Engineering stable peptide toxins by means of backbone cyclization: stabilization of the alpha-conotoxin MII

Abstract

Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo. By cyclizing the alpha-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xenopus oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.

Fig. 1.

Sequences of MII, cMII-5, cMII-6, and cMII-7. Disulfide bonds are shown as thin lines. The thick line joining the N and C termini of cMII-5, cMII-6, and cMII-7 indicates backbone cyclization.

Fig. 2.

Cyclization/oxidation RP-HPLC profiles of the cyclic MII analogues. Profiles are shown for the analogues containing a five-residue linker (GGAAG) (a), a six-residue linker (GGAAGG) (b), and a seven-residue linker (GAGAAG) (c). The peaks corresponding to the peptides subsequently named cMII-5, cMII-6, and cMII-7 are indicated by asterisks.

Fig. 3.

A comparison of the αH secondary shift values of residues 2–15 for MII (solid line, circles), cMII-5 (dashed line, triangles), cMII-6 (solid line, squares), and cMII-7 (solid line, diamonds). The values were calculated by subtracting the observed values from the random coil values (33).

Fig. 4.

The 3D structures of cMII-6 and cMII-7. (a) Stereroview of the superposition of backbone heavy atoms of 20 NMR-derived structures for cMII-6. (b) Stereroview of the superposition of backbone heavy atoms of 20 NMR-derived structures for cMII-7. (c) Superposition over the backbone heavy atoms of native MII (red), cMII-6 (blue), and cMII-7 (green), with disulfide bonds shown in yellow. (d) Ribbon representation of cMII-6. (e) Ribbon representation of cMII-7. (f) Ribbon representation of native MII (PDB ID code 1MII).

Fig. 5.

Retained biological activity for the cyclic analogues cMII-6 and cMII-7. (a) Superimposed nicotine (100 μM)-evoked currents recorded from isolated bovine chromaffin cells voltage-clamped at –70 mV in the absence (control) and presence of 1 μM native and cyclic derivatives of MII. (b) Concentration–response curves for inhibition of nicotine (5 μM)-evoked catecholamine release from isolated bovine chromaffin cells by increasing concentrations of MII, cMII-5, cMII-6, and cMII-7.

Fig. 6.

The stability of MII and its cyclic analogues against biological degradation. (a) The stability of MII (red), cMII-6 (blue), and cMII-7 (green) against proteolytic attack by EndoGluC. (b) The stability of MII, cMII-6, and cMII-7 in human blood plasma demonstrating that the stability of cMII-6 and cMII-7 is improved by 15–20% over native MII over 24 h.