Dioscorine: Structure Activity Relationship

 

Dioscorea sansibarensis

Continuing on the alkaloid dioscorine of the Zanzibar yam (Dioscorea sansibarensis), here are its bulbils, or the 'air-potatoes'. The bulbils of Dioscorea are well evolved to float and colonise new habitats by water. Each and every one of them will grow into a new plant. In fact, the Zanzibar yam was first introduced to Malaysia (and parts of Singapore) by British colonisers, but now the plant threatens to smother our native ecosystem. To make things worse, it is near impossible to eradicate Zanzibar yam without thoroughly up rooting its yam, and destroying the bulbils. 

 

The alkaloid dioscorine is very toxic when ingested. It is known to cause altered consciousness, followed by massive convulsion, and muscle paralysis to the human body. Death occurs when the body loses its ability to breathe. This is because dioscorine mimics two neurotransmitters of our brain called acetylcholine (ACh) and gamma amino butyric acid (GABA). I have talked about ACh in the post regarding atropine and its structure activity relationship, make sure you read it before continuing. Dioscorine is also an antagonist of ACh, but unlike atropine, it antagonises the nicotinic acetylcholine receptor (nAChR). nAChR is made up of five protein subunits (pentameric) that merge to form a pore, which gates positive ions like sodium or calcium (Figure 1). In other words, nAChR forbids positive ions from entering cells, and allows them entry only in the presence of ACh. We call this type of receptor an ion channel. Unlike its mAChR counterpart, nAChR has two binding sites for ACh, and two molecules of ACh are required to activate it. When the brain commands, ACh binds to nAChR and the ion channel changes shape to opens its gate. Positive ions like sodium rush into the cell (neuron or muscle), creating an electrical current called an action potential (physics: moving ions is a current). In voluntary muscles, the ACh induced action potential called EPSP causes other ion channels to open up and create an even bigger electrical current that stimulates muscle cells to release calcium, which changes the structure of muscle fibers, leading to contraction. The effects of nAChRs are extremely fast and they happen in milliseconds, literally a split second thought just as your brain commands.

 

Figure 1: Structure of nAChR.

Note: The five subunits that make up the nAChR are labelled as alpha, beta, gamma, and delta, respectively. The two ACh binding sites are located at the interface between the two alpha and gamma subunits. The inside of the pore is charged with water, so it tends to attract ions. When the pore opens, ions will naturally diffuse in and out of the pore according to their concentration gradients. Rang, H. P.; Ritter, J. M.; Flower, R. J.; Henderson, G. Rang & Dales’s Pharmacology; Elsevier: Edinburgh, 2012; pp 26. 


There are about five subtypes of nAChRs, and they are most abundant in the central nervous system, as well as the skeletal or voluntary muscle (neuromuscular junction). Dioscorine potentially antagonises many different nAChRs of the body, leading to altered consciousness and widespread muscle paralysis. As a receptor antagonist, dioscorine prevents the natural messenger ACh from binding to nAChR. I am not sure if two molecules of dioscorine are required to antagonise nAChR, but it appears that one is enough because it already forbids the binding of another ACh molecule. Hence, no action potential will be generated regardless of how desperate the brain sends its command. That net effect is paralysis, and it comes quickly given high enough dose of dioscorine. On the other hand, the other messanger GABA is responsible for the ‘slowing down’ actions of the brain. It does so by activating the GABA_A ion channel, which works in a similar way as nAChR. However, instead of eliciting an excitatory response, GABA_A channel allows the entry of negative ions like chloride, which make cells in the central nervous system chill and relax. Thus, if dioscorine also antagonises GABA, it can cause brain cells to get easily excitable, to point of convulsion and seizure. The convulsant activity of dioscorine is likened to that of picrotoxin, which is a known GABA antagonist. However, I emphasise that the GABA antagonistic effect of dioscorine is not well understood. This is probably because dioscorine is a ‘dirty’ poison that affects multiple targets at a same time. The overall toxicity or symptoms could be a result of its antagonism on both nAChR and GABA ion channels.

 

Now let’s examine the structure activity relationship of dioscorine (Figure 2). Dioscorine is an alkaloid, and it contains a nitrogen atom that has alkaline property. When dioscorine gets into the human body, its nitrogen atom will be neutralised by acids and becomes positively charged, which turns out to be very important. In a same way as atropine (please read the associated post), dioscorine also mimics the structure of ACh, but they differ in the distance between the positively charged nitrogen atom and the ester group. The distance for discorine is a bit further than that of atropine, and this makes dioscorine more selective on nAChR instead of mAChR. You may wonder why ACh can activate both receptors because compared to both atropine and dioscorine, ACh is small and flexible. ACh can twist and turn around to fit both nAChR and mAChR, but the more rigid alkaloids can’t. Guess what? Pharmacologists named the two receptors as nicotinic and muscarinic because they were selectively activated by the alkaloids nicotine (from tobacco) and muscarine (from the mushroom Amanita muscaria), respectively. Also in analogy to atropine, dioscorine is a competitive antagonist. Hence, dioscorine binds to nAChR in a similar way as ACh but it does not activate the receptor. Discorine will dissociate from the receptor with time, and it can be competitively displaced by increasing the dosage of ACh. That also means we can potentially give an antidote that is a nAChR agonist, or one that increases the level of ACh in the body. I have overlapped the structure of dioscorine with that of ACh and GABA in Figure 2, and you can compare their similarities. It is most plausible that dioscorine also allows extra intermolecular interactions with the affected receptors such that they do not change shape as they would when they are activated by ACh. 

 

Figure 2: Structure activity relationship of dioscorine and toxins affecting the nAChR.

Lastly,  here’s your homework before I end. I have also attached the chemical structures of a few other toxins that affect the nAChR in Figure 2. Nicotine as aforementioned is a competitive agonist of nAChR, and in low dose it causes excitation and addiction. In toxic dose however, nicotine can excite the nerve terminals until they become unresponsive (neuromuscular blockade), causing a paradoxical flaccid paralysis following initial spasm. Compare the chemical structure of nicotine, which is a pyridine alkaloid with that of ACh. Can you see the similarities, and predict the crucial intermolecular interactions that may take place with nAChR? The second molecule is the piperidine alkaloid lobeline, the toxic principle of Lobelia spp. and Hippobroma longiflora. Lobeline is another ‘dirty’ toxin than can affect multiple neurotransmitters, primarily ACh at nAChR. In fact, we aren’t quite sure if lobeline is an agonist or antagonist yet as it appears to act differently at different nAChR subtypes. It is even possible that lobeline has antimuscarinic action like that of atropine. Can you see the reason behind by comparing the structure of lobeline with ACh? Finally, we have anatoxin-a, perhaps the most toxic of all alkaloids acting on nAChR. This microbial toxin produced by cyanobacteria is responsible for massive mortality during algae blooms. Anatoxin-a is an irreversible agonist of nAChR, so once it binds to the receptor, the receptor will be permanently activated until it becomes totally unresponsive. Victims first develop muscle twitching, followed rapidly by paralysis, and death by respiratory failure. Remember I mentioned that irreversible agonist/antagonist are very bad? Anatoxin-a is so bad, it was called the ‘very fast death factor’ when it was first isolated. In fact, some of Nature’s most powerful toxins affect the nAChR, which include botulinum toxin, various venoms of snakes, spiders, and scorpions, as well as marine invertebrates. Who knows, we will meet them again in the future.