Inhibition fasciculins

Bourne, Y., Taylor, P. and Marchot, P. Acetylcholinesterase inhibition by fasciculin. Crystal structure of the complex. Cell 83 503-512,1995. 

Onchidal and fasciculins are natural toxins, which produce their toxicity in mammalian systems by virtue of primarily acetylcholinesterase (AChE) inhibition. AChE hydrolyzes and inactivates acetylcholine, thereby regidating the concentration of the transmitter at the synapse. Termination of activation is normally dependent on dissociation of acetylcholine from the receptor and its subsequent diffusion and hydrolysis, except in diseases where acetylcholine levels are limiting or under AChE inhibition, conditions that increase the duration of receptor activation (Silver, 1963). 

The fasciculins are a family of closely related peptides that are isolated from the venom of mambas and exert their toxic action by inhibiting AChE. The crystal structure of fasciculin 2 from green mamba Dendroaspis angusticeps) snake venom was first resolved in 1992 (Le Du et al., 1992). The three-dimensional (3D) structure of fasciculin 1 obtained from the US National Library of Medicine, National Center for Biotechnology Information, MMDB database is illustrated in Figure 11.2. 

A large number of organic compounds reversibly or irreversibly inhibit AChE (Long, 1963), which bind either to the esteratic or the anionic subsite of AChE catalytic site or to the peripheral site of the enzyme. Most of them are synthetic substances, sometimes bearing insecticidal properties. Few natural inhibitors of AChE are known and, to date, fasciculins are the only known proteinic AChE... 

Fasciculins inhibit AChE from mammals, electric fish, and some snake venoms with Ki values in the pico- to nanomolar range in contrast, avian, insect, and some other snake venom AChEs are relatively resistant, and high micromolar concentrations are required to inhibit mammalian butyrylcholinesterases (BuChE) (Marchot et al, 1993). Dissociation constants of Fasl and Fas3 are two-fold and 60-fold lower, respectively, than that of Fas2 for synaptosomal rat brain. 

Fasciculin inhibition of AChE is prevented by chemical modification of the enzyme at a peripheral site (Duran et al, 1994). The specific interaction of fasciculin 2 with peripheral sites present in Electrophorus electricus AChE Ki, 0.04 nM fasciculin) was investigated by chemical modification with A,A-dimethyl-2-phenylaziridium (DPA) in the presence of active or peripheral anionic site protective agents. An enzyme was obtained that compared to the native AChE and was 10 times less sensitive to fasciculin 2. This enzyme was fully inhibited by edrophonium and tacrine, and was 25-170 times less sensitive to several peripheral site ligands. It seems fasciculin 2 binding to an AChE peripheral site partially overlaps the site of other peripheral site ligands including acetylcholine. 

Onchidal and fasciculins are interesting natural compounds and it is difficult to predict their toxicity. In the case of onchidal, in silico computational predictive modeling for toxic endpoints of interest may prove useful for risk assessment decision support. Likewise, it is a challenge to predict the mditary potential and human impact of these natural toxins since their affinity for enzyme inhibition depends upon the amount and duration of the human exposure. 

Duran, R., Cervenansky, C., Dajas, F., Tipton, K.F. (1994). Fasciculin inhibition of acetylcholinesterase is prevented by chemical modification of the enzyme at a peripheral site. Biochim. Biophys. Acta 1201 381-8. 

Dajas, F., Bolioli, B., Castello, M.E., Silveira, R. (1987). Rat striatal acetylcholinesterase inhibition by fasciculin (a polypeptide fi-om green mamba snake venom). Neurosci. Lett. 77 87-91. 

Several of the most poisonous snakes in the world use inhibition of AChE as their means of killing. For example the Black Mamba (Dendroapsis polx/lepis) from Africa secretes fasciculins into its venom. The fasciculins are potent AChE inhibitors and make the Black Mamba s venom amongst the most potent venoms known only 21 mg (equivalent to about three grains of salt) would be needed to kill an average sized person. Indeed, a Hospital in South Africa reported treating seven patients for Mamba bites — all of whom died within 24 hours. The symptoms associated with a Mamba bite are tetany. 

