Science: Resiniferatoxin, Structure Activity Relationship

 

Teaser for our upcoming experiment

Here are some ultra hot ghost peppers (Capsicum sinensis hort. cultivar Bhut Jholokia) that I have prepared for an upcoming experiment, to isolate pure capsaicin. I never featured chili as a poisonous plant, but no doubt it is. After all, chilli (genus Capsicum) belongs to the nightshade family Solanaceae. Today, we are going to explore the structure activity relationship of capsaicin and its ultra potent analog, resiniferatoxin. 

 

Figure 1: Chemical structures of vanilloids and their biosynthesis.

Both capsaicin and resiniferatoxin (RTX) are called vanilloids. Chemically speaking, vanilloids refer to various derivatives of the natural product vanillin, which contains a 4-hydroxy-3-methoxy substituted aryl group (Figure 1). Vanillin was first isolated from the seedpod of the vanilla orchid (Vanilla planifolia, Orchidaceae), and it orginates from the amino acid L-phenylalanine. Vanillin itself is pretty fragrant and benign, but it can be transformed into highly pungent (spicy) compounds by certain plants such as chilli and ginger. In chilli, the aldehyde group (HC=O) of vanillin is transformed into an amine (-CH₂NH₂), which is then incorporated as an amide (CH₂NHC=O) into a ten carbons hydrocarbon chain to produce capsaicin. It is interesting to note that capsaicin contains a nitrogen atom, and it can rightfully be classified as an alkaloid, although phytochemists usually do not endorse it. On the other hand, the spicy component of ginger, namely, gingerol also contains a vanilloid fragment. However, gingerol does not contain any nitrogen atom, and it originates from phenylalanine and an upstream precursor called ferulic acid. As for resiniferatoxin, it’s vanilloid fragment is called a homovanillate ester, as it contains one more carbon atom than typical vanillin derivatives. I’m not sure if it arises from vanillin or even phenylalanine. The exact biosynthesis pathway of RTX still awaits delineation. 

 

Figure 2: Structure of the TRPV-1 receptor.
Reference: Elokely, K.; Velisetty, P.; Delemotte, L.; Palovcak, E.; Klein, M. L.; Rohacs, T.; Carnevale, V. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 137–145.

How do vanilloids make us feel spicy? Vanilloids are agonists that activate a specific receptor in our body (particularly sensory neurons) called  TRPV-1, V for vanilloid. The TRPV-1 receptor is a transmembrane homotetrameric (contains 4 similar sub-units) protein that forms a channel to guard positive ions like sodium, potassium and calcium from entering a cell (Figure 2) . When TRPV-1 gets activated, positive ions rush into sensory cell, producing a Transient (quick but short lasting) Receptor induced electric current (Potential) in the cell membrane, which basically says 'HOT, HEAT, DANGER!'. It is our body's own protective mechanism against harmful environmental stimuli such as intense heat (> 42 degrees Celsius), strong acid (< pH 1), and vanilloids like capsaicin. We call receptors like TRPV-1, nociceptors, noci is Latin for nocere, meaning harmful.  In some people, the body responds to TRPV-1 activation by sending out other chemical signals like noradrenaline to induce a 'fight or flight' response, to prepare us for escaping. In an overdose of capsaicin or perhaps during RTX toxicity, this sympathetic response can get so overwhelming, it leads to panic attack. Besides, vanilloids can increase the production of stomach acid, leading to dyspepsia. In severe toxicity, massive influx of calcium ions due to TRPV-1 activation can cause neurotoxic effects because brain cells literally die of over-excitation. As far as I know, death by capsaicin is rare and it is usually associated with very young patients or people with pre-existing medical conditions. While there are no documented cases of RTX poisoning, numerous instances of eye damage due to Euphorbia latex are known. Euphorbia latex contains phorbol esters which are highly corrosive, and I would expect ingestion of Euphorbia resinifera to be a potentially harmful if not fatal event. Hence, ultra hot chilli, pure capsaicin and  RTX are no picnic, except for some superhumans, which I’ll explain later.

 

Figure 3: Simplified mechanism of action of vanilliod on TRPV-1 receptor.

