Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone's B-chain C-terminus for its IR-B specificity.

Institute of Organic Chemistry and Biochemistry the Czech Academy of Sciences v v i Flemingovo n 2 166 10 Praha 6 Czech Republic

York Structural Biology Laboratory Department of Chemistry University of York Heslington York YO10 5DD United Kingdom

Zobrazit více v PubMed

Taniguchi C. M., Emanuelli B. & Kahn C. R. Critical nodes in signalling pathways: insights into insulin action. Nat. Rev. Mol. Cell Biol. 7, 85–96 (2006). PubMed

Cohen P. Timeline - the twentieth century struggle to decipher insulin signalling. Nat. Rev. Mol. Cell Biol. 7, 867–873 (2006). PubMed

Ward C. W., Menting J. G. & Lawrence M. C. The insulin receptor changes conformation in unforeseen ways on ligand binding: sharpening the picture of insulin receptor activation. Bioessays 35, 945–954 (2013). PubMed

Cabail M. Z. et al. The insulin and IGF1 receptor kinase domains are functional dimers in the activated state. Nat. Commun. 6, 6406 (2015). PubMed PMC

Seino S. & Bell G. I. Alternative splicing of human insulin receptor messenger RNA. Biochem. Biophys. Res. Commun. 159, 312–316 (1989). PubMed

Moller D. E., Yokota A., Caro J. F. & Flier J. S. Tissue-specific expression of two alternatively spliced insulin receptor mRNAs in man. Mol. Endocrinol. 3, 1263–1269 (1989). PubMed

Mosthaf L. et al. Functionally distinct insulin-receptors generated by tissue-specific alternative splicing. EMBO J. 9, 2409–2413 (1990). PubMed PMC

Serrano R. et al. Differential gene expression of insulin receptor isoforms A and B and insulin receptor substrates 1, 2 and 3 in rat tissues: modulation by aging and differentiation in rat adipose tissue. J. Mol. Endocrinol. 34, 153–161 (2005). PubMed

Jonietz E. Diabetes. Nature 485, S1–S19 (2012). PubMed

Boucher J., Kleinridders A. & Kahn C. R. Insulin receptor signaling in normal and insulin-resistant states. Cold Spring Harbor Perspect. Biol. 6, a009191 (2014). PubMed PMC

Pollak M. Insulin and insulin-like growth factor signalling in neoplasia. Nat. Rev. Cancer 9, 224–224 (2009). PubMed

Talbot K. et al. Demonstrated brain insulin resistance in Alzheimer's disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline. J. Clin. Invest. 122, 1316–1338 (2012). PubMed PMC

Herring R., Jones R. H. & Russell-Jones D. L. Hepatoselectivity and the evolution of insulin. Diabetes Obes. Metab. 16, 1–8 (2014). PubMed

Menting J. G. et al. How insulin engages its primary binding site on the insulin receptor. Nature 493, 241–245 (2013). PubMed PMC

Menting J. G. et al. Protective hinge in insulin opens to enable its receptor engagement. Proc. Natl. Acad. Sci. USA. 111, E3395–E3404 (2014). PubMed PMC

Zakova L. et al. Insulin analogues with modifications at position B26. Divergence of binding affinity and biological activity. Biochemistry 47, 5858–5868 (2008). PubMed

Jiracek J. et al. Implications for the active form of human insulin based on the structural convergence of highly active hormone analogues. Proc. Natl. Acad. Sci.USA. 107, 1966–1970 (2010). PubMed PMC

Zakova L. et al. Structural integrity of the B24 site in human insulin is important for hormone functionality. J. Biol. Chem. 288, 10230–10240 (2013). PubMed PMC

Zakova L. et al. Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex. Acta Crystallogr. D 70, 2765–2774 (2014). PubMed PMC

Tornoe C. W., Christensen C. & Meldal M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057–3064 (2002). PubMed

Rostovtsev V. V., Green L. G., Fokin V. V. & Sharpless K. B. A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem.-Int. Edit. 41, 2596–2599 (2002). PubMed

Meldal M. & Tornoe C. W. Cu-catalyzed azide-alkyne cycloaddition. Chem. Rev. 108, 2952–3015 (2008). PubMed

Pedersen D. S. & Abell A. 1,2,3-Triazoles in Peptidomimetic Chemistry. Eur. J. Org. Chem., 2399–2411 (2011).

Angell Y. L. & Burgess K. Peptidomimetics via copper-catalyzed azide-alkyne cycloadditions. Chem. Soc. Rev. 36, 1674–1689 (2007). PubMed

Park J. H. & Waters M. L. Positional effects of click cyclization on beta-hairpin structure, stability, and function. Org. Biomol. Chem. 11, 69–77 (2013). PubMed PMC

Kawamoto S. A. et al. Design of triazole-stapled BCL9 alpha-helical peptides to target the beta-catenin/B-cell CLL/lymphoma 9 (BCL9) Protein-protein interaction. J. Med. Chem. 55, 1137–1146 (2012). PubMed PMC

Scrima M. et al. Cu-I-catalyzed azide-alkyne intramolecular i-to-(i + 4) side-chain-to-side-chain cyclization promotes the formation of helix-like secondary structures. Eur. J. Org. Chem., 446–457 (2010).

