Fig. 1: Development and characterization of daLUXendin544 and daLUXendin660.

a,b, Tirzepatide, modified at the C-terminus with S39C to allow maleimide conjugation with either Cy3 or Cy5 (model obtained from cryo-electron microscopic structure of non-acyl tirzepatide bound to activated GLP1R in complex with G_s) (PDB 7VBI)²⁶. c, cAMP responses to GLP1, GIP, tirzepatide (TZP), daLUXendin544 (daLUX544) and daLUXendin660 (daLUX660) in AD293 cells expressing either hGLP1R, mGLP1R, hGIPR or mGIPR (n = 4 independent repeats). d,e, Binding affinity of daLUXendin660 against hGLP1R and hGIPR (d), with K_d values (e) in a separate bar graph (one-site fit specific binding) (n = 3 independent repeats). f,g, Competition binding for daLUXendin660 (f) and either Ex4-Cy5 or GIP-Cy5 (g) at hGLP1R and hGIPR versus GLP1 and GIP (one-site fit K_i) (n = 3 independent repeats). h, pK_i values for daLUXendin660, Ex4-Cy5 and GIP-Cy5 at hGLP1R and hGIPR versus GLP1 and GIP (n = 3 independent repeats). i, Mice were fasted 3 h before the onset of the dark phase. At dark onset, mice were injected subcutaneously with either vehicle, daLUXendin660 (daLUX; 10 nmol kg⁻¹) or tirzepatide (10 nmol kg⁻¹), and then food was returned. Graph showing cumulative food intake 12 h and 24 h post injection (n = 8 mice per investigated state) (repeated-measures two-way ANOVA with Šídák’s multiple comparisons test). j, Tirzepatide and daLUXendin660 similarly lower glucose in GLP1R^WT/WT and GLP1R^WT/KO mice during glucose tolerance testing (n = at least five mice per investigated state). k,l, Efficacy of tirzepatide and daLUXendin660 is reduced in GLP1R^KO/KO mice (k), shown also by area under the curve (AUC) (l) (n = at least five mice per investigated state) (one-way ANOVA with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli). m, mGLP1R:mGIPR selectivity is lower for daLUXendin660 versus tirzepatide (selectivity is calculated using the mean AUC ratio from at least five GLP1R^WT/WT and five GLP1R^KO/KO mice). Bar and line graphs show means; error bars, s.e.m. ***P < 0.001. Exact P values are displayed on each graph.

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