TRPV-1 is an ion-channel, much like the nicotinic acetylcholine receptor, which we have previously examined. Note that TRPV-1 is a fat-loving protein embedded in the cell membrane, which is made of fats called phospholipids (Figure 3). The fatty cell membrane keeps water and ions out because fat and water don’t mix, so when the TRPV-1 opens its gate, ions will rush it. If we examine the gross structure of vanilloids like capsaicin and RTX, they are also hydrophobic (fat-loving) molecules. In chemistry, fat loves fat, so capsaicin and RTX can freely diffuse across cell membrane and bind to TRPV-1 receptor like two sumos hugging each other. When that happens, the TRPV-1 protein changes shape (when two sumos hug, they're going to squeeze), and the ion-channel opens. This is also the reason why people recommend you to drink milk to mitigate spicy feeling because the fats in milk are going to wash vanillods away. If you just drink plain water, it’s not going to help at all. Remember, like dissolves like. 

 

Figure 4: Computer modeled, induced-fit binding of capsaicin and RTX onto TRPV-1 receptor.
Reference: Elokely, K.; Velisetty, P.; Delemotte, L.; Palovcak, E.; Klein, M. L.; Rohacs, T.; Carnevale, V. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 137–145.

In terms of structure activity relationship, the TRPV-1 receptor is quite versatile and adaptive. By computer modelling studies, scientists found that TRPV-1 can change it shape to accomodate different vanilloid receptor agonists. After all, capsaicin and RTX are drastically different sturcutres. It was found that the 4-hydroxy and 3-methoxy aryl substituents are crucial to capsaicin’s activity, but not to RTX. Besides, when they tempered the diterpene ring, RTX’s spiciness diminishes. This means that despite being agonists of TRPV-1, capsaicin and RTX have different binding interactions with the receptor. In other words, the receptor changes its shape to best-fit a particular agonist, which in the case of RTX is way better than capsaicin, hence its high potency. The computer modelling study also posits that inside the binding pocket, water molecules play a part to stablise the drug-receptor complex. We call this type of drug-receptor binding the induced fit model (Figure 4). 

 

My resin spurge (Euphorbia resinifera) is still young and growing, we will certain give the isolation of RTX a try when it's time.

The tinya plant (Euphorbia poissonii) also contains RTX and its less potent analog tinyatoxin. Tinyatoxin is the 3-demethoxy derivative of RTX. Replacing the 3-OMe substituent with 3-OH decreases the potency of RTX by half, but even then tinyatoxin is still 300X more spicy than pure capsaicin.

There's more to vanilloid’s pharmacology. When humans are exposed to vanilloids for a long period, the TRPV-1 receptor can become desensitised. Remember I mentioned that capsaicin overdose can cause calcium induced neurotoxicity? In local tastebuds or certain nerve terminals, huge calcium influx induced by prolonged TRPV-1 activation can cause nerve endings to degenerate, which may take days or weeks to recover! Hence, ultra hot chilli can become less spicy as you become more tolerant, and I predict RTX can permanently fry your nerve terminals following initial suffering. There are also evidences that vanilloids can induce human brain to release happy-feeling chemicals called endorphins amongst various neuropeptide (substance P and CGRP). Perhaps it’s an innate protection against pain as your body makes its own pain-killer to numb itself? Some people find this process absolutely pleasurable akin to taking psychoactive drugs because they get ‘high’ by eating super hot chilli! 

 

In recent years, scientists are trying to develop capsaicin and RTX as a pain-killer, or reverse engineer 'anti-capsaicin' TRPV-1 antagonists for the same purpose! Rest assured, they will numb you with anaesthesia before they fry your neurons with ultra potent vanilloids like RTX.  After that, you won’t feel pain anymore and it’s a great news for patients suffering from chronic pain by cancer and degenerative illnesses. Even more remarkably, vanilloids can be applied to the human bladder to desensitise its afferent (C-fiber) nerve terminals. This can mitigate urinary incontinence caused by an over-active bladder due to stroke or spinal injury. Scientists also found that the expression of TRPV-1 nociceptor is increased during inflammation or carcinogenesis, which increases sensitivity to pain. Regardless of agonist or antagonist, the vanilloids and TRPV-1 remain an exciting area of pharmaceutical research today.

 

That’s all, and before I end here’s one question for you to think about. Is it only the plant toxin that can affect TRPV-1 receptors? Are there any other toxins from Nature that can make us feel like on fire? Here’s a clue for you in the picture below.

What's up, who mentioned TRPV-1 receptor?