Where’s the drug? You might be hearing this a lot – and/or wondering it yourself. And there’s a common misconception that, if you’re diagnosed with covid19 (the respiratory disease caused by the novel Coronavirus, SARS-Cov-2) your doctors will give you a specific drug to target it – but this isn’t the case – at least not yet. 

When you have an infection that’s caused by bacteria, doctors can prescribe drugs called antibiotics that target bacterial Achilles’ heels. But when an infection is caused by a virus, there’s less doctors can do to specifically treat the infection – instead they normally rely on providing supportive measures to test the symptoms – things like fever reducers if you have a high fever or oxygen if you’re having trouble breathing. 

It’s harder to target the actual viruses in part because there’s really not much to them – just their genetic blueprint (viral genome) with some packaging. Different viruses have different compositions, but SARS-Cov-2 is a single-stranded RNA virus of the Coronaviridae family. Instead of double-stranded DNA like we have, it holds its genetic info in a single strand of RNA, wrapped up around nucleocapsid (N) proteins to form a compact, protected “nucleocapsid” and enveloped by a membrane coat embedded with proteins with spike proteins jutting out to help it bind to receptors on cells to sneak inside where it can hijack the cell to make lots of copies of itself (replicate), break out, and then infect new cells. Speaking of sneaky, despite the havoc they can wreak, viruses typically aren’t even considered “living” because they can’t really do anything on their own, instead relying on host cells for basic functioning. 

Bacteria, on the other hand, may be small, but they are whole, living, organisms. Single-celled ones, yes – but that only ups the pressure on them to make sure that each and every cell makes everything it needs to grow, thrive, and divide – and each of those daughter cells has everything it needs. So it has to make, and hold genetic instructions for, a lot more things, which offers a lot more potential drug targets. 

To put things into perspective, the SARS-Cov-2 genome is only about 30,000 letters long, the bacteria E. Coli has a genome that is about 4.6 million letters long and our own genome is about 3 billion letters long. In order to keep its genome so small, the virus uses its host cells’ machinery whenever possible, and, for the machinery it needs that we can’t provide, it uses clever space-saving tricks to write the instructions in as few letters as possible. This allows it to more easily carry out its non-life’s purpose: making lots more copies of its genome quickly, packaging them up in small packages, and shipping them out to infect new cells. Since all that’s taking place inside a cell, the virus, including its genome, has to be really small. 

Bacteria genomes don’t have this size restriction, so they can have more genetic instructions and make more things. This includes making some structures we don’t have. For example, since it’s not like it has skin to protect itself, each bacterial cell needs to be hardy – so they have thicker cell coatings than our cells. In addition to the fatty membranes like we have, bacteria have “cell walls” made up of sugars and peptides (short strands of protein letters). Since our cells don’t have such cell walls, they and the enzymes responsible for constructing them, are great targets for antibiotics. This, for example, is how penicillin, ampicillin, and related β-glycan antibiotics work. 

But viruses don’t have these cell walls  – and, to a cell’s perspective they look a lot like a neighboring cell because when the virus buds out of its host cell, it gets coated in the host cell’s membrane coat – told you they were sneaky!

So let’s look elsewhere… other antibiotics target other pieces of bacterial machinery like bacterial ribosomes – ribosomes are protein-making complexes made up of proteins and ribosomal RNAs. Before a protein gets made, a messenger RNA (mRNA) copy of its gene recipe gets made. And the ribosomes travel along this mRNA, following the mRNA’s instructions on which amino acid (protein letter) to add in which order (3 RNA letter “words” called codons “spell” one amino acid and a helper molecule called tRNA brings that letter to the ribosome to link up, using a complementary “anticodon” to provide specificity). 

We have ribosomes too, but our ribosomes are different enough from bacterial ones that we can use drugs to target the bacterial ones while not hurting our own. An example of this type of antibiotic is kanamycin. 

Viruses, too, rely on ribosomes to make their viral proteins, but they use their host cells’ ribosomes, so there aren’t “viral ribosomes” to target – instead, if we targeted viral protein writing, we’d target our own protein writing. 

I said “writing” there and not “making” because, although the peptide “writing” machinery is the same, there’s an important distinction between viral protein making and our protein making. The “writing” process of ribosomes using mRNA to link up amino acids into a long “polypeptide” is called translation. And that’s the same for us and for viruses.  For most of our proteins, the polypeptide that results from translation is a single protein (it gets folded and might get a few post-translational modifications, but, it’s ONE protein.) But viruses, with that constraint to keep their genome super small and packable, often write multiple proteins in one long “polypeptide” that they then cut (using enzymes called endoproteases) into several proteins. 

