If you heard about Pfizer’s new COVID *pill* and wondering what it is… it’s an inhibitor of the coronavirus main protease (MPro). The virus needs this protease in order to make functional proteins, so if a drug can stop the protease, they can stop the virus from doing too much damage. The drug can stop the protease, but our liver can “stop it” by metabolizing it too quickly (modifying it so it gets excreted more easily). So the pill also includes riponavir, which inhibits the responsible liver protein, CYP3A4. In the video I start with a “long story short” version and then dive in deep on the biochemistry and the research article about reporting its development and pharmacology.
The virus uses a lot of our cells’ proteins, and, by providing the instructions in its RNA, it gets our cells to make the proteins it needs but we don’t have. It makes some of these, the nonstructural proteins aka Nsps) as a long polyprotein (pp1a or pp1ab depending on where the protein-making complex (ribosome) stops reading). This polyprotein, as the name suggests, has many (poly) proteins (around a dozen) that are all connected because the ribosome makes them as one continuous chain. So it’s up to the virus’ proteases (protein “scissors”) to recognize where one protein ends and another starts and cut them apart into individual proteins. The virus needs to get us to make these proteases too – as part of the polyprotein, in fact, but they’re able to cut themselves out. There are 2 viral proteases, the main protease Mpro (nsp5) and a papain-like protease (PLpro), which is responsible for the first few cuts (separating nsp1/2, 2/3, and 3/4), with Mpro handling the rest⠀more on MPro: https://bit.ly/mproinhibitors ⠀
Since our cells don’t make these proteases, or even any very similar ones, they represent good potential drug targets since, if scientists can design some compound that binds and inhibits these proteases, it could be used to hurt the virus without hurting us. This strategy isn’t new. In fact, some of the components in the treatment mixtures for HIV are protease inhibitors.
In the active sites (where these protein scissors’ “blades” are) the cutting occurs thanks to the teamwork of a couple of the amino acids whose side chains stick out into the active site: a Cis-His catalytic dyad (Cys145 & His41). Each promoter has one of these active sites, so the protease has 2 sites open for binding substrate (thing to cut) or inhibitor. If an inhibitor could get there first, it could prevent the polyprotein from binding – and – if an inhibitor could get stuck onto that catalytic Cys through covalent bonding, it could blunt the proteases’ scissors for a longer time.
The paper I’m discussing is open-access (so free to read) and was published in Science. (and FYI lot of the geeky goodies are in the supplemental material)
Owen et. al, An oral SARS-CoV-2 M pro inhibitor clinical candidate for the treatment of COVID-19. Science, November 2, 2021
They started with an initial “hit” from SARS-CoV-1 experiments (the OG SARS). It was potent (strongly able to inhibit MPro) but it had poor bioavailability so had to be administered via IV. They wanted a pill so they looked at the structures of the drug & the enzyme and their knowledge of biochemistry and started modifying it, adding little pieces here or there or changing functional groups to try to give it better solubility, lower toxicity, etc., all the while using that structural information to know which parts they can’t change without messing up its ability to inhibit the protease’s activity.
They could screen using a FRET-based reporter peptide (chain of amino acids) that mimics one of the protease’s natural cut sites (in this case the N-terminal “auto-cleavage site” that the protease uses to cut itself out of the polypeptide chain). On one end of the peptide is a fluorophore (a part that can let off light) and near the other end is a quencher that can prevent light from being given off – but only if they’re close together. This is because of this phenomenon called Forster Resonance Energy Transfer and it’s really cool but too complicated to go into detail here so check out this post if you’re interested: http://bit.ly/fretdyes ⠀
For now, just know that if this peptide gets cut by the protease, the fluorophore and the quencher will be separated so you won’t see light – and this tells you that the protease is active. If you add an inhibitor that works, you should keep seeing light. ⠀
PF-07321332 passed this test. How does it work?
That nitrile nitrogen is really negative – atoms (like those individual carbons and oxygens) join together to form molecules by sharing pairs of negatively-charged subatomic particles called electrons, but some don’t share fairly. Nitrogen is a major electron hog (it’s highly electronegative) so, in the carbonyl, it draws the shared electrons closer to it, making it partly negative and the carbon partly positive. The carbon thus “wants” more electrons so it’s called electrophilic.
The active site’s catalytic Cysteine has a sulfur that wants to share electrons (is electrophilic) thanks to the Histidine pulling off its proton, so the sulfur attacks the electrophilic carbon, leading the compound to get stuck on.
It gets covalently stuck through a thioimidite adduct, as you can see in the structure of it bound to MPro, PDB 7si9 https://www.rcsb.org/structure/7si9
but it’s reversible inhibition. So it’s strongly stuck but it can still come off since enzymes can catalyze reactions in both directions and you literally have it stuck in the active site, where enzymes do their biochemical magic. But the forward direction is greatly favored, so it stays stuck pretty well.
It also passed additional tests for bioavailibilty and stuff.
But it wasn’t lasting long enough once the liver had a say. Instead, it was being modified by CYP enzymes, leading to them being excreted quickly. So that’s why they added ritonavir – it inhibits a CYP enzyme in our body that they show to be responsible.
more Covid-19 resources: https://bit.ly/covid19bbresources ⠀
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: https://thebumblingbiochemist.com