Proteolytic stability in gastrointestinal tract and blood plasma is the major obstacle for oral peptide drug development. Inhibitor cystine knots (ICKs) are linear cystine knot peptides which have multifunctional properties and could become promising drug scaffolds. ProTx-I, ProTx-II, GTx1-15, and GsMTx-4 were spider-derived ICKs and incubated with pepsin, trypsin, chymotrypsin, and elastase in physiological conditions to find that all tested peptides were resistant to pepsin, and ProTx-II, GsMTx-4, and GTx1-15 showed resistance to all tested proteases. Also, no ProTx-II degradation was observed in rat blood plasma for 24 hours in vitro and ProTx-II concentration in circulation decreased to half in 40 min, indicating absolute stability in plasma and fast clearance from the system. So far, linear peptides are generally thought to be unsuitable in vivo, but all tested ICKs were not degraded by pepsin and stomach could be selected for the alternative site of drug absorption for fast onset of the drug action. Since spider ICKs are selective inhibitors of various ion channels which are related to the pathology of many diseases, engineered ICKs will make a novel class of peptide medicines which can treat variety of bothering symptoms.

Tarantula venom glands produce a large variety of bioactive peptides. Here we present the identification of venom components obtained by sequencing clones isolated from a cDNA library prepared from the venom glands of the Chilean common tarantula,Grammostola rosea. The cDNA sequences of about 1500 clones out of 4000 clones were analyzed after selection using several criteria. Forty-eight novel toxin-like peptides (GTx1 to GTx7, and GTx-TCTP and GTx-CRISP) were predicted from the nucleotide sequences. Among these peptides, twenty-four toxins are ICK motif peptides, eleven peptides are MIT1-like peptides, and seven are ESTX-like peptides. Peptides similar to JZTX-64, aptotoxin, CRISP, or TCTP are also obtained. GTx3 series possess a cysteine framework that is conserved among vertebrate MIT1, Bv8, prokineticins, and invertebrate astakines. GTx-CRISP is the first CRISP-like protein identified from the arthropod venom. Real-time PCR revealed that the transcripts for TCTP-like peptide are expressed in both the pereopodal muscle and the venom gland. Furthermore, a unique peptide GTx7-1, whose signal and prepro sequences are essentially identical to those of HaTx1, was obtained.

Inhibitor cystine knot (ICK) peptides are knotted peptides with three intramolecular disulfide bonds that affect several types of ion channels. Some are proteolytically stable and are promising scaffolds for drug development. GTx1-15 is an ICK peptide that inhibits the voltage-dependent calcium channel Ca_v3.1 and the voltage-dependent sodium channels Na_v1.3 and Na_v1.7. As a model molecule to develop an ICK peptide drug, we investigated several important pharmaceutical characteristics of GTx1-15. The stability of GTx1-15 in rat and human blood plasma was examined, and no GTx1-15 degradation was observed in either rat or human blood plasma for 24 h in vitro. GTx1-15 in blood circulation was detected for several hours after intravenous and intramuscular administration, indicating high stability in plasma. The thermal stability of GTx1-15 as examined by high thermal incubation and protein thermal shift assays indicated that GTx1-15 possesses high heat stability. The cytotoxicity and immunogenicity of GTx1-15 were examined using the human monocytic leukemia cell line THP-1. GTx1-15 showed no cytotoxicity or immunogenicity even at high concentrations. These results indicate that GTx1-15 itself is suitable for peptide drug development and as a peptide library scaffold.

Kv2.1, the voltage-gated ion channel, is ubiquitously expressed in variety of tissues and dysfunction of this ion channel is responsible for multiple diseases. Electrophysiological properties of ion channels are so far characterized with eukaryotic cells using the manual patch clamp which requires skilful operators and expensive equipments. In this research, we created a simple and sensitive drug screen method using bacterial giant spheroplasts and the automated patch clamp which does not require special skills. We expressed a eukaryotic voltage-gated ion channel Kv2.1 in Escherichia coli using prokaryotic codon, and prepared giant spheroplasts large enough for the patch clamp. Human Kv2.1 currents were successfully recorded from giant spheroplasts with the automated system, and Kv2.1-expressed E. coli spheroplasts could steadily reacted to the dose-response assay with TEA and 4-AP. Collectively, our results indicate for the first time that the bacterial giant spheroplast can be applied for practical pharmaceutical assay using the automated patch clamp.