Synthesis, and functional properties of a modified human insulin A-Chain: implication in a ‘Mini-Insulin’ structure determination

Flem, Guillaume Le; Dupradeau, François‐Yves; Pujol, Jean-Pierre; Monti, Jean‐Pierre; Bogdanowicz, Patrick

doi:10.1016/s0968-0896(02)00080-9

Cited by 4 publications

(3 citation statements)

References 37 publications

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“…It is worth noting that in previous NMR studies of a human insulin A-chain analogue (C7S, C20S), carried out in 100% DMSO, a single conformation was observed, 25 while studies carried out with a mutant human insulin in aqueous buffer reported the presence of at least two conformations. 23,24 These studies provide further support that the redox-dependent conformational states observed for the KR A1-15 peptide are intrinsic properties of the insulin A-chain.…”

Section: Resultsmentioning

confidence: 95%

Evaluating the Intrinsic Cysteine Redox-Dependent States of the A-Chain of Human Insulin Using NMR Spectroscopy, Quantum Chemical Calculations, and Mass Spectrometry

et al. 2009

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Previous functional studies have proposed that solution-phase loading of human insulin A-chain peptides into cell surface Class II molecules may be limited by the redox state of intrinsic cysteine residues within the A-chain peptide. T cell functional studies of a human insulin A-chain analogue (KR A1-15) comprised of residues 1-15 of the A-chain peptide as well as an amino-terminal lysinearginine extension have been carried out in a reducing environment. These data suggest that free thiol moieties within this peptide may participate in major histocompatibility complex (MHC) II/ peptide interactions. Two-dimensional 1 H NMR spectroscopy data partnered with quantum chemical calculations identified that KR A1-15 exists in a conformational flux sampling heterogeneous redox dependent conformations including: one reduced and two oxidized states. These findings were further supported by mass spectrometry analysis of this peptide that confirmed the presence of a redox state dependent conformational equilibrium. Interestingly, the presence of a free thiol ( 1 H γ ) resonance for cysteine 8 in the oxidized state supports the existence of the third redox dependent conformation represented as a mixed disulfide conformation. We believe these data support the presence of a redoxdependent mechanism for reg ulating the a ctivity of huma n insulin and provides a better understanding of redox chemistry that may be extended to other protein systems.

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Section: Resultsmentioning

confidence: 95%

Evaluating the Intrinsic Cysteine Redox-Dependent States of the A-Chain of Human Insulin Using NMR Spectroscopy, Quantum Chemical Calculations, and Mass Spectrometry

et al. 2009

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“…FastMoc solid phase synthesis of bis(thiol) peptides (2,5,8,11) Peptides (2, 5, 8, 11, Figure 1) were synthesized on a SASRIN resin 1 (333 mg, 0.3 mmol/g, synthesis scale ¼ 0.1 mmol) by solid phase synthesis using an Applied Biosystem 433A peptide synthesizer according to the manufacturer's instructions. 16 The following steps were performed automatically: Fmoc protective group removal was achieved using piperidine in NMP; activation of amino acids was performed with the HBTU/HOBt/DMF/DIEA mixture in NMP. Each coupling reaction was realized with 10 equiv.…”

Section: Discussionmentioning

confidence: 99%

“…2 We previously found that a synthetic A-chain derived peptide, named analog (3) was able to mimick insulin stimulation effects on cell DNA synthesis, glucose uptake, and glycogen synthesis albeit with lower efficiency than native insulin. 16 In order to determine which parts of this molecule were responsible for its biological activity, we synthesized three new analogs (truncated in C, or in N or in N and C termini parts). We then tested their in vitro effects, using cell culture and conventional assays, with native insulin as a positive control.…”

