Structure–Activity Studies of Cysteine‐Rich α‐Conotoxins that Inhibit High‐Voltage‐Activated Calcium Channels via GABA_B Receptor Activation Reveal a Minimal Functional Motif

Abstract

α-Conotoxins are disulfide-rich peptides that target nicotinic acetylcholine receptors. Recently we identified several α-conotoxins that also modulate voltage-gated calcium channels by acting as G protein-coupled GABA(B) receptor (GABA(B)R) agonists. These α-conotoxins are promising drug leads for the treatment of chronic pain. To elucidate the diversity of α-conotoxins that act through this mechanism, we synthesized and characterized a set of peptides with homology to α-conotoxins known to inhibit high voltage-activated calcium channels via GABA(B)R activation. Remarkably, all disulfide isomers of the active α-conotoxins Pu1.2 and Pn1.2, and the previously studied Vc1.1 showed similar levels of biological activity. Structure determination by NMR spectroscopy helped us identify a simplified biologically active eight residue peptide motif containing a single disulfide bond that is an excellent lead molecule for developing a new generation of analgesic peptide drugs.

Cyclic analogues of α‐conotoxin Vc1.1 inhibit colonic nociceptors and provide analgesia in a mouse model of chronic abdominal pain

“…To translate our ex vivo and in vitro findings to an in vivo model, we recorded the VMR, a nociceptive brainstem reflex consisting of the contraction of the abdominal muscles, in response to CRD (Ness and Gebhart, ). By using abdominal EMG, we were able to assess the effect of intra‐colonically administered Vc1.1 or cVc1.1 on visceral sensitivity in vivo in fully awake healthy or CVH mice (Christianson and Gebhart, ; Deiteren et al, ; Carstens et al, ). We chose Vc1.1 and cVc1.1 as they had the least and highest anti‐nociceptive actions in our colonic nociceptor recordings respectively.…”

“…The algesic ion channels and receptors, Na V 1.1 (Osteen et al, ), TRPV1 (Brierley et al, ), TRPA1 (Hughes et al, ; Brierley et al, ), TRPV4 (Brierley et al, ), P2X3 (Brierley et al, ), bradykinin B 1 (Brierley et al, ), Na V 1.8 (Beyak, ) and TNFR1 (Hughes et al, ), are highly expressed in these afferents. In addition, serosal afferents become mechanically hypersensitive in models of CVP (Hughes et al, ) and have a nociceptor phenotype (Brierley et al, ; Castro et al, ; de Araujo et al, ; Carstens et al, ; Osteen et al, ; Castro et al, ). In the present study, they are therefore referred to as colonic ‘nociceptors’.…”

“…Mechanical sensitivity was then re‐tested in response to application of 3 × 3 s 2 g vfh probes to the afferent receptive field. This process was then repeated for 10, 100 and 1000 nM of the respective peptide and mechanical sensitivity re‐tested after each concentration (Castro et al, ; de Araujo et al, ; Carstens et al, ; Osteen et al, ; Castro et al, ).…”

Host defense peptide‐derived privileged scaffolds for anti‐infective drug discovery

“…The activity of Peptide γ was similar to full-length HBD3, although both peptides were considerably less potent than the antibiotic gentamicin. The control peptide [S 23 ]HBD3 [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] [27] was inactive.…”

Cyclisation of Disulfide‐Rich Conotoxins in Drug Design Applications

“…The solution structure of cVc1.1–6 suggests that the linker might interfere with binding of Ser4 and Asp5, which have both been shown to be important for activity at nAChR. This same region of the sequence was recently reported to be crucial for activity on HVA calcium channels …”