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

Long-acting insulin analogues represent the most prescribed class of therapeutic proteins. An innovative design strategy was recently proposed: diselenide substitution of an external disulfide bridge. This approach exploited the distinctive physicochemical properties of selenocysteine (U). Relative to wild type (WT), Se-insulin[C7U^A, C7U^B] was reported to be protected from proteolysis by insulin-degrading enzyme (IDE), predicting prolonged activity. Because of this strategy's novelty and potential clinical importance, we sought to validate these findings and test their therapeutic utility in an animal model of diabetes mellitus. Surprisingly, the analogue did not exhibit enhanced stability, and its susceptibility to cleavage by either IDE or a canonical serine protease (glutamyl endopeptidase Glu-C) was similar to WT. Moreover, the analogue's pharmacodynamic profile in rats was not prolonged relative to a rapid-acting clinical analogue (insulin lispro). Although [C7U^A, C7U^B] does not confer protracted action, nonetheless its comparison to internal diselenide bridges promises to provide broad biophysical insight.     

One-Pot Chemical Protein Synthesis Utilizing Fmoc-Masked Selenazolidine to Address the Redox Functionality of Human Selenoprotein F**

Human SELENOF is an endoplasmic reticulum (ER) selenoprotein that contains the redox active motif CXU (C is cysteine and U is selenocysteine), resembling the redox motif of thiol-disulfide oxidoreductases (CXXC). Like other selenoproteins, the challenge in accessing SELENOF has somewhat limited its full biological characterization thus far. Here we present the one-pot chemical synthesis of the thioredoxin-like domain of SELENOF, highlighted by the use of Fmoc-protected selenazolidine, native chemical ligations and deselenization reactions. The redox potential of the CXU motif, together with insulin turbidimetric assay suggested that SELENOF may catalyze the reduction of disulfides in misfolded proteins. Furthermore, we demonstrate that SELENOF is not a protein disulfide isomerase (PDI)-like enzyme, as it did not enhance the folding of the two protein models; bovine pancreatic trypsin inhibitor and hirudin. These studies suggest that SELENOF may be responsible for reducing the non-native disulfide bonds of misfolded glycoproteins as part of the quality control system in the ER.     

Chemical Synthesis of Interleukin‐2 and Disulfide Stabilizing Analogues

Chemical protein synthesis allows the construction of well‐defined structural variations and facilitates the development of deeper understanding of protein structure–function relationships and new protein engineering strategies. Herein, we report the chemical synthesis of interleukin‐2 (IL‐2) variants on a multimilligram scale and the formation of non‐natural disulfide mimetics that improve stability against reduction. The synthesis was accomplished by convergent KAHA ligations; the acidic conditions of KAHA ligation proved to be valuable for the solubilization of the hydrophobic segments of IL‐2. The bioactivity of the synthetic IL‐2 and its analogues were shown to be equipotent to recombinant IL‐2 and exhibit improved stability against reducing agents.     

Total Chemical Synthesis of an Intra‐A‐Chain Cystathionine Human Insulin Analogue with Enhanced Thermal Stability

Despite recent advances in the treatment of diabetes mellitus, storage of insulin formulations at 4 °C is still necessary to minimize chemical degradation. This is problematic in tropical regions where reliable refrigeration is not ubiquitous. Some degradation byproducts are caused by disulfide shuffling of cystine that leads to covalently bonded oligomers. Consequently we examined the utility of the non‐reducible cystine isostere, cystathionine, within the A‐chain. Reported herein is an efficient method for forming this mimic using simple monomeric building blocks. The intra‐A‐chain cystathionine insulin analogue was obtained in good overall yield, chemically characterized and demonstrated to possess native binding affinity for the insulin receptor isoform B. It was also shown to possess significantly enhanced thermal stability indicating potential application to next‐generation insulin analogues.     