Section: Introductionmentioning

confidence: 99%

Human insulin A‐chain peptide analog(s) with in vitro biological activity

Flem¹,

Pêcher²,

Flem-Bonhomme³

et al. 2009

Cell Biochemistry & Function

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In a previous study, we showed that a synthetic human insulin 1-chain analog, named analog (3) was capable of mimicking in vitro effects of native insulin, including stimulation of cell proliferation, glucose uptake and glycogen synthesis. Here, we have synthesized three new analogs (6, 9, 12) of the human A-chain, bearing or not their N- or C-terminal residue, to determine the structural features which are responsible for their biological properties. In vitro experiments clearly demonstrated that the N-terminal part of the peptides is required for the biological activity of the molecules, suggesting its crucial role in the mechanism underlying the cellular effect. Our findings may help to better understand the mechanism of interaction between insulin and its receptor. In addition, the present data demonstrate that some mini-insulin derived from the A-chain can exert similar effects as native insulin. These small peptides may offer specific advantages over insulin in the definition of new strategies for diabetes treatment.

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4-(Tetralin-1-yl)- and 4-(Naphthalen-1-yl)alkyl Derivatives of 1-Cyclohexylpiperazine as σ Receptor Ligands with Agonist σ₂ Activity

et al. 2004

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Several 1-cyclohexylpiperazine derivatives related to sigma(2) receptor ligand 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine (33, K(i) = 0.34 nM) were synthesized and tested in radioligand binding assays, to attempt a structure-affinity relationship study. Intermediate alkyl chain length and methoxyl group position on the tetralin nucleus were varied. A few naphthalene analogues were also prepared. High affinities were found in sigma(2) receptor binding for almost all compounds, some of which displayed K(i) values in subnanomolar range, but low sigma(2)/sigma(1) selectivities were found. The highest sigma(2) affinities were displayed by compounds with an intermediate alkyl chain of three (32 and 43) or five methylenes (39 and 46). Quite high sigma(1) receptor affinity was found for compounds with a four-methylene chain; 36 (K(i) = 0.036 nM) and 45 (K(i) = 0.22 nM) displaying good sigma(1)/sigma(2) selectivity (406- and 139-fold, respectively). Moreover, homologues of compound 33 displayed also satisfactory selectivities over dopamine D(2)-like, serotonin 5-HT(3), and adrenergic alpha(1) receptors. These compounds and a few others were tested in the inhibition of the electrically evoked contractions in guinea pig bladder and were demonstrated to be full sigma(2) agonists. The activity values correlated well to the affinity scale (EC(50) in microM range). 33 and related compounds are proposed as a class of potential antineoplastic and PET diagnosis agents.

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Synthesis, and functional properties of a modified human insulin A-Chain: implication in a ‘Mini-Insulin’ structure determination

Cited by 4 publications

References 37 publications

Evaluating the Intrinsic Cysteine Redox-Dependent States of the A-Chain of Human Insulin Using NMR Spectroscopy, Quantum Chemical Calculations, and Mass Spectrometry

Evaluating the Intrinsic Cysteine Redox-Dependent States of the A-Chain of Human Insulin Using NMR Spectroscopy, Quantum Chemical Calculations, and Mass Spectrometry

Human insulin A‐chain peptide analog(s) with in vitro biological activity

4-(Tetralin-1-yl)- and 4-(Naphthalen-1-yl)alkyl Derivatives of 1-Cyclohexylpiperazine as σ Receptor Ligands with Agonist σ₂ Activity

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Synthesis, and functional properties of a modified human insulin A-Chain: implication in a ‘Mini-Insulin’ structure determination

Cited by 4 publications

References 37 publications

Evaluating the Intrinsic Cysteine Redox-Dependent States of the A-Chain of Human Insulin Using NMR Spectroscopy, Quantum Chemical Calculations, and Mass Spectrometry

Evaluating the Intrinsic Cysteine Redox-Dependent States of the A-Chain of Human Insulin Using NMR Spectroscopy, Quantum Chemical Calculations, and Mass Spectrometry

Human insulin A‐chain peptide analog(s) with in vitro biological activity

4-(Tetralin-1-yl)- and 4-(Naphthalen-1-yl)alkyl Derivatives of 1-Cyclohexylpiperazine as σ Receptor Ligands with Agonist σ2 Activity

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4-(Tetralin-1-yl)- and 4-(Naphthalen-1-yl)alkyl Derivatives of 1-Cyclohexylpiperazine as σ Receptor Ligands with Agonist σ₂ Activity