1. Lactam-stabilized helical analogues of the analgesic μ-conotoxin KIIIA
Keith K Khoo, Michael J Wilson, Brian J Smith, Min-Min Zhang, Joszef Gulyas, Doju Yoshikami, Jean E Rivier, Grzegorz Bulaj, Raymond S Norton J Med Chem. 2011 Nov 10;54(21):7558-66. doi: 10.1021/jm200839a. Epub 2011 Oct 12.
μ-Conotoxin KIIIA (μ-KIIIA) blocks mammalian voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. Previous structure-activity studies of μ-KIIIA identified a helical pharmacophore for VGSC blockade. This suggested a route for designing truncated analogues of μ-KIIIA by incorporating the key residues into an α-helical scaffold. As (i, i+4) lactam bridges constitute a proven approach for stabilizing α-helices, we designed and synthesized six truncated analogues of μ-KIIIA containing single lactam bridges at various locations. The helicity of these lactam analogues was analyzed by NMR spectroscopy, and their activities were tested against mammalian VGSC subtypes Na(V)1.1 through 1.7. Two of the analogues, Ac-cyclo9/13[Asp9,Lys13]KIIIA7-14 and Ac-cyclo9/13[Lys9,Asp13]KIIIA7-14, displayed μM activity against VGSC subtypes Na(V)1.2 and Na(V)1.6; importantly, the subtype selectivity profile for these peptides matched that of μ-KIIIA. Our study highlights structure-activity relationships within these helical mimetics and provides a basis for the design of additional truncated peptides as potential analgesics.
2. Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels
Keith K Khoo, et al. Biochemistry. 2012 Dec 11;51(49):9826-35. doi: 10.1021/bi301256s. Epub 2012 Nov 28.
In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of μ-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely related μ-conotoxins. The two major isomers of synthetic μ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of Na(V)1.2 (K(d) values of 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on nuclear magnetic resonance data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the μ-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an α-helix spanning residues 7-12. In addition, the major folding isomers of μ-KIIIB, an N-terminally extended isoform of μ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as μ-KIIIA, and both blocked Na(V)1.2 (K(d) values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA and μ-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of μ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail's repertoire of active peptides.
3. Structure of the analgesic mu-conotoxin KIIIA and effects on the structure and function of disulfide deletion
Keith K Khoo, Zhi-Ping Feng, Brian J Smith, Min-Min Zhang, Doju Yoshikami, Baldomero M Olivera, Grzegorz Bulaj, Raymond S Norton Biochemistry. 2009 Feb 17;48(6):1210-9. doi: 10.1021/bi801998a.
Mu-conotoxin mu-KIIIA, from Conus kinoshitai, blocks mammalian neuronal voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. We have determined its solution structure using NMR spectroscopy. Key residues identified previously as being important for activity against VGSCs (Lys7, Trp8, Arg10, Asp11, His12, and Arg14) all reside on an alpha-helix with the exception of Arg14. To further probe structure-activity relationships of this toxin against VGSC subtypes, we have characterized the analogue mu-KIIIA[C1A,C9A], in which the Cys residues involved in one of the three disulfides in mu-KIIIA were replaced with Ala. Its structure is quite similar to that of mu-KIIIA, indicating that the Cys1-Cys9 disulfide bond could be removed without any significant distortion of the alpha-helix bearing the key residues. Consistent with this, mu-KIIIA[C1A,C9A] retained activity against VGSCs, with its rank order of potency being essentially the same as that of mu-KIIIA, namely, Na(V)1.2 > Na(V)1.4 > Na(V)1.7 >or= Na(V)1.1 > Na(V)1.3 > Na(V)1.5. Kinetics of block were obtained for Na(V)1.2, Na(V)1.4, and Na(V)1.7, and in each case, both k(on) and k(off) values of mu-KIIIA[C1A,C9A] were larger than those of mu-KIIIA. Our results show that the key residues for VGSC binding lie mostly on an alpha-helix and that the first disulfide bond can be removed without significantly affecting the structure of this helix, although the modification accelerates the on and off rates of the peptide against all tested VGSC subtypes. These findings lay the groundwork for the design of minimized peptides and helical mimetics as novel analgesics.
