Science: Mitotic Poison, Colchicine

 

Gloriosa superba var. carsonii (Colchicaceae)

Today, we are going to explore two plant toxins that target the human body's process of cell division, migration and signalling. Both toxins are alkaloids, namely, colchicine and vincristine, and we call them cytotoxic (toxic to cell) drugs or mitotic poisons. Despite their toxicity, both alkaloids are vital to cancer chemotherapy, gout, and inflammatory diseases. This is where the line between poison and medicine converges, and that a narrow window of therapy exists.

 

Figure 1: Chemical structure of colchicine,

Colchicine is an old drug and as its name suggest it was first isolated in 1820 by two French chemists Pelletier and Caventou from the bulbs of autumn crocus (Colchicum autumnale, Colchicaceae). Despite its well-known toxicity and medicinal values, the structure of colchicine took chemists more than 100 years to solve! That's because early chemists did not have modern spectroscopy techniques, and they had to pain painstakingly break down the colchicine molecule to deduce its structure. Today,  people like me would take barely half an hour to figure colchicine out, so consider yourself lucky. Unlike all other alkaloids we have encountered, colchicine is unusual because it does not have it's nitrogen atom incorporated into any ring systems. Colchicine is made up of two distinctive seven-membered rings that are merged back to back (labelled A' and C in Figure 1). Ring A' is called tropolone and it is aromatic, while ring C is merged with a benzene ring, as well as a tertiary acetamide group, which contains the alkaloidal nitrogen atom. However, the amide nitrogen atom is non-basic, and colchicine is yet another non-alkaline alkaloid. In 3D space (Figure 1), both ring A and D are planar, while ring C adopts a half 'boat' configuration. Besides, there is a stereocenter in ring C, and it causes the amide group to point downwards into the plane, hence a an (S)-enantiomer, or (–)-colchicine, one that rotates polarised right to the left hand side. Nature only favors (–)-colchicine because plants can only recognise amino acids of particular stereochemical properties. I've talked about stereochemistry in the atropine article, check that out if you're interested. 

 

Figure 2: Simplified biosynthesis pathway of colchicine.

 

Remember I mentioned that solving colchicine's structure was hard? Now let me show you how it feels in the biosynthesis pathway, bear in mind that early chemists got to work backwards from what I'm about to describe. Colchicine is only known to occur in plants of the family Colchicaceae, and we classify colchicine as a phenethylisoquinoline alkaloid because plants make it from alkaloids containing a phenethylisoquinoline skeleton (Figure 1). Examine Figure 2 carefully. We start off from two amino acids, namely, tyrosine, and phenylalanine, which give two upstream precursors, dopamine and 4-hydroxycinnamaldehyde, respectively. These two condense via Pictet-Spengler's reaction (cf. aristolochic acid biosynthesis) to give autumnaline, which is a phenethylisoquinoline alkaloid. I've labelled the three rings in autumnaline as A, B, and C, so that you can visualuse how plants change it into colchicine. First, some radical oxidation steps connect ring A and C to give the seven membered ring D. This is followed by another enzyme catalysed oxidation to generate a highly unstable cyclopropane ring, which results in a rearrangement reaction that destroys from B, and expands ring A into a tropolone ring (A'). That gives us a proto-colchicine alkaloid, demecolchicine, when is then acetylated (amide) to produce colchicine. If you don't understand it, please try to make sense of it because you'll come to appreciate the genius of early chemists. 

 

Gloriosa superba is widely cultivated in the tropics for its colchicine rich tubers. There is more than enough colchicine here to dispatch three adult humans.