Chemoselective Derivatization of Folded Synthetic Insulin Variants with Potassium Acyltrifluoroborates (KATs)

Synthetic folded insulin variants containing an ornithine‐hydroxylamine residue are readily modified in aqueous buffers by amide‐forming ligations with potassium acyltrifluoroborates (KATs). The synthetic insulin analogs were prepared by Fmoc‐SPPS, α‐ketoacid‐hydroxylamine (KAHA) ligation, and a prosthetic C‐peptide that delivers the correct disulfide pattern and allows facile incorporation at the B0 position of Glargine M2 of a new ornithine hydroxylamine protected with a photolabile group. The folded insulin is readily modified by photo‐deprotection followed by amide‐forming KAT ligation to give insulin variants labeled with dyes, lipids, and PEGs, as well as the formation of a covalent dimer.     

Palladium in the Chemical Synthesis and Modification of Proteins

The field of site‐specific modification of proteins has drawn significant attention in recent years owing to its importance in various research areas such as the development of novel therapeutics and understanding the biochemical and cellular behaviors of proteins. The presence of a large number of reactive functional groups in the protein of interest and in the cellular environment renders modification at a specific site a highly challenging task. With the development of sophisticated chemical methodologies it is now possible to target a specific site of a protein with a desired modification, however, many challenges remain to be solved. In this context, transition metals in particular palladium‐mediated C−C bond‐forming and C−O bond‐cleavage reactions gained great interest owing to the unique catalytic properties of palladium. Palladium chemistry is being explored for protein modifications in vitro, on the cell surface, and within the cell. Very recently, palladium complexes have been applied for the rapid deprotection of several widely utilized cysteine protecting groups as well as in the removal of solubilizing tags to facilitate chemical protein synthesis. This Minireview highlights these advances and how the accumulated knowledge of palladium chemistry for small molecules is being impressively transferred to synthesis and modification of chemical proteins.     

Synthesis of PAF,an Antifungal Protein from P. chrysogenum,by Native Chemical Ligation: Native Disulfide Pattern and Fold Obtained upon Oxidative Refolding

The folding of disulfide proteins is of considerable interest because knowledge of this may influence our present understanding of protein folding. However, sometimes even the disulfide pattern cannot be unequivocally determined by the available experimental techniques. For example, the structures of a few small antifungal proteins (PAF, AFP) have been disclosed recently using NMR spectroscopy but with some ambiguity in the actual disulfide pattern. For this reason, we carried out the chemical synthesis of PAF. Probing different approaches, the oxidative folding of the synthetic linear PAF yielded a folded protein that has identical structure and antifungal activity as the native PAF. In contrast, unfolded linear PAF was inactive, a result that may have implications concerning its redox state in the mode of action.     

新型胰岛素增敏剂的合成及其生物活性评价

以川芎嗪为结构单元,与噻唑烷二酮类降糖药物的药效团组合,设计并合成了一个新的胰岛素增敏剂——2-甲氧基-5-[(2,4-二氧代-5-噻唑烷基)-甲基]-N-[(3,5,6-三甲基-2-吡嗪基)-甲基]-苯甲酰胺(Ⅰ),其结构经1H NMR和MS表征。体外活性测试结果表明,Ⅰ在用药量为1×10-5mol·L-1时对PPAR-γ的激动活性与吡格列酮相当;体内生物活性测试结果表明,Ⅰ有抑制血糖升高的倾向,有一定的血管内皮细胞保护作用。     

Preparation and Analysis of Acylated Insulin with Dehydrocholic Acid

Insulin was chemically modified with dehydrocholic acid without the use of protecting agents and the main monoacylated insulin, ε-N^BZ9-Dehydrocholyl insulin was obtained selectively and analyzed by PAGE, HPLC and MALDI-TOF-MS.     

