Insulin-like Growth Factor 1 Analogs Clicked in the C Domain: Chemical Synthesis and Biological Activities

Macháčková, Kateřina; Collinsová, Michaela; Selicharová, Irena; Pícha, Jan; Budêšínský, Miloś; Vaněk, Václav; Žáková, Lenka; Brzozowski, A.M.; Jiráček, Jiřı́

doi:10.1021/acs.jmedchem.7b01331

Cited by 19 publications

(24 citation statements)

References 76 publications

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“…Insulin analogs were prepared by the solid-phase peptide synthesis of A and B chains and biomimetic recombination of their disulfide bridges (27–29). IGF-1 and IGF-2 analogs were prepared in Escherichia coli cells as previously (26, 30).…”

Section: Resultsmentioning

confidence: 99%

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

Macháčková

Mlčochová

Potalitsyn

et al. 2019

Journal of Biological Chemistry

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Information on how insulin and insulin-like growth factors 1 and 2 (IGF-1 and -2) activate insulin receptors (IR-A and -B) and the IGF-1 receptor (IGF-1R) is crucial for understanding the difference in the biological activities of these peptide hormones. Cryo-EM studies have revealed that insulin uses its binding sites 1 and 2 to interact with IR-A and have identified several critical residues in binding site 2. However, mutagenesis studies suggest that Ile-A10, Ser-A12, Leu-A13, and Glu-A17 also belong to insulin's site 2. Here, to resolve this discrepancy, we mutated these insulin residues and the equivalent residues in IGFs. Our findings revealed that equivalent mutations in the hormones can result in differential biological effects and that these effects can be receptor-specific. We noted that the insulin positions A10 and A17 are important for its binding to IR-A and IR-B and IGF-1R and that A13 is important only for IR-A and IR-B binding. The IGF-1/IGF-2 positions 51/50 and 54/53 did not appear to play critical roles in receptor binding, but mutations at IGF-1 position 58 and IGF-2 position 57 affected the binding. We propose that IGF-1 Glu-58 interacts with IGF-1R Arg-704 and belongs to IGF-1 site 1, a finding supported by the NMR structure of the less active Asp-58–IGF-1 variant. Computational analyses indicated that the aforementioned mutations can affect internal insulin dynamics and inhibit adoption of a receptor-bound conformation, important for binding to receptor site 1. We provide a molecular model and alternative hypotheses for how the mutated insulin residues affect activity.

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Section: Resultsmentioning

confidence: 99%

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

Macháčková

Mlčochová

Potalitsyn

et al. 2019

Journal of Biological Chemistry

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“…Another interesting observation is that GIV and SGIV dcVILPs have higher affinity to bind and stimulate signaling via IGF1R than insulin, since these ligands are missing the C-domain that is important for IGF-1:IGF1R interaction [43][44][45][46] . Even though dcVILPs are completely missing the C-domain, they bind to IGF1R with 7x (GIV dcVILP) and 10x (SGIV dcVILP) higher affinity compared to insulin.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Viral Insulins Reveals White Adipose Tissue Specific Effects in Mice

Marchand

Noh

Páníková

et al. 2020

Preprint

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Members of the insulin/IGF superfamily are well conserved across the evolutionary tree. We recently showed that four viruses in the Iridoviridae family possess genes that encode proteins highly homologous to human insulin/IGF-1. Using chemically synthesized single chain (sc), i.e. IGF-1-like, forms of the viral insulin/IGF-1 like peptides (VILPs), we previously showed that they can stimulate human receptors. Because these peptides possess potential cleavage sites to form double chain (dc), i.e. more insulin-like, VILPs, in this study, we have characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1). GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A, IR-B) and to the IGF1R, and for the latter show higher affinity than human insulin. These dcVILPs stimulate IR and IGF1R phosphorylation and post-receptor signaling in vitro and in vivo. Both GIV and SGIV dcVILPs stimulate glucose uptake in mice. In vivo infusion experiments in awake mice revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75nmol/kg/min) stimulated a comparable glucose uptake in heart, skeletal muscle and brown adipose tissue, GIV dcVILP stimulated ~2 fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This was associated with increased Akt phosphorylation and glucose transporter type 4 (GLUT4) gene expression compared to insulin. Taken together, these results show that GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action, design new analogues that specifically target the tissues, and provide new insights into their potential role in disease.

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“…The only differences are in Tyr B16 and Ser B9 in insulin, which are replaced with Gln 15 and Ala 8 in IGF-1, respectively. In addition, Met 59 in IGF-1, which does not have equivalent receptor-binding residue in insulin, was shown to interact with Arg 704 IGF-1R αCT peptide, and its mutation abolishes receptor binding ( 34 ). Ala 8 in IGF-1 was shown to interact with both the L1 domain (Glu 91 ) and αCT peptide (Glu 693 ) of IGF-1R, whereas insulin Ser B9 interacts only with the IR αCT peptide (His 710 ) ( 6 , 8 ).…”

Section: Discussionmentioning

confidence: 99%

“…Ligand–dose response IGF-1R autophosphorylation levels for selected analogs were determined, using the In-Cell Western assay adapted for chemiluminescence as described ( 34 ). Data were subtracted from background values and expressed as the contribution of phosphorylation relative to the 10 n m IGF-1 signal.…”

Section: Methodsmentioning

confidence: 99%

A versatile insulin analog with high potency for both insulin and insulin-like growth factor 1 receptors: Structural implications for receptor binding

Žáková

Marek

Socha

et al. 2018

Journal of Biological Chemistry

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Insulin and insulin-like growth factor 1 (IGF-1) are closely related hormones involved in the regulation of metabolism and growth. They elicit their functions through activation of tyrosine kinase–type receptors: insulin receptors (IR-A and IR-B) and IGF-1 receptor (IGF-1R). Despite similarity in primary and three-dimensional structures, insulin and IGF-1 bind the noncognate receptor with substantially reduced affinity. We prepared [d-HisB24, GlyB31, TyrB32]-insulin, which binds all three receptors with high affinity (251 or 338% binding affinity to IR-A respectively to IR-B relative to insulin and 12.4% binding affinity to IGF-1R relative to IGF-1). We prepared other modified insulins with the aim of explaining the versatility of [d-HisB24, GlyB31, TyrB32]-insulin. Through structural, activity, and kinetic studies of these insulin analogs, we concluded that the ability of [d-HisB24, GlyB31, TyrB32]-insulin to stimulate all three receptors is provided by structural changes caused by a reversed chirality at the B24 combined with the extension of the C terminus of the B chain by two extra residues. We assume that the structural changes allow the directing of the B chain C terminus to some extra interactions with the receptors. These unusual interactions lead to a decrease of dissociation rate from the IR and conversely enable easier association with IGF-1R. All of the structural changes were made at the hormones' Site 1, which is thought to interact with the Site 1 of the receptors. The results of the study suggest that merely modifications of Site 1 of the hormone are sufficient to change the receptor specificity of insulin.

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Insulin-like Growth Factor 1 Analogs Clicked in the C Domain: Chemical Synthesis and Biological Activities

Cited by 19 publications

References 76 publications

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

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

Characterization of Viral Insulins Reveals White Adipose Tissue Specific Effects in Mice

A versatile insulin analog with high potency for both insulin and insulin-like growth factor 1 receptors: Structural implications for receptor binding

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