Reassessment of an Innovative Insulin Analogue Excludes Protracted Action yet Highlights the Distinction between External and Internal Diselenide Bridges

Dhayalan, Balamurugan; Chen, Yen‐Shan; Phillips, Nelson B.; Swain, Mamuni; Rege, Nischay; Mirsalehi, Ali; Jarosinski, Mark A.; Ismail‐Beigi, Faramarz; Metanis, Norman; Weiss, Michael A.

doi:10.1002/chem.202000309

Cited by 13 publications

(12 citation statements)

References 49 publications

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“…We found that the substitution of native/non-native disulfide with diselenide bonds typically enhances protein folding and yield, preserves protein structure and often confers no significant effect on function. Based on these results, the current study of the selenohirudin analogues extends the previous studies on selenocysteinecontaining analogues of BPTI 34,35 , conotoxins [28][29][30][31] and insulin [53][54][55] , which together demonstrate the high potential of disulfide-todiselenide substitutions in basic and applied research. In addition to its obvious advantage in the study and preparation of disulfiderich proteins, such an approach may also prove useful for a wide range of applications, including the rational improvement of the stability, activity and specificity of this unique and medically important family of proteins.…”

Section: Discussionsupporting

confidence: 75%

Diselenide crosslinks for enhanced and simplified oxidative protein folding

et al. 2021

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The in vitro oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and is viewed as a folding model for a wide range of disulfide-rich proteins. Hirudin’s folding pathway is notorious for its highly heterogeneous intermediates and scrambled isomers, limiting its folding rate and yield in vitro. Aiming to overcome these limitations, we undertake systematic investigation of diselenide bridges at native and non-native positions and investigate their effect on hirudin’s folding, structure and activity. Our studies demonstrate that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the corresponding hirudin analogues, while reducing the complexity and heterogeneity of the process. Moreover, crystal structure analysis confirms that the diselenide substitutions maintained the overall three-dimensional structure of the protein and left its function virtually unchanged. The choice of hirudin as a study model has implications beyond its specific folding mechanism, demonstrating the high potential of diselenide substitutions in the design, preparation and characterization of disulfide-rich proteins.

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Section: Discussionsupporting

confidence: 75%

Diselenide crosslinks for enhanced and simplified oxidative protein folding

et al. 2021

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“…4 We found that the substitution of native/non-native disulfide with diselenide bonds typically enhances protein folding and yield, preserves protein structure, and often confers no significant effect on function. Based on these results, the current study of the seleno-hirudin analogs extends the previous studies on selenocysteine-containing analogs of BPTI, 33,34 conotoxins, [27][28][29][30] and insulin, [52][53][54] which together demonstrate the high potential of disulfide-to-diselenide substitutions in basic and applied research. In addition to its obvious advantage in the study and preparation of disulfide-rich proteins, such an approach may also prove useful for a wide range of applications, including the rational improvement of the stability, activity and specificity of this unique family of proteins.…”

Section: Discussionsupporting

confidence: 74%

Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

Mousa

Hidmi

Pomyalov

et al. 2020

Preprint

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The oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and is viewed as a folding model for a wide range of disulfide-rich proteins. Hirudin’s folding pathway is notorious for its highly heterogeneous intermediates and scrambled isomers, which plague its folding rate and yield in vitro. Aiming to overcome these limitations, we undertook a systematic investigation of diselenide bridges at native and non-native positions and investigated their effect on hirudin’s folding, structure and activity. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the corresponding hirudin analogs, while reducing the complexity and heterogeneity of the process. Moreover, crystal structure analysis confirmed that the diselenide substitutions maintained the overall structure of the protein and left the function virtually unchanged. The choice of hirudin as a study model has implications beyond its specific folding mechanism, demonstrating the high potential of diselenide substitutions in the design, preparation and characterization of disulfide-rich proteins.

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“…The degradation of A7–B7 diselenide insulin with insulin-degrading enzyme was reported to be significantly increased and thus the lifetime enhanced. However, a more recent evaluation indicated that the diselenide insulin analogue, in fact, did not have increased stability . This requires further examination and clarification.…”

Section: Chemically Diverse Analoguesmentioning

confidence: 97%

“…However, a more recent evaluation indicated that the diselenide insulin analogue, in fact, did not have increased stability. 175 This requires further examination and clarification. An intra-A-chain diselenide variant of human insulin, A6− A11 diselenide insulin, was also prepared by Metanis and colleagues (Scheme 26).…”

Section: Diselenide Insulinsmentioning

confidence: 99%

The Chemical Synthesis of Insulin: An Enduring Challenge

2021

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The pancreatic peptide hormone insulin, first discovered exactly 100 years ago, is essential for glycemic control and is used as a therapeutic for the treatment of type 1 and, increasingly, type 2 diabetes. With a worsening global diabetes epidemic and its significant health budget imposition, there is a great demand for new analogues possessing improved physical and functional properties. However, the chemical synthesis of insulin’s intricate 51-amino acid, two-chain, three-disulfide bond structure, together with the poor physicochemical properties of both the individual chains and the hormone itself, has long represented a major challenge to organic chemists. This review provides a timely overview of the past efforts to chemically assemble this fascinating hormone using an array of strategies to enable both correct folding of the two chains and selective formation of disulfide bonds. These methods not only have contributed to general peptide synthesis chemistry and enabled access to the greatly growing numbers of insulin-like and cystine-rich peptides but also, today, enable the production of insulin at the synthetic efficiency levels of recombinant DNA expression methods. They have led to the production of a myriad of novel analogues with optimized structural and functional features and of the feasibility for their industrial manufacture.

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Reassessment of an Innovative Insulin Analogue Excludes Protracted Action yet Highlights the Distinction between External and Internal Diselenide Bridges

Cited by 13 publications

References 49 publications

Diselenide crosslinks for enhanced and simplified oxidative protein folding

Diselenide crosslinks for enhanced and simplified oxidative protein folding

Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

The Chemical Synthesis of Insulin: An Enduring Challenge

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