A structurally minimized yet fully active insulin based on cone-snail venom insulin principles


Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone-snail venom with moderate human insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins—a human des-octapeptide insulin analog—as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24–PheB25–TyrB26 aromatic triplet and opens a new direction for therapeutic insulin development.

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Fig. 1: Overview of crystal structures of Con-Ins–G1-bound Fv83-7-bound μIR and of human insulin-bound Fv83-7-bound μIR.
Fig. 2: Structural biology of Con-Ins–G1 and human insulin within their respective μIR cocomplexes.
Fig. 3: Side-chain substitution of human insulin PheB24 by Con-Ins–G1 TyrB15.
Fig. 4: Structure–activity studies on Con-Ins–G1 and DOI.
Fig. 5: Structure–activity studies on DOI.
Fig. 6: Biochemical, in vitro and in vivo characterization of mini-Ins.
Fig. 7: Probing the interaction of mini-Ins with hIR.

Data availability

Coordinates and structures factors for the structures presented here have been deposited in the Protein Data Bank as follows: Cons-Ins–G1-bound Fv83-7-bound μIR, PDB 6VEQ; human insulin-bound Fv83-7-bound μIR, PDB 6VEP and mini-Ins, PDB 6VET. Supplementary Methods and Source Data for Figs. 4a,b,d, 5b–d, 6 and 7a,b and Extended Data Figs. 2 and 3 are available with the paper online.

Change history

  • 12 June 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.


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X.X. is a Juvenile Diabetes Research Foundation Postdoctoral Fellow. N.A.S. acknowledges receipt of an Australian Research Training Scholarship. R.S.N acknowledges fellowship support from the Australian National Health and Medical Research Council. Part of this work was undertaken using resources from the National Computational Infrastructure, which is supported by the Australian Government and provided through Intersect Australia Ltd, and through the HPC-GPGPU Facility, which was established with the assistance of a Linkage Infrastructure, Equipment and Facilities grant (LE170100200). We thank the CSIRO Protein Production Facility for the production under contract of cIR485, the precursor of IR310.T. Crystallization screening was undertaken at the CSIRO Collaborative Crystallisation Centre (www.csiro.au/C3), Melbourne, Australia. This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of the Australian Nuclear Science and Technology Organisation, and made use of the ACRF detector. We thank M. Margetts for production of the heavy and light chain fragments of Fv83-7. This work is supported by NIDDK (DK120430 to D.H.C.), NIGMS (GM125001 to D.H.C.), the Juvenile Diabetes Research Foundation (5-CDA-2018-572-A-N to D.H.C. and 1-INO-2017-441-A-N to H.S.H.), the Australian National Health and Medical Research Council (NHMRC) Project grant nos. APP1143546 (to M.C.L., R.S.N., B.J.S., B.E.F. and D.H.C.) and APP1099595 (to M.C.L.). M.C.L.’s research is also made possible at The Walter and Eliza Hall Institute of Medical Research through Victorian State Government Operational Infrastructure Support and the Australian NHMRC Independent Research Institutes Infrastructure Support Scheme.

Author information




X.X., J.G.M., R.S.N., H.S.-H., B.O., M.C.L. and D.H.-C.C. designed the study. M.C.L. and D.H.-C.C. wrote the manuscript with input from all authors. M.M.D. and J.G. generated Con-Ins–G1 analogs and M.M.D. performed related in vitro assays with assistance from G.G., and N.A.S. and B.J.S. performed modeling studies. C.D. and B.E.F. performed receptor binding and signaling studies. R.A. and S.J.F. performed in vivo bioactivity assays. X.W. and X.H. performed antibody response assays. C.A.M. and R.S.N. performed analytical ultracentrifugation studies. J.G.M and M.C.L performed crystallographic studies, and J.G.M. produced protein.

Corresponding authors

Correspondence to Michael C. Lawrence or Danny Hung-Chieh Chou.

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Competing interests

M.C.L.’s laboratory has a funded Agreement with Eli Lilly and Company (USA) to conduct research not connected to this publication. Patents associated with part of this work were licensed to Monolog LLC, which aims to develop new fast-acting insulin analogs.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Stereo views of sample (2mFobs-DFcalc) electron density for the structures presented in the manuscript.

(a) L1 domain residues 32-35 within monomer 1 of the Con-Ins-G1-bound μIR + Fv 83-7 crystal structure. (b) L1 domain residues 32-35 within monomer 1 of the human-insulin- bound μIR + Fv 83-7 crystal structure. (c) B-chain residues 14-16 within monomer 1 of the mini-Ins crystal structure. All maps are contoured at the 1.0 σ level.

Extended Data Fig. 2 Sedimentation equilibrium analysis of mini-Ins.

Sedimentation equilibrium analysis of mini-Ins was performed at 35,000 rpm with the best fit (curves) to a single species of apparent mass 5080 ± 45 Da. The molecular weight of mini-Ins is 5067. Detailed procedure can be found in ref. 2. Source data are available with the paper online. Source data

Extended Data Fig. 3 Antibody response of 21-day immunization of bovine insulin, human insulin and mini-Ins.

Data are the average of 4 independent animals. Error bar represents S.E.M. Source data are available with the paper online. Source data

Extended Data Fig. 4 Isothermal titration calorimetry.

Representative ITC thermograms for the titration against IR485 + IR-A704–719 αCT peptide of (a) mini-Ins; (b) hIns; (c) Con-Ins G1 and (d) human DOI.

Extended Data Fig. 5 Separation of insulin and IR amino acid pairs at the secondary binding site during 1 ns MD simulation.

(a) Distance between GluB10 carboxylate carbon (Cδ) and Arg539 guanyl carbon (Cζ). This salt pair remain closely associated (~4 Å) throughout the simulation. (b) Distance between HisA8 imidazole Nε2 nitrogen and Asp574 carboxylate carbon. (c) Distance between ArgA9 guanyl carbon and Glu575 carboxylate carbon (Cγ): this salt bridge forms (separation ~4 Å) following ~6ns MD. The salt bridge is observed to dissociate and reform several times throughout the simulation. Dissociation of this interaction correlates with increase in separation of the HisA8-Asp574 pair, reflecting mobility in the Phe572-to-Tyr579 loop of the FnIII-1.

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Supplementary Tables 1–4 and methods.

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Source Data Extended Data Fig. 2

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Xiong, X., Menting, J.G., Disotuar, M.M. et al. A structurally minimized yet fully active insulin based on cone-snail venom insulin principles. Nat Struct Mol Biol 27, 615–624 (2020). https://doi.org/10.1038/s41594-020-0430-8

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