St. John’s wort is an herb, long used in folk medicine for the treatment of mild depression. Its antidepressant constituent, hyperforin, has properties such as chemical instability and induction of drug-drug interactions that preclude its use for individual pharmacotherapies. Here we identify the transient receptor potential canonical 6 channel (TRPC6) as a druggable target to control anxious and depressive behavior and as a requirement for hyperforin antidepressant action. We demonstrate that TRPC6 deficiency in mice not only results in anxious and depressive behavior, but also reduces excitability of hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Using electrophysiology and targeted mutagenesis, we show that hyperforin activates the channel via a specific binding motif at TRPC6. We performed an analysis of hyperforin action to develop a new antidepressant drug that uses the same TRPC6 target mechanism for its antidepressant action. We synthesized the hyperforin analog Hyp13, which shows similar binding to TRPC6 and recapitulates TRPC6-dependent anxiolytic and antidepressant effects in mice. Hyp13 does not activate pregnan-X-receptor (PXR) and thereby loses the potential to induce drug-drug interactions. This may provide a new approach to develop better treatments for depression, since depression remains one of the most treatment-resistant mental disorders, warranting the development of effective drugs based on naturally occurring compounds.
Cone snails produce a fast-acting and often paralyzing venom that is usually injected into their prey or predator through a hypodermic needle-like modified radula tooth. Many diverse compounds are found in their venom including small molecules, peptides and enzymes. However, peptidic toxins called conotoxins (10–40 residues and 2–4 disulfide bonds) largely dominate these cocktails. These disulfide rich toxins are very valuable pharmacological tools for investigating the function of ions channels, G-protein coupled receptors, transporters and enzymes. Here, we report on the synthesis, structure determination and biological activities of two α-conotoxins, CIA and CIB, found in the predatory venom of the piscivorous species Conus catus. CIA is a typical 3/5 α-conotoxin that blocks the rat muscle type nAChR with an IC50 of 5.7 nM. Interestingly, CIA also inhibits the neuronal rat nAChR subtype α3β2 with an IC50 of 2.06 μM. CIB is a 4/7 α-conotoxin that blocks rat neuronal nAChR subtypes, including α3β2 (IC50 = 128.9 nM) and α7 (IC50 = 1.51 μM). High resolution NMR structures revealed typical α-conotoxin folds for both peptides. We also investigated the in vivo effects of these toxins on fish, since both peptides were identified in the predatory venom of C. catus. Consistent with their pharmacology, CIA was highly paralytic to zebrafish (ED50 = 110 μg/kg), whereas CIB did not affect the mobility of the fish. In conclusion, CIA likely participates in prey capture through muscle paralysis, while the putative ecological role of CIB remains to be elucidated.
Peptides derived from animal venoms provide important research tools for biochemical and pharmacological characterization of receptors, ion channels, and transporters. Some venom peptides have been developed into drugs (such as the synthetic ω-conotoxin MVIIA, ziconotide) and several are currently undergoing clinical trials for various clinical indications. Challenges in the development of peptides include their usually limited supply from natural sources, cost-intensive chemical synthesis, and potentially complicated stereoselective disulfide-bond formation in the case of disulfide-rich peptides. In particular, if extended structure–function analysis is performed or incorporation of stable isotopes for NMR studies is required, the comparatively low yields and high costs of synthesized peptides might constitute a limiting factor. Here we investigated the expression of the 4/7 α-conotoxin TxIA, a potent blocker at α3β2 and α7 nicotinic acetylcholine receptors (nAChRs), and three analogs in the form of maltose binding protein fusion proteins in Escherichia coli . Upon purification via nickel affinity chromatography and release of the toxins by protease cleavage, HPLC analysis revealed one major peak with the correct mass for all peptides. The final yield was 1–2 mg of recombinant peptide per liter of bacterial culture. Two-electrode voltage clamp analysis on oocyte-expressed nAChR subtypes demonstrated the functionality of these peptides but also revealed a 30 to 100-fold potency decrease of expressed TxIA compared to chemically synthesized TxIA. NMR spectroscopy analysis of TxIA and two of its analogs confirmed that the decreased activity was due to an alternative disulfide linkage rather than the missing C-terminal amidation, a post-translational modification that is common in α-conotoxins. All peptides preferentially formed in the ribbon conformation rather than the native globular conformation. Interestingly, in the case of the α7 nAChR, but not the α3β2 subtype, the loss of potency could be rescued by an R5D substitution. In conclusion, we demonstrate efficient expression of functional but alternatively folded ribbon TxIA variants in E. coli and provide the first structure–function analysis for a ribbon 4/7-α-conotoxin at α7 and α3β2 nAChRs. Computational analysis based on these data provide evidence for a ribbon α-conotoxin binding mode that might be exploited to design ligands with optimized selectivity.
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