several things that need to be followed up on, and I’ll be going into more detail on those in future posts and as more information comes out. For now, congratulations to this big team for moving so quickly and getting this out to the research community!

37 comments on “What the Coronavirus Proteins Are Targeting”

  1. Barry says:

    I thought this sort of screen was best for soluble/cytosolic proteins and not so much for the membrane-integral ones? But Ace-1 is membrane integral, no? Does it shed all its aggregate (but not the viral tag) upon ionization in the MS?

    1. Christophe Verlinde says:

      ACE2 is not an integral membrane protein, it is a single pass type I membrane protein.

      1. steve says:

        If it is single pass then it IS an integral membrane protein (which only means it’s integrated in the membrane). Many integral membranes are single pass – antibodies, TCRs, etc.

      2. Barry says:

        Whatever distinction you’re making is too subtle for me.
        Before I can ask what cell components associate with the tagged viral proteins, I gotta lyse the cells and filter out the insoluble debris. Seems to me a protein like ACE2 would stay in among the cell membrane solids, and wouldn’t get the chance to dance with my probes?

        1. MrXYZ says:

          SIngle-pass membrane proteins (at least Type 1 proteins) can be made as soluble ectodomain constructs that maintain some, if not all, their binding capabilities. In other words, the structure and function of the protein are not completely determined by its integration into the membrane. As opposed to integral membrane proteins such as GPCRs where there structure and function is determined by how they are integrated into the membrane.

          1. Barry says:

            thanks for the clarification!
            But if I’m looking at a cell lysate rather than at an engineered construct, I still expect ACE2 to be filtered out with the cell-membrane debris?

  2. S says:

    Pharma companies have already started gathering compounds (that they believe they could quickly scale up) for physical screening too – https://www.gatesfoundation.org/Media-Center/Press-Releases/2020/03/Life-Science-Companies-Commit-to-the-Fight-Against-COVID-19-Pandemic-alongside-Gates-Foundation

  3. Aaron Groen says:

    Niclosamide is an anthelmintic that inhibits viral replication of coronavirus in vitro (see https://lnkd.in/eVdZ-fw).

    Another anthelmintic, nitazoxanide was in a clinical trial that successfully reduced duration of flu symptoms (see https://lnkd.in/dHabGK8).

    Am I missing something? Why aren’t we trying these compounds to treat COVID-19? Seems like there is more evidence for these compounds than for Chloroquine.

    1. Pajas says:

      Totally agree. Niclosamide was independently reported by Gassen et al. last year to reduce MERS-Corona infection (https://www.nature.com/articles/s41467-019-13659-4) by direct inhibition of SKP2. This is very interesting as it is a host protein, and hence treatment with niclosamide should be less prone the occurrence of resistance as I see it. To me this compound is a no-brainer to go straight into animal testing.

    2. anon the II says:

      Niclosamide is an anthelmintic that inhibits X in vitro.

      I’ve randomly come across at least 3 completely independent drug discovery efforts that came up with that discovery. I think it’s just a more potent resveratrol. Buyer beware!

  4. Lane Simonian says:

    Interesting that chloroquine makes the list as a sigma-1 binder. Under oxidatives stress, sigma-1 receptor agonists are suppose to limit intracellular calcium release, which reduces further oxidative stress and inflammation.

    http://jpet.aspetjournals.org/content/319/3/1355

    This is why sigma-1 agonists are being studied for a series of neurodegenerative diseases.

    https://www.sciencedirect.com/science/article/pii/S1347861314000267

    Perhaps, a similar mechanism exists for cholorquine.

    https://www.ncbi.nlm.nih.gov/pubmed/21883227

    However, if a compound’s only mechanism of action is sigma-1 activation that alone is probably insufficient to deal with a series of neurological and viral diseases. Some other mechanism likely also needs to be present.

  5. JB says:

    Ironically, this may be the perfect disease for those much maligned stem cell therapies. The idea is that they secrete a whole bunch of anti-inflammatory factors, and when infused IV go to first pass organs like the lungs. People appear to be dying from ARDS related to cytokine storm. Stem cells to the rescue?

