Biophysical Optimization of a Therapeutic Protein by Nonstandard Mutagenesis

Pandyarajan, Vijay; Phillips, Nelson B.; Cox, Gabriela P.; Yang, Yanwu; Whittaker, Jonathan; Ismail‐Beigi, Faramarz; Weiss, Michael A.

doi:10.1074/jbc.m114.588277

Cited by 27 publications

(46 citation statements)

References 78 publications

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“…All of the variants caused similar reductions in blood glucose upon subcutaneous injection into diabetic mice (Fig. 1D) 2, 20–22 . RAIs cannot be distinguished from ProI in rodent models 23 .…”

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confidence: 72%

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4S-Hydroxylation of Insulin at ProB28 Accelerates Hexamer Dissociation and Delays Fibrillation

et al. 2017

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Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of non-canonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. Crystal structures of dimeric and hexameric insulin preparations suggest that a hydrogen bond between the hydroxyl group of Hzp and a backbone amide carbonyl positioned across the dimer interface may be responsible for the altered behavior. The effects of hydroxylation are stereospecific; replacement of ProB28 by (4R)-hydroxyproline (Hyp) causes little change in the rates of fibrillation and hexamer disassociation. These results demonstrate a new approach that fuses the concepts of medicinal chemistry and protein design, and paves the way to further engineering of insulin and other therapeutic proteins.

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confidence: 72%

“…Glucose levels were measured post-injection via tail vein sampling. ProI, AspI, HzpI, HypI or vector were formulated as described 22 . Error bars denote one standard deviation (n = 3).…”

Section: Figurementioning

confidence: 99%

4S-Hydroxylation of Insulin at ProB28 Accelerates Hexamer Dissociation and Delays Fibrillation

et al. 2017

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“…Indeed, substitution of Tyr B26 by 3-I-Tyr in the rapid-acting analog insulin lispro enhances its stability and resistance to fibrillation while maintaining its biological activity (45). The present crystal structure has demonstrated how the modified side chain pivots to enable burial of the iodine atom in the hydrophobic core.…”

Section: Discussionmentioning

confidence: 72%

“…2C). Given these features, the present study focused on the effects of iodination of Tyr B26 , long known to enhance the affinity of insulin for the IR (7)(8)(9) and recently shown to enhance the pharmaceutical properties of a rapid-acting clinical analog, including its stability and resistance to physical degradation (45). 12 Such findings raise salient questions regarding the role of the iodo-aromatic modification on the structure of the free hormone and its potential role at the hormone-receptor interface.…”

Section: -[Iodo-tyrmentioning

confidence: 99%

“…Potential structural differences between isoforms IR-A and IR-B (110) are also not well understood. 12 Biophysical effects of the iodo-Tyr B26 modification were recently studied in the context of insulin lispro (containing substitutions Pro B28 3 Lys and Lys B29 3 Pro; the active component of Humalogா (Lilly) (45). Although the structure of the modified analog was not determined, iodo-Tyr B26 was found to mitigate the loss of stability associated with the paired B28 -B29 substitutions.…”

Section: Rigid-body Modeling Distinguished Opposite Edges Of the B26 mentioning

confidence: 99%

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Extending Halogen-based Medicinal Chemistry to Proteins

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Pandyarajan²,

Phillips³

et al. 2016

Journal of Biological Chemistry

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Edited by Norma AllewellInsulin, a protein critical for metabolic homeostasis, provides a classical model for protein design with application to human health. Recent efforts to improve its pharmaceutical formulation demonstrated that iodination of a conserved tyrosine (Tyr B26 ) enhances key properties of a rapid-acting clinical analog. Moreover, the broad utility of halogens in medicinal chemistry has motivated the use of hybrid quantum-and molecularmechanical methods to study proteins. Here, we (i) undertook quantitative atomistic simulations of 3-[iodo-Tyr B26 ]insulin to predict its structural features, and (ii) tested these predictions by X-ray crystallography. Using an electrostatic model of the modified aromatic ring based on quantum chemistry, the calculations suggested that the analog, as a dimer and hexamer, exhibits subtle differences in aromatic-aromatic interactions at the dimer interface. Aromatic rings (Tyr B16 , Phe B24 , Phe B25 , 3-ITyr B26 , and their symmetry-related mates) at this interface adjust to enable packing of the hydrophobic iodine atoms within the core of each monomer. Strikingly, these features were observed in the crystal structure of a 3-[iodo-Tyr B26 ]insulin analog (determined as an R 6 zinc hexamer). Given that residues B24 -B30 detach from the core on receptor binding, the environment of 3-I-Tyr B26 in a receptor complex must differ from that in the free hormone. Based on the recent structure of a "micro-receptor" complex, we predict that 3-I-Tyr B26 engages the receptor via directional halogen bonding and halogen-directed hydrogen bonding as follows: favorable electrostatic interactions exploiting, respectively, the halogen's electron-deficient -hole and electronegative equatorial band. Inspired by quantum chemistry and molecular dynamics, such "halogen engineering" promises to extend principles of medicinal chemistry to proteins.Insulin, a small protein critical to metabolic homeostasis (1), provides a model for studies of protein folding and design (2) with long-standing application to human therapeutics (3). The hormone contains two chains, A and B (Fig. 1A), linked by two disulfide bridges (cystines A7-B7 and A20 -B19); the A chain is further stabilized by cystine A6 -A11. In pancreatic ␤-cells, insulin is stored within the secretory granules as zinc-coordinated hexamers. This study has exploited insulin semi-synthesis (4) (simplified through the use of norleucine (Nle) 9 at position B29; arrow in Fig. 1A (5)) to investigate a site-specific modification of an aromatic ring by a single halogen atom (6). The modification, 3-iodo-Tyr at position B26 (3-I-Tyr B26 ), is associated with enhanced binding to the insulin receptor (IR)

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Strategies for Interference of Insulin Fibrillogenesis: Challenges and Advances

et al. 2022

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The discovery of insulin came with very high hopes for diabetic patients. In 2021, the world celebrated the 100th anniversary of the discovery of this vital hormone. However, external use of insulin is highly affected by its aggregating tendency that occurs during its manufacturing, transportation, and improper handling which ultimately leads to its pharmaceutically and biologically ineffective form. In this review, we aim to discuss the various approaches used for decelerating insulin aggregation which results in the enhancement of its overall structural stability and usage. The approaches that are discussed are broadly classified as either a measure through excipient additions or by intrinsic modifications in the insulin native structure.

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Biophysical Optimization of a Therapeutic Protein by Nonstandard Mutagenesis

Cited by 27 publications

References 78 publications

4S-Hydroxylation of Insulin at ProB28 Accelerates Hexamer Dissociation and Delays Fibrillation

4S-Hydroxylation of Insulin at ProB28 Accelerates Hexamer Dissociation and Delays Fibrillation

Extending Halogen-based Medicinal Chemistry to Proteins

Strategies for Interference of Insulin Fibrillogenesis: Challenges and Advances

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