Ingale S. & Dawson P. E. On resin side-chain cyclization of complex peptides using CuAAC. Org. Lett. 13, 2822–2825 (2011). PubMed

Holland-Nell K. & Meldal M. Maintaining biological activity by using triazoles as disufide bond mimetics. Angew. Chem.-Int. Edit. 50, 5204–5206 (2011). PubMed

Williams G. M., Lee K., Li X., Cooper G. J. S. & Brimble M. A. Replacement of the CysA7-CysB7 disulfide bond with a 1,2,3-triazole linker causes unfolding in insulin glargine. Org. Biomol. Chem. 13, 4059–4063 (2015). PubMed

Neumann H., Wang K. H., Davis L., Garcia-Alai M. & Chin J. W. Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 464, 441–444 (2010). PubMed

Zakova L. et al. The use of Fmoc-Lys(Pac)-OH and penicillin G acylase in the preparation of novel semisynthetic insulin analogs. J. Pept. Sci. 13, 334–341 (2007). PubMed

Nakagawa S. H. & Tager H. S. Importance of aliphatic side-chain structure at positions 2 and 3 of the insulin A chain in insulin-receptor interactions. Biochemistry 31, 3204–3214 (1992). PubMed

Chen X. Q., Khairallah G. N., O’Hair R. A. J. & Williams S. J. Fixed-charge labels for simplified reaction analysis: 5-hydroxy-1,2,3-triazoles as byproducts of a copper(I)-catalyzed click reaction. Tetrahedron Lett. 52, 2750–2753 (2011).

Ludvigsen S., Olsen H. B. & Kaarsholm N. C. A structural switch in a mutant insulin exposes key residues for receptor binding. J. Mol. Biol. 279, 1–7 (1998). PubMed

Glendorf T. et al. Engineering of insulin receptor isoform-selective insulin analogues. PLoS ONE 6, e20288 (2011). PubMed PMC

Isaad A. L., Papini A. M., Chorev M. & Rovero P. Side chain-to-side chain cyclization by click reaction. J. Pept. Sci. 15, 451–454 (2009). PubMed

Herrmann T., Guntert P. & Wuthrich K. Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol. 319, 209–227 (2002). PubMed

Guntert P. & Wuthrich K. Improved efficiency of protein structure calculations from NMR data using the program DIANA with redundant dihedral angle constraints. J. Biomol. NMR 1, 447–456 (1991). PubMed

Harjes E. et al. GTP-Ras disrupts the intramolecular complex of C1 and RA domains of Nore1. Structure 14, 881–888 (2006). PubMed

Koradi R., Billeter M. & Wuthrich K. MOLMOL: A program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55 (1996). PubMed

Bhattacharya A., Tejero R. & Montelione G. T. Evaluating protein structures determined by structural genomics consortia. Proteins 66, 778–795 (2007). PubMed

Kabsch W. Xds. Acta Crystallogr. D 66, 125–132 (2010). PubMed PMC

Kabsch W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D 66, 133–144 (2010). PubMed PMC

Otwinowski Z. & Minor W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997). PubMed

Winn M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011). PubMed PMC

Vagin A. & Teplyakov A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).

Murshudov G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011). PubMed PMC

Emsley P., Lohkamp B., Scott W. G. & Cowtan K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010). PubMed PMC

McNicholas S., Potterton E., Wilson K. S. & Noble M. E. M. Presenting your structures: the CCP4mg molecular-graphics software. Acta Crystallogr. D 67, 386–394 (2011). PubMed PMC

Morcavallo A. et al. Insulin and insulin-like growth factor II differentially regulate endocytic sorting and stability of insulin receptor isoform A. J. Biol. Chem. 287, 11422–11436 (2012). PubMed PMC

Kosinova L. et al. Insight into the structural and biological relevance of the T/R transition of the N-terminus of the B-chain in human insulin. Biochemistry 53, 3392–3402 (2014). PubMed PMC

A radioligand binding assay for the insulin-like growth factor 2 receptor

Mutations at hypothetical binding site 2 in insulin and insulin-like growth factors 1 and 2 result in receptor- and hormone-specific responses

Structural Perspectives of Insulin Receptor Isoform-Selective Insulin Analogs

Optimized syntheses of Fmoc azido amino acids for the preparation of azidopeptides

Probing Receptor Specificity by Sampling the Conformational Space of the Insulin-like Growth Factor II C-domain