    1. Carolyn Homan says:

      TIMPS to offset MMP’s down regulate immune response cytokine storm? Not professional. Sorry for non tech language

    2. Leslie says:

      Prograf (tacrolimus) could be palliative of cytokine storms for a near-term approach.

  6. Rob W. says:

    Minor correction on the purification:
    Proteins were tagged with a Strep-tag, which is a short peptide with high affinity to streptavidin.
    They were purified with a mutant streptavidin immobilized on magnetic beads.
    They were eluted from that streptavidin by competition with biotin.

    1. Derek Lowe says:

      Of course! Just reworked that part, thanks!

  7. See also:

    http://protinfo.compbio.buffalo.edu/cando/results/covid19/

    which uses a more direct approach, i.e., comprehensive docking of known drugs to all the structures. chloroquine isn’t predicted to bind to any of the SARS-CoV-2 proteins but it is similar in structure/behaviour to known SARS-CoV actives from two papers (Shen et al and Dyall et al).

    In terms of repurposing, the null hypothesis is that everything that works for SARS-CoV should work here given the similarity of the two proteomes (though there are some differences of course) and so any repurposing method should be able to do better than this “naive” guess.

    In terms of targets, the above approach is more direct compared to the UCSF work which is clever but an indirect one (but also less computational).

    1. Derek Lowe says:

      I’m glad to hear that you’re working on getting experimental validation – my bias is towards the less computational approaches, even though indirect, but data will sort everything out!

  8. I should mention we’re working with collaborators to do the validations in a BSL3+/4 lab – hopefully that goes well but on the web is the list that anyone can test and there should be a short paper appearing on Biorxviv soon. The list is a bit buried/obscured since we didn’t want just anyone to start popping those pills. Here are some highlights from our predictions:

    Some of the interesting predictions from the March 5, 2020 round include chloroquine and other antimalarials at rank 35-40 (approach 1), ACE inhibitors at rank 19-21 (approach 2), remdesivir at rank 46 (approach 2), and amprenavir and other HIV protease inhibitors at rank 48-50 (approach 2), all of which have either shown or believed to have efficacy against SARS-CoV-2 and are undergoing (multiple) clinical trials to demonstrate efficacy. Therefore, some of the other higher ranked drugs in our lists are also worth evaluating, with the potential payoff of choice, greater efficacy, and reduced cost if shown to inhibit SARS-CoV-2 in vitro.

  9. quinoline mensch says:

    Regarding hydroxychloroquine:
    https://justthenews.com/politics-policy/coronavirus/bahrain-hydroxychloroquine-success-response-covid-19
    Other sidechain hydroxylated 4-aminoquinolines, ie. amodiaquine https://en.wikipedia.org/wiki/Amodiaquine , and its analog FGI-104 https://en.wikipedia.org/wiki/FGI-104 , share antiviral properties that have been characterized as being broad spectrum, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776286/ .

    It is suggested that these compounds somehow interfere with the virus particles’ ability to hijack host cell’s membrane remodeling machinery ESCRTs https://en.wikipedia.org/wiki/ESCRT , needed to endocytize (internalize) the membrane attached virons into the cytoplasm , and then exocytosis or budding of newly formed virus particles from the host cell following replication.

  10. luysii says:

    ” interaction profile of 27 of the viral proteases”

    I think you mean “interaction profile of 27 of the viral proteins”

    1. Derek Lowe says:

      That I do! Typing that section while trying to do something else; I’m surprised it came out in English. Fixed!

  11. CL says:

    It’s certainly interesting to look more into the potential chloroquine/sigma1 connection. It’s been known for a while that sigma1 agonists like dextromethorphan have antitussive effects (or vice versa, that antitussive compounds bind to sigma1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3396511/). I’d assume that some of these compounds are being used right now to treat the cough of covid-19 patients. So maybe the in human data is already there?

  12. Joe Kelleher says:

    Would this approach necessarily identify the active mechanism of useful pharmaceutical interventions? I understood the purported antiviral mechanism of chloroquine was decreasing the acidity of the viral endosome rather than anything to do with sigma receptors, and of course it may turn out to be something else yet again, if indeed it is clinically useful at all.

