The toxins found in venomous animals have been optimized over time to aid in prey capture and digestion and also to help the animals defend themselves. While venomous animals receive their fair share of notoriety for the painful (and often deadly) effects of their bites and stings, their venoms have been harnessed for the treatment of human diseases for thousands of years. In recent years, venoms have been subjected to more rigorous scientific investigation as a potential source of new therapeutic entities. Currently, five venom-derived peptide drugs are on the market, and many more are in pre-clinical or clinical development for indications such as cancer, pain, heart disease, stroke, and diabetes 
. Animal venoms represent a valuable source of untested bioactive molecules, as the venoms of only a few hundred species have been studied to date.
Animal venom peptides that are active at GPCRs can be divided into two families 
. The members of the first family mimic the natural agonist at the target receptor. Peptides belonging to this family are snake sarafotoxins, which are functional analogs of the endogenous endothelins 
, the cone snail toxin conopressin, which is similar to the arginine-vasopressin peptide 
, and the cone snail toxin contulakin-G, which is similar to the neurotensin peptide 
. The second family of GPCR toxins consists of highly reticulated peptides with folds unrelated to those of natural ligands 
. We have discovered Bm
K-YA, the first scorpion venom peptide that displays a primary structure resembling that of the enkephalin-like peptides. Bm
K-YA thus belongs to the family of venom peptides that mimic the natural agonists and suggests that scorpion venom may represent a novel source of GPCRs ligands.
By analyzing the sequence of the protein encoding BmK-
YA, we found a polyprotein containing four Bm
K-YA (YGGYMNPA) and four His4
K-YA (YGGHMNPA). This polyprotein contains a typical signal sequence, which indicates that it is secreted. Furthermore Bm
K-YA and His4
K-YA can be amidated suggesting that they are bioactive. The organization of this precursor is reminiscent to that of the mammalian opioid peptide precursors where multiple sequence-related peptides within a single genomic transcript. Excluding the endomorphins, the classical opioid peptides are derived from three larger precursors: proopiomelanocortin (POMC), proenkephalin (PENK) and prodynorphin (PDYN), which encodes for one, seven and three enkephalin-containing sequences (YGGFM or YGGFL). Compared to these, the precursor encoding Bm
K-YA, contains eight copies of enkephalin-like sequences (YGGYM or YGGHM). Interestingly the core Bm
K-YA enkephalin-like sequences are followed by four conserved residues (NPAG), of which the glycine residue serves as amide donor. This amidation is thought to be mediated by a specific amidation enzyme 
. Indeed, two amidated enkephalin-like peptides, amidorphin and metorphamide, have been reported in mammals 
. A data base search did not yield any significant hits in mammalian genomes although eight repeats of the sequence (…RGGYVNPAG…) are found as part of the TBC1 domain family member 14 protein. This is however a non secreted protein.
We show that Bm
K-YA in vitro
interacts with the three subtypes of opioid receptors, μ, δ and κ, but with preference to the δ subtype. Its selectivity to the δ-subtype is 6.8 times higher than that to the μ, and 12 times higher than that to the κ subtype. It therefore displays a pharmacological profile that is different from morphine. Bm
K-YA is a full agonist at the δ receptor with an EC50
of 2.5 µM while morphine is only a partial agonist with an EC50
of 15 µM 
. Although both molecules can activate δ receptors with low potency, morphine cannot stimulate δ receptors as effectively as Bm
K-YA at high concentrations. On the other hand, morphine is a full agonist at the μ receptor with an EC50
of 180 nM 
, while Bm
K-YA is only a partial agonist with an EC50
of 17 µM. Thus Bm
K-YA might induce fewer of the side effects associated with μ receptors. This may serve as a starting point for structure-function relationship studies leading to design specific antinociceptive drugs.
Whether BmK-YA acts at opioid receptors in the scorpion is not known but not expected. The fact that BmK-YA is encoded in a precursor that also contains His4-BmK-YA leads us to hypothesize that both peptides should act at the same receptors. However, His4-BmK-YA is inactive at the opiod receptors. Indeed, it is the specific His4 substitution that is responsible for the lack of activity since Phe4-BmK-YA (YGGFMNPA), which contains a copy of Met-enkephalin, exhibits high affinity for the opioid receptors.
Several lines of evidence indicate that BmK-YA is the first member of a new bioactive peptide family in scorpions. First, BmK-YA is encoded by a precursor that can be secreted. Second, the organization of this precursor is similar to that of the mammalian opioid peptide precursors with multiple sequence-related peptides within a single genomic transcript. Third, BmK-YA and His4-BmK-YA are flanked by processing cleavage sites and can be amidated. Fourth, the NH2- tripeptide YGG sequence of BmK-YA and His4-BmK-YA is identical to the core sequence YGGF of the opioid peptides () and thus suggest evolutionary conservation. Whether they act as bioactive peptides in vivo will however await the identification of their receptor(s).
The discovery of Bm
K-YA and its identification as an enkephalin-like peptide demonstrates that relatively “primitive” organisms may possess opioid-like systems. The present study supports previous work that have characterized opioid peptides (enkephalin-containing) in invertebrate, for example, the mussel Mytilus edulis
and the digestive system of the scallop Chalmys farreri
. It has also been reported on the basis of binding and immunocytochemical analyses that δ opioid receptors subtypes may exist in invertebrates 
K-YA is the first invertebrate peptide that displays a similar but not identical enkephalin sequence. Because enkephalin sequences are found in invertebrates and vertebrates while the Bm
K sequence is not, it is reasonable to assume that enkephalins served as templates for Bm
K-YA. Since the Bm
K-YA gene is not found in other species by database bank search it may be unique to the scorpion. The final appearance of Bm
K-YA gene may be the result of a genome duplication 
which started with PENK. Over time one copy of a duplicated PENK gene may retain the PENK organization scheme and the function of the ancestral gene, while the other copy would accumulate amino acid substitution and diverge into a unique member of the Bm
K-YA gene family. Because we did not, in our purification, identify other enkephalin-containing peptides such as true opioid peptides (N terminus:YGGF), we expect that they do not exist in scorpion. Thus Bm
K-YA might have evolved to carry role(s) distinct from classical opioid function. This is reinforced by the coexistence of His4
K-YA, which does not exhibit activity at mammalian opioid receptors. Also, the presence of these peptides in the venom of the scorpion is counterintuitive to them displaying an analgesic activity. Consequently we propose that these peptides must interact with receptors that are divergent of the mammalian opioid receptors and that, in the venom, Bm
K-YA and His4
K-YA may have evolved for specialized use, such as prey capture, defense or immune response.