One of the notable things about the current pandemic is the way that all our modern biology and analytical techniques are on display. Molecular biology, structural biology, bioinformatics, technologies like cryo-EM structure determination, fast sequencing, protein interaction screening and more – this is a real-time look at how basic biomedical research gets done. I’ve been reading a new preprint that starts off with protein sequence analysis and ends up with an actionable clinical recommendation (skip to the end for that one!), and I think it’s a good illustration.
This team from Germany and the UK has been analyzing the new pathogen with a technique they’d already developed to look at differentially conserved amino acids among related strains. Comparing the current SARS-CoV-2 and the earlier SARS-CoV, they find that most of the amino acid changes are conservative (one hydrophobic residue swapped for another), with some polar/nonpolar swaps and very few instances of swapping charged residues. That’s about what you’d expect, of course, but the question is whether any of these are functionally relevant. A closer look shows that these differentially conserved positions are found much more in the notorious “spike” protein region than (say) in the envelope proteins, and that overall 92% of these changes are on protein surfaces rather than buried residues, which makes you think that they are indeed important.
It’s already been noted that several residues involved with the spike (S) protein’s interaction with the human ACE2 protein vary between the older SARS virus and the new one. This group went on to look at various human cell lines and their susceptibility to viral infection, and found that while the presence of the ACE2 protein in general is important, there are more things at work. For example, 293 cells (kidney-derived) don’t express ACE2 at all, and had been shown during earlier work on SARS to be resistant to infection. The new coronavirus doesn’t infect them either – but if you engineer the cells to express ACE2, original SARS will then infect them, but the new virus doesn’t do so nearly as well. Across several other cell lines, there is no good correlation between the amount of ACE2 present (as long as it’s there in some amount) and the ability of SARS-CoV2 to infect them, so it’s not just a simple “the more ACE2 the worse” situation. We don’t know what the other factors are, but there clearly are more than just ACE2 levels.
They also looked at transmembrane serine protease 2 (TMPRSS2), another human enzyme that is hijacked to allow viral entry (and which is a target of the serine protease inhibitor camostat, a drug that many readers will have heard of by now and which is in clinical trials against the current virus). As with ACE2, susceptibility to SARS-CoV2 didn’t correlate with TMPRSS2 levels, either, though – more evidence that things aren’t as simple as you might hope. The paper goes on to look at camostat itself and another similar protease inhibitor, nafamostat, which is also approved in Japan for pancreatitis. Both drugs are more active against SARS-CoV2 infection in cell culture than they are against the first SARS virus, and nafamosat is more active than camostat. Unfortunately, the concentrations that are needed in the cell assay (0.5 and 1.2 micromolar respectively) are still above what these drugs appear to achieve in vivo (reported plasma concentrations of 0.2 micromolar), so we’ll have to see from the human trial data if that’s enough to show efficacy. It’s not wildly far off, particularly for nafamostat, but you’d still rather have it the other way around, for sure.
But there’s some possible good news as well. The team also looked at the serine protease inhibitor aprotinin, a small protein inhibitor of serine proteases that has had an up-and-down history in human therapy and has been looked at as an antiviral as well (via its TMPRSS2 inhibition). As Trasylol, it was used to slow down bleeding during surgical procedures (by inhibiting several proteases in the fibrinolysis pathway), but as with all drugs that either enhance or reduce blood clotting, you’re walking a fine line between benefit and trouble. Apronitin was temporarily taken off the market in 2007 because of possible association with clotting events, but this suspension was lifted by the EMA in 2012 after further review of the data. In this work, though, apronitin was not only more potent in the cell assays, it displayed much stronger effects on the formation of double-stranded RNA (a marker of viral infection), and did so at levels below the known blood levels on human administration. It’s more effective on the new coronavirus than it is on SARS, which seems to be partly explained by the sequence differences noted above. The authors say:
Since aprotinin interferes with SARS-CoV-2 in therapeutic concentrations and displays more pronounced direct antiviral effects than camostat and nafamostat, it seems to have a greater potential for the treatment of SARS-CoV-2-infected individuals based on our data.
You’d want to be careful with this drug because of its past history, but the authors note that there is actually an aerosol formulation of apronitin that’s been approved in Russia, so that mode of administration looks feasible and might have less systematic risk.
The paper goes on to look at another approved drug that I haven’t seen getting as much attention: omeprazole, the well-known proton pump inhibitor for acid reflux. It has been reported as having some antiviral activity in the past, possibly by increasing the pH in lysosomal compartments. In their assays, it did interfere with viral infection, but at levels too high for realistic human dosing. But here’s the interesting part: they found that simultaneous treatment with omeprazole (at human therapeutic concentrations) increased the activity of apronitin by 2.7 fold and increased the activity of remdesivir by 10-fold. That seems like a very useful observation! As far as I can see, the paper did not check for an interaction of omeprazole and camostat/nafamostat, which would be interesting to know as well. The same group had noted in 2019 that the drug increased the activity of acyclovir against the herpes virus.
So while we’re still figuring out if remdesivir has efficacy by itself, there’s an opportunity to administer a widely used, well-tolerated drug to give it a better chance. And both the beneficial interaction of omeprazole with apronitin and the earlier acyclovir/herpes result suggests that this could be a more general effect with many other drug candidates, which seems well worth investigating.