The inhibitor cystine knot (ICK) fold is an evolutionarily conserved structural motif shared by a large group of polypeptides with diverse sequences and bioactivities. Although found in different phyla (animal, plant, and fungus), ICK peptides appear to be most prominent in venoms of cone snail and spider. Recently, two scorpion toxins activating a calcium release channel have been found to adopt an ICK fold. We have isolated and identified both cDNA and genomic clones for this family of ICK peptides from the scorpion Opistophthalmus carinatus. The gene characterized by three well-delineated exons respectively coding for three structural and functional domains in the toxin precursors illustrates the correlation between exon and module as suggested by the "exon theory of genes." Based on the analysis of precursor organization and gene structure combined with the 3-D fold and functional data, our results highlight a common evolutionary origin for ICK peptides from animals. In contrast, ICK peptides from plant and fungus might be independently evolved from another ancestor.

Although cyclic diketopiperazines have been known since the beginning of the century, only now have they attracted considerable interest with respect to their biological activity. The aim of this study was to determine if the diketopiperazines cyclo(L-histidyl-L-phenylalanyl) (cyclo(His-Phe)) and cyclo(L-histidyl-L-tyrosyl) (cyclo(His-Tyr)) have significant biological activity relevant to the treatment of cardiovascular-related disease states, cancer and infectious diseases. Haematological studies were performed, including thrombin substrate binding, blood clotting time, platelet adhesion, platelet aggregation and fibrinolysis assays. A cytotoxicity screening utilizing a tetrazolium-based assay on the cell lines HeLa, WHCO3, and MCF-7 was performed. The whole-cell patch-clamp technique was used to investigate ion-channel activity in ventricular myocytes of rats, and isolated rat heart studies were performed to investigate the cardiac effects involving heart rate and coronary flow rate. Cyclo(His-Tyr) produced a significant prolongation of blood clotting time, slowing of clot lysis and inhibition of ADP-induced platelet adhesion and aggregation (P < 0.05). Cyclo(His-Phe) showed significant (P < 0.05) anti-tumour activity, causing greatest reduction of cell viability in cervical carcinoma cells. Preliminary results from patch-clamp studies indicate that both diketopiperazines caused blocking of sodium and calcium ion channels, but opening of inward rectifying potassium ion channels. In the rat isolated heart studies, cyclo(His-Phe) caused a gradual reduction in heart rate (P = 0.0027) and a decrease in coronary flow rate (P = 0.0017). Cyclo(His-Tyr) significantly increased the heart rate (P = 0.0016) but did not cause any significant change of coronary flow rate (P > 0.05). Cyclo(His-Tyr) showed notable (P < 0.05) antibacterial activity and both diketopiperazines showed excellent antifungal activity (P < 0.05). These observations reveal diketopiperazines to be ideal lead compounds for the rational design of an agent capable of preventing metastasis, inhibiting tumour growth, and as potential chemotherapeutic, antiarrhythmic and antihypertensive agents, as well as potential antibacterial and antifungal agents.

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Scorpion alpha-K(+) channel toxins are a large family of polypeptides with a similar structure but diverse pharmacological activities. Despite many structural and functional data available at present, little progress has been made in understanding the toxin's molecular basis responsible for the functional diversification. In this paper, we report the first complete cDNA sequences of toxins belonging to subfamily 6 and identify five new members, called alpha-KTx 6.6-6.10. By analyzing the rates of mutations that occurred in the corresponding cDNAs, we suggest that accelerated evolution in toxin-coding regions may be associated with the functional diversification of this subfamily. To pinpoint sites probably involved in the functional diversity of alpha-KTx family, we analyzed this family of sequences using the evolutionary trace method. This analysis highlighted one channel-binding surface common for all the members. This surface is composed of one conserved lysine residue at position 29 assisted by other residues at positions 10, 26, 27, 32, 34, and 36. Of them, the positions 29, 32, and 34 have been reported to be the most major determinants of channel specificity. Interestingly, another contrary surface was also observed at a higher evolutionary time cut-off value, which may be involved in the binding of ERG (ether-a-go-go-related gene) channel-specific toxins. The good match between the trace residues and the functional epitopes of the toxins suggested that the evolutionary trace results reported here can be applied to predict channel-binding sites of the toxins. Because, the side-chain variation in the trace positions is strongly linked with the functional alteration and channel-binding surface transfer of alpha-KTx family, we conclude that our findings should also be important for the rational design of new toxins targeting a given potassium channel with high selectivity.

A novel peptidyl inhibitor of voltage-gated K + channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called Ôa-KTx1.1Õ). PBTx3 is the tenth member (called Ôa-KTx1.10Õ) of subfamily 1 of K + channel-blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels. The dissociation constants (K d ) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 lM, 547 nM and 492 nM. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor X a (fX a ) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The threedimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel b sheet and a single a-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other a-KTx scorpion toxins.