Chemical Synthesis of Proteins with Non‐Strategically Placed Cysteines Using Selenazolidine and Selective Deselenization

Although native chemical ligation has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. The challenging protein, 125‐residue human phosphohistidine phosphatase 1 (PHPT1), has three cysteines near the C‐terminus, which are not strategically placed for ligation. Herein, we report the first sequential native chemical ligation/deselenization reaction. PHPT1 was prepared from three unprotected peptide segments using two ligation reactions at cysteine and alanine junctions. Selenazolidine was utilized as a masked precursor for N‐terminal selenocysteine in the middle segment, and, following ligation, deselenization provided the native alanine residue. This approach was used to synthesize both the wild‐type PHPT1 and an analogue in which the active‐site histidine was substituted with the unnatural and isosteric amino acid β‐thienyl‐l ‐alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site.     

Synthesis of Disulfide Surrogate Peptides Incorporating Large‐Span Surrogate Bridges Through a Native‐Chemical‐Ligation‐Assisted Diaminodiacid Strategy

The use of synthetic bridges as surrogates for disulfide bonds has emerged as a practical strategy to obviate the poor stability of some disulfide‐containing peptides. However, peptides incorporating large‐span synthetic bridges are still beyond the reach of existing methods. Herein, we report a native chemical ligation (NCL)‐assisted diaminodiacid (DADA) strategy that enables the robust generation of disulfide surrogate peptides incorporating surrogate bridges up to 50 amino acids in length. This strategy provides access to some highly desirable but otherwise impossible‐to‐obtain disulfide surrogates of bioactive peptide. The bioactivities and structures of the synthetic disulfide surrogates were verified by voltage clamp assays, NMR, and X‐ray crystallography; and stability studies established that the disulfide replacements effectively overcame the problems of disulfide reduction and scrambling that often plague these pharmacologically important peptides.     

An Amphiphilic Selenide Catalyst Behaves Like a Hybrid Mimic of Protein Disulfide Isomerase and Glutathione Peroxidase 7

Protein disulfide isomerase (PDI) and glutathione peroxidase 7 (GPx7) cooperatively promote the oxidative folding of disulfide (SS)‐containing proteins in endoplasmic reticulum by recognizing the nascent proteins to convert them into the native folds by means of SS formation and SS isomerization and by catalyzing reoxidation of reduced PDI with H₂O₂, respectively. In this study, new amphiphilic selenides with a long‐chain alkyl group were designed as hybrid mimics of PDI and GPx7 and were applied to the refolding of reduced hen egg‐white lysozyme (HEL‐R). Competitive SS formation at pH 4 using HEL‐R and glutathione (GSH) in the presence of the selenide catalyst and H₂O₂ showed that the amphiphilic selenides can preferentially catalyze SS formation of HEL‐R, probably on account of hydrophobic interactions between the protein and the catalyst. In contrast, simple water‐soluble selenides did not exhibit such behavior. In addition, when the pH of the solution was adjusted to 8.5 after the SS formation, surviving GSH promoted the SS isomerization of misfolded HEL to recover the native SS linkages. Thus, the amphiphilic selenides designed here could mimic the function of the PDI‐GPx7 system. The combination of a water‐soluble selenide and a long‐chain alkyl group would be a useful motif in designing medicines for both protein misfolding diseases and antioxidant therapy.     

Diaminodiacid Bridges to Improve Folding and Tune the Bioactivity of Disulfide‐Rich Peptides

Disulfide‐rich peptides containing three or more disulfide bonds are promising therapeutic and diagnostic agents, but their preparation is often limited by the tedious and low‐yielding folding process. We found that a single cystine‐to‐diaminodiacid replacement could significantly increase the folding efficiency of disulfide‐rich peptides and thus improve their production yields. The practicality of this strategy was demonstrated by the synthesis and folding of derivatives of the μ‐conotoxin SIIIA, the preclinical hormone hepcidin, and the trypsin inhibitor EETI‐II. NMR and X‐ray crystallography studies confirmed that these derivatives of disulfide‐rich peptide retained the correct three‐dimensional conformations. Moreover, the cystine‐to‐diaminodiacid replacement enabled structural tuning, thereby leading to an EETI‐II derivative with higher bioactivity than the native peptide.