Science: Metabolic Poison, Carboxyatractyloside

The cocklebur (Xanthium strumarium) is one of the plants that produce carboxyatractyloside. 

The Chilean jessamine (Cestrum parqui) contains carboxyparquin.

Today, in my last article on metabolic poison, we are going to examine two deadly plant toxins called carboxyatractyloside and its structural analog carboxyparquin. Both toxins are potent inhibitors of the mitochondrial  transmembrane protein ADP-ATP translocase, which is crucial to cellular respiration.To understand how they work, we must first examine their chemical structures. 

Figure 1: Chemical structure of carboxyatractyloside.

Figure 2: 3D structure of carboxyatractyloside.

Figure 3: Chemical structure of carboxyparquin.

 

Carboxyatractyloside is comprised of two distinct chemical moieties, namely, a kaurene and a glycoside (sugar). Kaurene is a diterpene, which means that it contains twenty carbon atoms arranged into four rings merged back to back with one another (labelled as A, B, C, and D in Figure 1).  Ring A contains two carboxylic acid functional groups (highlighted in red), which gave the prefix of carboxyatractyloside. Besides, a glycoside (sugar) moiety is also attached ring A, and it contains an two sulfate (pink), and one isovalerate ester groups (green). On the other hand, carboxyparquin is almost similar to carboxyatractyloside, they differ only in the glycoside moiety. Carboxyparquin has a carboxylic acid (red) and gamma-lactone group (pink) in place of two sulfate esters (Figure 2).

 

Figure 4: Simplified biosynthesis pathway of kaurene (diterpene).

Carboxyatractyloside is found is various plants of the daisy family (Asteraceae), particularly the cocklebur (Xanthium spp.) and the Mediterranean distaff thistle (Atractylis spp.). Carboxyparquin is found exclusively in the Cestrum genus of the nighshade family (Solanaceae). Plants synthesise kaurene from an important twenty carbon precursor called geranylgeranyl phosphate (GGPi), which originates from the mevalonate (acetyl-CoA) pathway. All phytochemicals that are synthesised from GGPi are called diterpene and we will encounter more of them in the future. GGPi contains 4 X 5 isoprene units = 20 carbon atoms, with  4 double bonds that would allow intramolecular cyclisation following protonation to produce an intermediate called copalyl PP.  Examine Figure 4 carefully as I have arranged and numbered the carbon atoms of GGPi and copalyl PP into the backbone of a kaurene. Follow the coloured arrows and connect the bonds that would produce a kaurene molecule. 

 

Figure 5: ADP-ATP translocase and its inhibition by carboxyatractyloside.

Carboxyatractyloside and carboxyparquin are highly toxic, less than 0.3 grams of the pure toxin is fatal to a 70 kg human. That makes the two at least as toxic as hydrogen cyanide! Both toxins are strongly hepatotoxic, which means that they destroy the liver. The liver is usually the first to be poisoned because it is the organ that carries out detox on most drugs and poisons (first-pass metabolism). In chronic carboxyatractyloside poisoning, victims often exhibit symptoms such as depression, weakness, jaundice, ascites (build up of fluid in the abdominal cavity), convulsions and death within a few weeks. Liver damage results in a build up of toxic metabolites such as ammonia, which causes fatal brain damage (hepatic encephalitis). In contrast, victims of acute poisoning can collapse suddenly due to low blood sugar and then die. This is typical of metabolic poisons that inhibit cellular respiration such as rotenone and cyanide. Fatal instances of poisoning by carboxyatractyloside and carboxyparquin are well-known to occur in humans and livestock, which are usually due to accidental ingestion of associated plants. 

 

Figure 6: Structure comparison between ADP, carboxyatractyloside, and carboxyparquin

Carboxyatractyloside and carboxyparquin are non-competitive inhibitors of ADP-ATP translocase, which is one of the most abundant surface protein of mitochondria. The ADP-ATP translocase is a cone shaped protein pump that exports ATP out of, and imports ADP into the mitochondria matrix (Figure 5). Remember that mitochondria has its own membrane and it doesn't allow ADP and ATP passage because both are hydrophilic charged molecules (mitochondria membrane is hydrophobic). Thus, ADP-ATP translocase exploits the negative charges of ADP (3-) and ATP (4-) to facilitate binding onto itself. Count the negative charges on the phosphate group of ADP and ATP in Figure 5, respectively. Once that happens, the protein changes shape and it flips sides, pushing the ADP or ATP in and out of the mitochondrial membrane. The process requires energy, but it's more than sufficed by the electron transport chain. If we examine the structures of carboxyparquin and carboxyatractyloside, they also contain prominent negative charges such as sulfate and carboxylate (Figure 6). That is why the two toxins can fool the ADP-ATP translocase into thinking that they are ADP, and binds with it. In fact, scientists have crystalised ADP-ATP translocase with carboxyatractyloside and showed that the toxin's negative charges of sulfate and carboxylate bind to positively charged amino acids such as protonated arginine, asparagine and lysin (Figure 7). Moreover, the binding of carboxyatractyloside and carboxyparquin are stronger than ADP, and they cannot be displaced by increasing concentration of ADP (non-competitive). As a result, the ADP-ATP translocase is permanently locked in it 'ADP'-bound state, becoming malfunctional. Even though ATP is still produced by the electron transport chain, they cannot be exported out of mitochondria. The net effect is  still cellular suffocation. The body tries to compensate for a lack of ATP by promoting glycolysis, which happens outside of mitochondria. However, increased glycolysis will rapidly deplete blood sugar concentration, causing sudden collapse as observed in acute carboxyatratyloside toxicity. There is currently no antidote available for carboxyatractyloside and carboxyparquin poisoning, as both toxins are associated with high mortality (> 70%). 

 

Protein crystal structure of carboxyatractyloside bound ADP-ATP translocase. Source:

Pebay-Peyroula, E.; Dahout-Gonzalez, C.; Kahn, R.; Trézéguet, V.; Lauquin, G. J. M.; Brandolin, G. Nature 2003, 426, 39–44.

 

At last, we have come to an end for the discussion on three metabolic poisons, i.e., rotenone, cyanide and carboxyatractyloside. There are more toxins that belong to this group such as fluoroacetate and bonkrekic acid, but we will deal with them next time. In my subsequent post, I'm thinking of toxins that prevent cells from growing.