ARE there any natural P2Y1/12 inhibitors?

What happens when the natural product we need isn’t on the list?

Last week, we left off on our adventure in trying to find natural products that replicate Triple Anticoagulant Therapy after having identified two of the three we needed.

Here’s a reminder of where we are so far:

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P2Y1 and P2Y12 Inhibitors as Platelet Activation Inhibitors

The last component that we need to find is a natural P2Y1 and P2Y12 receptor inhibitor.

Why are we looking for P2Y1 and P2Y12 inhibitors?

A couple of reasons:

1. That’s the mechanism of action of clopidogrel, the last component of Triple Anticoagulant Therapy that we are trying to find a natural version of and

2. The P2Y1 and P2Y12 receptors play a key role in platelet activation.

Here’s the mechanistic picture of those receptors in platelets and how the activation of the receptors contributes to platelet activation:

The P2Y1 and P2Y12 receptors are highlighted in the blue circle.

As you can see, the P2Y1 and P2Y12 receptors are activated by ADP (adenosine diphosphate) and that activation of these two receptors in turn activates signal transduction pathways inside the platelet, leading to platelet hyperactivation.

To find a natural inhibitor of the P2Y1 and P2Y12 receptors, we need to examine our list of natural products below to see if we can find a platelet activation inhibitor with the correct mechanism of action: inhibition of the P2Y1 and P2Y12 receptors.

Having sifted through this list twice, I already had a good idea which of the natural products are platelet activation inhibitors. I just had to continue the deep dive to determine which of those platelet activation inhibitors worked by inhibiting the P2Y1/12 receptors.

And that’s when I discovered that we have a problem…

I went through the whole list and NONE of the natural products on that list inhibits the P2Y1 or P2Y12 receptors.

Undeterred, I went through the list again to double check. Same result. No P2Y1 or P2Y12 receptor inhibitors.

So now what?

What do we do when the natural product we need isn’t on our comprehensive list of anticoagulants and platelet activation inhibitors?

At this point, I decided to turn the tables on things and start with the biological targets instead of the natural products.

So I did the normal scientific database and literature search that I would do for inhibitors of the P2Y1 and P2Y12 receptors if I was trying to find a starting scaffold to develop a new pharmaceutical drug.

I ended up having to wade through a ton of literature on clopidogrel and other existing pharmaceutical drugs that inhibit the P2Y1 and P2Y12 receptors.

But I finally found a natural product that does inhibit these receptors - Salvianolic acid.

Salvianolic acid is a component of a Traditional Chinese Medicine called Dan shen, which is an extract of the Salvia miltiorrhiza plant.

I actually found a really good paper (Ref 3) with excellent data showing that Salvianolic acid does inhibit the two receptors we are interested in:

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Salvianolic Acids as P2Y1 & P2Y12 Inhibitors - The Data

The salvianolic acids - salvianolic acid A (SAA), salvianolic acid B (SAB), and salvianolic acid C (SAC) [see structures below] - were originally identified as P2Y1 and P2Y12 receptors inhibitors by a computational Molecular Dynamics simulation using the protein crystal structures of the P2Y1 (PDB: 4XNW) and P2Y12 receptors (PDB: 4PXZ and 4NTJ) receptors, following by a radioligand binding assay. (Ref 3)

In the radioligand binding assay, eleven natural products were tested at a single dose (100 uM) for the ability to bind to the P2Y12 receptor using a tritium ([3H]) labeled derivative of the natural receptor ligand ADP ([3H]-2-methylthio-adenosine-5′-diphosphate ([3H]2MeSADP)). (Ref 3)

In this assay, salvianolic acids A, B, and C all showed >50% inhibition of radiolabeled-ADP binding, meaning these compounds were the best at binding to the P2Y12 receptor. (see data below)

Individual binding experiments were then carried out for each receptor - P2Y12 & P2Y1 - with each of the three salvianolic acids. (Ref 3) The dose-response curves and binding affinities (Ki) are shown below.

Functional assays were also carried out showing that each of the three salvianolic acids inhibited downstream signaling of the P2Y12 & P2Y1 receptors. (Ref 3) These assays were carried out in the U2OS cell line, which expresses the P2Y12 & P2Y1 receptors.

All three salvianolic acids - salvianolic acid A (SAA), salvianolic acid B (SAB), and salvianolic acid C (SAC) - are components in Den Shen extract, which is the component we can now use in the natural version of Triple Anticoagulant Therapy.

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So now we have all three natural components that we need to create a nutraceutical that will have the same mechanisms of action as Triple Anticoagulant Therapy but will be available over the counter (OTC) as a supplement.

Here is our completed table:

The first run of the natural version of Triple Anticoagulant Therapy is being manufactured RIGHT NOW (November 2023).

If you’re interested in getting a bottle when Presale Ordering opens next week, go to www.getLongCovidhelpnow.com and put your name on the notification list.

If you want to hear more about this natural supplement, come join the Road to Long Covid Recovery Facebook Group.

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Reference

1. Treatment of Long COVID symptoms with triple anticoagulant therapy. Research Square Preprint 2023, rs-2697680/v1. https://doi.org/10.21203/rs.3.rs-2697680/v1

2. Platelet Physiology. Semin. Thromb. Hemost. 2016, 42, 191–204. http://dx.doi.org/10.1055/s-0035-1564835.

3. Salvianolic acids from antithrombotic Traditional Chinese Medicine Danshen are antagonists of human P2Y1 and P2Y12 receptors. Scientific Reports. 2018, 8, 8084. https://doi.org/10.1038/s41598-018-26577-0