In order to maximise the toxic potential of their venoms, many snakes have several toxins in their venoms which act by different biochemical mechanisms. This is an ingenious ploy which means that more than one of the bod/s vital systems is hit by the venom so making death more certain than if only one were hit. The Black Mamba is an excellent example of a snake with multiple toxic components in its venom. In addition to the fasciculins. Mamba venom has dendrotoxins which inhibit neurotransmission by blocking the exchange of + and - ions across the neuronal membrane. This prevents passage of the nerve impulse. If the impulse is en route to the big toe the toe will be paralysed — this is certainly not life-threatening. However, if the impulse is to the pulmonary muscles, respiratory failure and death will result. The dendrotoxins from the Black Mamba are very much less toxic than the fasciculins (it would take 1.6 g to kill a person), however the combined effect of the two toxins is far more toxic than the toxicities of the individual components (this is termed synergy) which is why the Black Mamba is lethal to humans. 

The venom of the mamba snake (Dendroaspis angusticeps, Dendroaspis polylepis, Dendroaspis viridis, Dendroaspis jamesoni) contains a mixture of neurotoxic compounds, including postsynaptic cholinoreceptor a-neurotoxins, dendrotoxins, fasciculins, and muscarinic toxins (Hawgood and Bon, 1991). Effects at the NMJ include AChE inhibition by fasciculins and increased presynaptic release of ACh by dendrotoxins (polypeptides that facilitate ACh release in response to nerve stimulation) together with the high ACh content of mamba toxin (6-24 mg/g), these effects are synergistic and enhance neurotoxicity and lethality. Moreover, the venom may contain other components that have a synergistic action with dendrotoxin. 

A large number of organic compounds reversible or irreversibly inhibits AChE (Long, 1963), which bind either to the esteratic or the anionic subsite of AChE catalytic site or to the peripheral site of the enzymes. Most of them are S5mthetic substances, sometimes with insecticidal properties. Few natural inhibitors of AChE are known and, to date, fasciculins are the only known proteinic AChE inhibitors. They have been shown to display a powerful inhibitory activity toward mammalian AChE. lodination of Fas3 provided a fully active and specific probe of fasciculin-binding sites on rat brain AChE (Marchot et al., 1993). These authors demonstrate that fasciculins bind on a peripheral site of AChE, distinct from the catalytic site and, at least partly, common with the sites on which some cationic inhibitors and the substrate in excess bind since phosphorylation of the catalytic serine (esteratic subsite) by [l,3- H]diisopro-pyl fluorophosphate can still occur on the Fas3. In the... 

Fasciculins inhibit AChE from mammals, electric fish, and some snake venoms with Ki values in the picomolar... 

Administration of Fasl and Fas2 to mice at doses of l-3mg/kg and 0.05-2.0mg/kg, respectively after intra-peritoneal (i.p.) injection caused severe, generalized, and long-lasting fasciculations (5-7 h), followed by gradual recovery to normal behavior. In vitro preincubation with fasciculins at concentrations of O.Olpg/mL inhibited... 

The toxin binds to acetylcholinesterase and renders acetylcholine unhydrolyzed. This causes continuous excitement of the muscle. The inhibition of acetylcholinesterase is seen not only in vitro, but also in vivo. For instance, 80% of the acetylcholinesterase activity in the locus coeruleus was inhibited by the injection of fasciculin 2 in rats (Abo et al., 1989). The inhibition of the enzyme by fasciculin is longlasting, and a 74% inhibition five days after injection was observed (Quillfeldt et al., 1990). 

By inhibiting acetylcholinesterase, fasciculin increased the amplitude and time course of the endplate potential (Lee et al., 1985). Fasciculin also increased the amplitude of the miniature endplate potential (Cervenansky et al., 1991). 

Because of the inhibition of acetylcholinesterase, dendrotoxins or other facilitatory toxins enhance the release of acetylcholine. Thus, dendrotoxins and fasciculins have synergistic action that enhances the lethality. 

Abo, V, Viera, L., Silveira, R., and Dajas, F. (1989). Effects of local inhibition of locus caeruleus acetylcholinesterase by fasciculin in rats. Neurosci. Lett. 98 253-257. 

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