Colchicine is extremely toxic, less than half a gram is fatal to adult humans. Instances of poisoning due to Colchicaceae plants such as Gloriosa spp. and Colchicum are well-documented. Victims usually develop severe dehydration from vomiting and diarrhea, followed by bone marrow suppression, pancytopenia (all blood cells count low, until nil), loss of hair and nails, multi-organ failure, massive internal bleeding, neurotoxicity, and death. Basically, every cells of the body dies, and there is no antidote available for colchicine poisoning. Remember I mentioned cytotoxic? The faster a cell grows like in hair, gastrointestinal mucosa, or blood (bone marrow), the faster it dies of colchicine. The same applies to malignant cancer cells, but unfortunately colchicine is way too toxic to be used to treat cancer. You can certainly kill cancer with colchicine, but you'll kill the patient too. Less toxic derivatives or analogs of colchicine (called trimethoxyphenyl-leads) like demecolcine are still being investigated as novel anticancer drugs. In a lower dose (microgram), colchicine can be used to treat gout and certain inflammatory diseases. That's because low dose colchicine can stop immune cells like neutrophils from migrating and generating further pro-inflammatory signals . In fact, plants containing colchicine like autumn crocus were used to treat gout since ancient times. We can see a narrow therapeutic window  exists for colchicine, any dosage above this window gives toxicity, whereas below would be ineffective. Sometimes, this window is so narrow, toxicity is observed together with medicinal outcome. In the case of colchicine, patients often experience diarrhea before they obtain relief from gout, and more unfortunate gout patients have died from an overdose. Pharmacists call dangerous drugs like colchicine narrow-index drug, as their prescriptions are mandated by vigilant patient monitoring and counseling. Despite its high toxicity, colchicine remains an affordable and valuable drug that helps countless gout patients everyday. 

Figure 3: Mechanism of action of colchicine and vincristine.

Colchicine acts by binding to a protein found in most cells called tubulin. Tubulin is a protein of about 50 kDa, and there are two types of tubulins called alpha-tubulin (blue) and beta-tubulin (red), which bind together to form a heterodimer (a-b). Tubulin heterodimers can link together into a long chain of protein called a protofilament, 13 of which can fold up side by side to from a cellular structure called microtubule. A microtubule is basically a strong tube or cylinder that is used by cells to pull components. During cell division, that is when a cell wants to duplicate itself, the chromosomes condense and replicate. Microtubules start to form at organelles called centrioles located at both sides of the cell. A centriole acts as an anchoring site for microtubules formation, and microtubles assemble from individual tubulin heterodimers starting at the beta-tubulin position (+ site). As more and more tubulin heterodimers associate, the protofilaments merge and fold to produce a functional microtubule. Many microtubules then grab the chromosomes or relevant cellular components, and pull at the opposite ends, which split the cell into two. Once cell division is completed, microtubules will degrade back into individual tubulin proteins. This is a highly controlled processed governed by a principle called dynamic instability because beta-tubulin unit has intrinsic (GTPase) activity to break apart from alpha-tubulin. It is the speed at which the dimers associate, and constant replenishing of degraded units that dictate microtuble formation or destruction. It is like driving a car that constantly breaks down, and you have to replenish the broken components with things you collect along your journey. As long as you can keep up with the maintenance, you can get going. If you don't, you car breaks down completely and you'll have to start all over again. Colchicine binds to the interface between alpha and beta tubulin heterodimer. This causes the colchicine bound dimer to be unable to 'replenish' our intrinsically unstable microtubule car. As a result, colchicine prevents microtubules formation, halting cell division. When cell division is severely disrupted, a cell usually dies by committing suicide. On the other hand, microtubules are also used to pull immune cells like white blood cells at one end (promote cell polarisation) when they migrate. Note that immune cells can literally move about into every nooks and crannies of your body thanks to microtubules. However, in the presence of low dose colchicine, immune cells become 'frozen' and unable to move, thus cannot carry out pro-inflammation activities. Lucky for us, neutrophil or immune cells that are involved in gout attack (migrate into joints) are particularly inhibited by colchicine. 

In my next article on mitotic poisons, we will meet another alkaloid that is strongly cytotoxic but more forgiving than colchicine. It has made it to become one of the most life-saving anticancer drugs today, and if you think colchicine chemistry is hard, wait until you meet vincristine.