Facile synthesis of insulin fusion derivatives through sortase A ligation

Disotuar, Maria M.; Smith, Jake A.; Li, Jinze; Alam, Steven L.; Lin, Nai-Pin; Chou, Danny Hung‐Chieh

doi:10.1016/j.apsb.2020.11.011

Cited by 12 publications

(14 citation statements)

References 35 publications

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“…Another potential approach to circumvent the solubility issue of B chain intermediates is using sortase A (SrtA)-mediated ligation to semi-synthesize insulin analogs with B chain C-terminal modifications ( Disotuar et al, 2021 ). However, SrtA ligation needs a specific recognition sequence around the ligation site, which becomes a functionless region between insulin and the ligated peptide.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and Characterization of Phenylboronic Acid-Modified Insulin With Glucose-Dependent Solubility

et al. 2022

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Glucose-responsive insulin represents a promising approach to regulate blood glucose levels. We previously showed that attaching two fluorophenylboronic acid (FPBA) residues to the C-terminal B chain of insulin glargine led to glucose-dependent solubility. Herein, we demonstrated that relocating FPBA from B chain to A chain increased the baseline solubility without affecting its potency. Furthermore, increasing the number of FPBA groups led to increased glucose-dependent solubility.

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Section: Resultsmentioning

confidence: 99%

Synthesis and Characterization of Phenylboronic Acid-Modified Insulin With Glucose-Dependent Solubility

et al. 2022

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“…Sedimentation velocity analytical ultracentrifugation (SV-AUC) was performed using a Beckman Coulter XL-I centrifuge. Desoctapeptide insulin (DOI) was prepared from human insulin (hIns; Gibco A11382IJ) by trypsin digestion as described 54 . hIns (775 μg·mL −1 ) and DOI (100 μg·mL −1 ) were run as oligomerizing and monomeric controls, respectively, in phosphate buffer (137 mM NaCl, 2.7 mM KCl, 5.3 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4) using the absorbance optical system.…”

Section: Methodsmentioning

confidence: 99%

Symmetric and asymmetric receptor conformation continuum induced by a new insulin

et al. 2022

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Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes, and biochemical properties. Here, we report a fully active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryoelectron microscopy and protein engineering to elucidate its interactions with the human insulin receptor ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of native insulin but also compromise therapeutic utility by delaying the onset action, suggesting approaches to develop improved therapeutic insulins. Curiously, a receptor conformation present in low abundance adopts a highly asymmetric structure that displays novel coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.

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“…Recently, we mutated the B27 position from Thr to Leu and the B29 position from Lys to Glu, which makes the C-terminal segment a recognition segment for sortase A (SrtA), an efficient ligating enzyme to allow the selective conjugation of peptide segments on B chain. 13 Inspired by this work, we sought to add the SrtArecognition sequence into the C-terminal of insulin A chain. In this work, we reported an insulin construct with a SrtArecognition sequence on the C-terminal A chain.…”

Section: ■ Introductionmentioning

confidence: 99%

Antagonistic Insulin Derivative Suppresses Insulin-Induced Hypoglycemia

et al. 2023

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Insulin derivatives provide new functions that are distinctive from native insulin. We investigated insulin modifications on the C-terminal A chain with insulin receptor (IR) peptide binders and presented a full and potent IR antagonist. We prepared insulin precursors featuring a sortase A (SrtA) recognition sequence, LPETGG, at the C-terminal A chain and used a SrtA-mediated ligation method to synthesize insulin derivatives. The insulin precursor exhibits full IR agonism potency, similar to native human insulin. We explored derivatives with linear IR binding peptides attached to the insulin C-terminal A chain. One insulin derivative with an IR binder (Ins-AC-S2) can fully antagonize IR activation by insulin, as confirmed by cell-based assays. This IR antagonist suppresses insulin-induced hypoglycemia in a streptozotocin-induced diabetic rat model. This study provides a new direction toward insulin antagonist development.

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Facile synthesis of insulin fusion derivatives through sortase A ligation

Cited by 12 publications

References 35 publications

Synthesis and Characterization of Phenylboronic Acid-Modified Insulin With Glucose-Dependent Solubility

Synthesis and Characterization of Phenylboronic Acid-Modified Insulin With Glucose-Dependent Solubility

Symmetric and asymmetric receptor conformation continuum induced by a new insulin

Antagonistic Insulin Derivative Suppresses Insulin-Induced Hypoglycemia

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