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Colubrid snake venoms potentially represent a vast source of novel biological actives and structural motifs owing to their diverse phylogeny. The present study describes the identification of rufoxin, a neurotoxin from the venom of Rhamphiophis oxyrhynchus (Rufous beaked snake) which is a member of the African colubrid lineage, the psammophiines. Rufoxin (1 microM) displayed reversible post-synaptic neurotoxic activity as evidenced by significant inhibition of indirect twitches and responses to exogenous nicotinic agonists in the chick biventer cervicis nerve-muscle preparation. Rufoxin (0.1-1.0 microM) also caused a rightward parallel shift of cumulative concentration-response curves to carbachol (CCh; 0.6-80 microM) without a significant depression of the maximum response, suggestive of classical competitive antagonism at the skeletal muscle nicotinic receptor. Rufoxin lacks NH(2)-terminal sequence homology to previously identified snake venom toxins. This work indicates a wider distribution of neurotoxins across the advanced snake superfamily than previously described.

In this study, we have pharmacologically characterised boigatoxin-A, a three finger toxin isolated from the venom of the colubrid, Boiga dendrophila (Mangrove catsnake). In the chick biventer cervicis nerve-muscle preparation boigatoxin-A (1 microM) displayed poorly reversible postsynaptic blockade as evidenced by the inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous acetylcholine (1 mM) and carbachol (20 microM). Boigatoxin-A (0.3-0.5 microM) caused a concentration-dependent depression of the maximum response of cumulative concentration response curves to CCh (0.6-80 microM). Boigatoxin-A (1 microM) induced readily reversible inhibition of electrically evoked (0.2 Hz, 0.3 ms, 70-100 V) twitches of the prostatic segment of the rat vas deferens. This inhibition was not significantly attenuated by 8-phenyltheophylline (20 microM) or idazoxan (1 microM). Boigatoxin-A (1 microM) did not affect alpha,beta-mATP (10 microM) or noradrenaline (25 microM) responses in unstimulated epididymal segments of the rat vas deferens. Our data suggests that this toxin has weak postsynaptic neurotoxicity in skeletal muscle and also prejunctional neurotoxic activity in the smooth muscle of the rat vas deferens to inhibit the release of neurotransmitter(s), but not via prejunctional purinergic or adrenergic receptors. This is the first report of such activity for a toxin isolated from snake venom and reinforces the largely untapped potential of colubrid venoms.

The venom of R. oxyrhynchus, a member of the psammophiine subfamily of the colubrid assemblage, was examined for biological activity using biochemical and pharmacological techniques. Venom displayed a high protein content, a complex electrophorectic profile and PLA2 activity but no detectable proteolytic or haematological activities. In the chick biventer cervicis nerve muscle preparation, venom (1-10 microg/ml) displayed postsynaptic neurotoxic activity as evidenced by inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous acetylcholine (1 mM) and carbachol (20 microM). This inhibitory effect was poorly reversible by washing. Venom (30-50 microg/ml) caused a rapid and readily reversible inhibition of direct (0.1 Hz, 2 ms, supramaximal V) twitches of the chick biventer cervicis nerve muscle preparation without morphological changes to the muscle fibers. Venom (30-100 microg/ml) inhibited electrically-evoked (0.2 Hz, 0.3 ms, 70-100 V) twitches of the prostatic segment of the rat vas deferens. This inhibitory effect was not significantly attenuated by 8-phenyltheophylline (8-PT; 20 microM), idazoxan (1 microM), a combination of ranitidine (0.2 microM) and thioperamide (10 microM) or capsazepine (10 microM). Venom (5 mg/kg) induced hypotension with subsequent cardiovascular collapse in the anaesthetised rat. The cardiovascular collapse was prevented by artificial respiration of the animals prior to venom administration. The biological activities demonstrated by R. oxyrhynchus venom may aid in prey envenomation strategies such as prey immobilisation. This study provides further evidence that colubrid venoms are comprised of multiple components which can display a variety of actions, some of which may be novel, therefore reinforcing the largely untapped potential of colubrid venoms.

In this study, venoms from species in the Colubrinae, Homalopsinae, Natricinae, Pseudoxyrhophiinae and Psammophiinae snake families were assayed for activity in the chick biventer cervicis skeletal nerve muscle preparation. Boiga dendrophila, Boiga cynodon, Boiga dendrophila gemincincta, Boiga drapiezii, Boiga irregularis, Boiga nigriceps and Telescopus dhara venoms (10 microg/ml) displayed postsynaptic neuromuscular activity as evidenced by inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches. Neostigmine (5 microM) reversed the inhibition caused by B. cynodon venom (10 microg/ml) while the inhibitory effects of Psammophis mossambicus venom (10 microg/ml) spontaneously reversed, indicating a reversible mode of action for both venoms. Trimorphodon biscutatus (10 microg/ml) displayed irreversible presynaptic neurotoxic activity. Detectable levels of phospholipase A2 activity were found only in T. biscutatus, T. dhara and P. mossambicus venoms. The results demonstrate a hitherto unsuspected diversity of pharmacological actions in all lineages which may have implications ranging from clinical management of envenomings to venom evolution.

The evolution of venom in advanced snakes has been a focus of long-standing interest. Here we provide the first complete amino acid sequence of a colubrid toxin, which we have called alpha-colubritoxin, isolated from the Asian ratsnake Coelognathus radiatus (formerly known as Elaphe radiata), an archetypal nonvenomous snake as sold in pet stores. This potent postsynaptic neurotoxin displays readily reversible, competitive antagonism at the nicotinic receptor. The toxin is homologous with, and phylogenetically rooted within, the three-finger toxins, previously thought unique to elapids, suggesting that this toxin family was recruited into the chemical arsenal of advanced snakes early in their evolutionary history. LC-MS analysis of venoms from most other advanced snake lineages revealed the widespread presence of components of the same molecular weight class, suggesting the ubiquity of three-finger toxins across advanced snakes, with the exclusion of Viperidae. These results support the role of venom as a key evolutionary innovation in the early diversification of advanced snakes and provide evidence that forces a fundamental rethink of the very concept of nonvenomous snake.