    Assuming chloroquine is active against sars-cov-2, I’m wondering if it’s mechanism may share something with the novel antivirals FGI-104 and FGI-106 (both on Wikipedia). These are supposedly active against other viruses, and FGI-104 looks like chloroquine with a substantial augmentation to its side chain. FGI-106 meanwhile looks like two chloroquine molecules back to back, this time with slightly shorter aliphatic chains. Would it be a simple experimental protocol to test small molecules like FGI-104 and FGI-106, and other molecules at a similar stage of development, against the virus, at least in vitro against relevant cell cultures?

  13. donorcure says:

    I’m glad that people are studying this matter. We need to unite as one to defeat this invisible monster. I’m pretty sure that the human data is already there.

  14. adam zweifach says:

    How does colchicine, a microtubule-binder, reduce inflammation in gout? None of us know. Maybe we should try to get the 25 clinically-approved candidates into some kind of human testing as soon as possible, under whatever emergency protocol makes sense. Worry about mechanism- which you will likely never really know- later.

    1. milkshake says:

      Nobody knows?? How about neutropenia??

  15. Cjones1 says:

    After reading this article, doctors may want to consider using anticoagulants. It might prevent having to use ventilators.

    https://t.co/TOnOd7FVI5

    I am curious about the effectiveness of beta glucans and other anti-virals found in mushrooms.

  16. Tom says:

    I saw metformin mentioned in the paper. I have read many times that diabetics are at higher risk of death from covid19. I wonder if metformin makes the disease worse or if it helps (and those who take it would be worse off without it)?

  17. Keith Doyle says:

    I’m curious– does data exist as to what medications existing Covid-19 infected individuals were already taking when they became infected, along with the ultimate outcomes of their disease? I’ve not yet heard of any studies of existing data the mining of which might help reveal drugs or drug combinations that could be effective against the virus. Is that because that data does not exist? Or that it’s considered unreliable?

  18. Cal_Bar says:

    First paragraph:

    “that the strep-tag binds do..”

    I think you meant “t”o instead of “do”?

  19. SK says:

    If the best candidates are repurposed off patent drugs, who is going to pay for the clinical trials? Is private industry/pharma incentivised at all to participate in finding a Covid-19 treatment? What about implementing prizes/bounties for successful clinical trials (protocol designed by company and carried out by independent CRO according to pre-defined criteria such as improvement over standard of care/reduction in viral load etc)?

    1. Barry says:

      A new indication could start a new 20yr clock for a “use patent” quite independent of a composition-of-matter patent that might have long since run out.
      Read the history of John Harrison’s clocks for the shortcomings of the “prize” incentive system that our USPTO was designed to supercede.

      1. SK says:

        You can’t build a decent business case based on a “new use” patent for an off-patent drug because you can’t enforce a monopoly: doctors can just prescribe the generic drug off label (and compounding pharmacies can also prepare). The only viable way to incentivise such “unmonopolisable therapies” is a well-designed prize fund (https://en.wikipedia.org/wiki/Health_Impact_Fund) or advance market commitment (see https://en.wikipedia.org/wiki/Advance_market_commitments). Longitude prizes were tried 400 years ago, a lot has happened since then (and they actually worked, Harrison solved the longitude problem and got his £20k, it’s just that the board were expecting a mathematical, not a mechanical solution – should have been more clear in drafting the conditions for the prize).

        Basically, under the current system I don’t see pharma funding any clinical trials based on second uses for generic drug treatments (although might fund a few small trials for CSR). I would be glad to be proved wrong.

      2. SK says:

        You can’t build a decent business case based on a “new use” patent for an off-patent drug because you can’t enforce a monopoly: doctors can just prescribe the generic drug off label (and compounding pharmacies can also prepare). The only viable way to incentivise such “unmonopolisable therapies” is a well-designed prize fund or advance market commitment. Longitude prizes were tried 400 years ago, a lot has happened since then (and they actually worked, Harrison solved the longitude problem and got his £20k, it’s just that the board were expecting a mathematical, not a mechanical solution – should have been more clear in drafting the conditions for the prize).

        Basically, under the current system I don’t see pharma funding any clinical trials based on second uses for generic drug treatments (although might fund a few small trials for CSR). I would be glad to be proved wrong.

  20. krishna p. says:

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