For example, SARS-Cov-2’s biggest gene is a “replicase” gene which contains instructions for making proteins the virus needs to replicate. And, in another of its space-saving tricks, the virus can make 2 different polyproteins from this one gene – pp1a and pp1ab. It’s able to do this because it uses a “slippery sequence” and a pseudo knot  (a stretch of the RNA folds into a “roadblock”) to stall the ribosome on its translation journey and get it to slip up, change reading frame (e.g read “aredcat” as “are dca t” instead of “a red cat”), and keep going past the rep1a stop sign to make pp1ab. 

As a result, pp1ab has the same stuff pp1a but it also has extra protein instructions – pp1a has nsps 1-11 and pp1ab has nsps 1-16. Nsps stands for Nonnstructural Proteins and they’re called that to distinguish them from the virus’s structural proteins (including the spike protein, S; envelope protein, E; membrane protein, M; and nucleocapsid protein, N) – which are important for packaging the viral genome and getting it to new cells. These structural proteins (and a few others) have their own individual genes downstream of the replicase gene. 

Nsps include things like enzymes (reaction mediators/speed-uppers) for helping copy the RNA and processing the polypeptides, which need to be chopped up into their individual proteins. Within the polyproteins are 2 proteases, with different sequence specificities, which cut the polyproteins into their individual nsps. A Papain-like protease (PLpro) is encoded within nsp3 and a serine type “main protease” (Mpro) is encoded by nsp5. PLpro is responsible for the first few cuts (separating nsp1/2, 2/3, and 3/4) and Mpro handles the rest. 

Since viruses need proteases to make functional proteins from polyproteins, some antivirals are protease inhibitors – including the HIV treatment combo Lopinavir/ritonavir (trademark Kaletra). Scientists had hoped that Kaletra also might help treat covid19 but the results of a small trial found it ineffective and side-effect-causing.  

So that was disappointing news. But there’s still hope for another treatment, remdesivir, which is is similar to Kaletra in that it was designed for treating a different disease (it was initially found in a screen for hepatitis C and was later found potentially useful against Ebola) – but the comparisons stop there. Remdesivir takes a whole different approach, acting as an adenosine analog (it mimics the RNA letter A) to mess up viral replication (genome copying). 

SARS-Cov-2’s small size makes copying less time-consuming, but the fact that it’s written in RNA, not DNA, means the virus needs to provide its own copier, since our cells don’t have an RNA to RNA copier (an RNA dependent RNA polymerase (RdRP)). So one of the nsps (nsp12) is an RdRP. Since we don’t have one of these it offers an attractive target. 

When you go to target an enzyme, and you really want to put a stake in its heart, you go to that heart – the “active site,” where reacting molecules come together and get changed. For an RNA polymerase, this active site is where the RNA letters bind to get linked. So if we want to mess things up we can use something that looks like an RNA letter (a nucleotide analog) but is modified in some way that either cripples the enzyme or screws up the product. 

But there’s a problem. The copier may be different, but the letters it’s using are the same – it’s using the same RNA letters our cells use, just linking them in the order that it wants based on its RNA template. Instead of what our cells do when writing RNA – linking letters in the order we want based on a DNA template (a process called transcription). So, when we target RdRP we have to be sure we’re only targeting RdRP and not our own DNA-dependent RNA polymerase. Thankfully, our own polymerase isn’t as easily fooled so the drug remdesivir doesn’t interfere with them. 

But how exactly does it interfere with the viral polymerase? Laurel Oldach wrote a really great article for ASBMB about remdesivir – and it’s a really cool format – you can click on various parts of the molecule to see how the different “functional groups” give the drug different chemical “superpowers”. http://bit.ly/2Wttg76 

I encourage you to check out her article to learn more, but here’s an overview. RNA letters (ribonucleotides) have 3 main parts – a ribose sugar linked up to phosphate groups (at the “5’ position”) and 1 of 4 unique nitrogenous bases at the 1’ position (A, U, C, or G). At the 3’ position on the ribose is a hydroxyl (OH) group. Letters link up 5’ to 3’ using the phosphate and hydroxyl groups. 

Unlike some nucleotide analogs, which act as obligate chain terminators (add one and you’re done) because they don’t have the 3’ hydroxyl (OH) group needed to latch onto another letter, remdesivir *does* have a 3’OH, so multiple messed up letters can get added on before it causes the chain to terminate, potentially because the slight differences in the fake letter’s shape make the RNA less comfortably held by the polymerase. And having a 3’ OH might allow it to sneak past the viral proofreaders, so the virus doesn’t realize it’s making messed up RNA and try to fix it. 

Remdesivir is a prodrug, meaning that our bodies have to tweak it a bit to get the active form. The changes that need to be made for remdesivir involve the 5’ phosphate – nucleotides are added as triphosphates, so three phosphate groups in a row (2 of which get kicked off to pay the energy cost of linking) but remdesivir just has one – and it’s “hidden” by a bulky protective group. Inside our bodies, that group gets cleaved off and the other two phosphates added on by enzymes our cells already have – so that activation is “like no big deal”. But could remdesivir be a big deal?

There are currently 5 clinical trials underway testing the efficacy of remdesivir against Covid-19. The NIH is doing one, as well as a WHO consortium and the drug’s maker, Gilead. And there are also lots of negotiations underway about pricing…

Another RdRP inhibitor in the running is favipiravir, aka Avigan. Unlike remdesivir, which mimics the sugar and the base part of an RNA letter, favipiravir only mimics the base, so it has to get both ribosylated and phosphorylated once inside of cells. But then it gets added by RdRP (and importantly not by any of our polymerases) and messes up the viral RNA. It isn’t approved by the FDA yet, but has been approved for a different use in Japan – treating influenza – and so there’s a lot of research into the molecule already (as well as production pipelines). 

A small trial out of China showed hopeful results, warranting further investigation, but not premature hype.  http://bit.ly/3ddpyVg 

And, finally, there’s one you might have heard about in a certain press briefing – and which despite claims – is NOT yet an FDA approved treatment for covid19 – chloroquine, or its more stable form, hydroxychloroquine. It’s an antimalarial drug that’s been around for over half a century. 

Malaria is a parasite, not a virus, but chloroquine also has some anti-viral activity. It is thought to prevent viruses from getting into and/or out of cells by messing with their modes of transportation – when the virus enters the cell, it does so by the cell kinda pinching in the part of the membrane that the virus had bound to (using that spike protein to latch onto a receptor on the cell surface). So when the virus gets inside the cell, it’s still “quarantined” inside of a membrane-bound pouch called an endosome. Chloroquine messes with the pH in endosomes, preventing the virus from getting out (and it does some other stuff too).

Results were recently published from a VERY small “open-label” study (patients and doctors knew which patients were being treated with what) which show initial promising results, including when chloroquine was combined with the antibiotic azithromycin (which might help combat secondary bacterial infections that can develop once the patient’s lungs are made vulnerable by the virus) BUT the study was really badly set up, multiple patients dropped out, and they used a really low threshold for success. A great thread about it by Jason Pogue can be found here: https://twitter.com/jpogue1/status/1241138975802359813?s=20 

This doesn’t mean that there’s no effect, but at this point, it’s basically just anecdotal evidence, and it’s too soon to really say this – or any of these drugs – are effective – and they can have SERIOUS side effects so it is NOT a “nothing to lose” situation. I encourage you to check out this Science article by Derek Lowe that lays out the results of studies to date on all these drugs  http://bit.ly/3ddpyVg 

Developing new therapeutics is a long process. The reason these drugs were able to go to trial so soon is that they’d already been approved or at least were close to approval for other purposes. So they were found to be “safe” (at least safe enough to be used compared to their benefit for a certain condition) they just needed to get the okay to be reused – sometimes through “compassionate use” exceptions and sometimes through official clinical trials. These official trials are important in order to know if the drug has unpredicted side effects specific to this new condition it’s being used for and thus which weren’t an issue when they were going through testing for their other uses. Additionally, the controlled trials are crucial to figure out if a drug really is more effective than the current standard of care or if patients on it are just getting better because they would have gotten better anyway. 

Speaking of patients that have gotten better – the “getting better” involves an adaptive immune response in which the patient’s immune system learns to recognize parts of the virus as foreign (usually parts of the spike proteins in the case of these types of viruses) by developing little proteins called antibodies that bind specifically to that spike protein and trigger an immune response.  Even after the infection is cleared, a low level of those antibodies stick around to “keep watch.” 

Scientists are hoping that they can isolate some of these antibodies from the blood of patients who have recovered from covid19, mass produce them, and use them to treat and/or vaccinate patients against covid19 by putting their immune system on watch and/or blocking its access into cells. 

Antibody-based therapies have been found to be effective against other viruses – Ebola for example – remdesivir was actually somewhat “abandoned” as an Ebola treatment before getting FDA approval after scientists found that antibody-based treatments were more effective. But these antibody-based treatments are a lot more time-consuming and expensive than making small molecule drugs like remdesivir. You can synthesize remdesivir by combining different chemicals under the right conditions (o-chem really is useful!). But “synthesizing” an antibody means making – and purifying – a whole protein – and a lot of it. 

And of course, any new drug will have to go through studies on safety and efficacy, which can be a lengthy process but it’s really important it be done correctly. 

One of the hopes is that, if people practice social distancing, in addition to spreading cases out over time so that we don’t overburden the health system, we can delay the outbreak’s peak and buy us some time to find effective therapeutics.

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

Leave a Reply

Your email address will not be published.