Semisynthesis of Aminomethyl-Insulin: An Atom-Economic Strategy to Increase the Affinity and Selectivity of a Protein for Recognition by a Synthetic Receptor.

Advancements in the molecular recognition of insulin by nonantibody-based means would facilitate the development of methodology for the continuous detection of insulin for the management of diabetes mellitus. Herein, we report a novel insulin derivative that binds to the synthetic receptor cucurbit[7]uril (Q7) at a single site and with high nanomolar affinity. The insulin derivative was prepared by a four-step protein semisynthetic method to present a 4-aminomethyl group on the side chain of the PheB1 position. The resulting aminomethyl insulin binds to Q7 with an equilibrium dissociation constant value of 99 nM in neutral phosphate buffer, as determined by isothermal titration calorimetry. This 6.8-fold enhancement in affinity versus native insulin was gained by an atom-economical modification (-CH2NH2). To the best of our knowledge, this is the highest reported binding affinity for an insulin derivative by a synthetic receptor. This strategy for engineering protein affinity tags induces minimal change to the protein structure while increasing affinity and selectivity for a synthetic receptor.

Synthesis and Identification of Biologically Active Mono-Labelled FITC-Insulin Conjugate.

Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery. Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesised conjugate identified using reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA. Syntheses were carried out at varying reaction times between 4 and 20 h. Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18 h and confirmed by electrospray mass spectroscopy. Reactions were studied across a pH range of 7-9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2 h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels. MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells. No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin. Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications.

Availability, affordability and price components of insulin products in different-level hospital pharmacies: Evidence from two cross-sectional surveys in Nanjing, China.

The essential medicine--insulin cannot be easily accessed and afforded in many countries. To help address this issue, we evaluated the availability, affordability and price of insulin products in Nanjing, eastern China. Two cross-sectional studies were conducted in 2016 and 2018. A total of 56 hospital pharmacies were sampled, using a simplified and adapted World Health Organization/Health Action International (WHO/HAI) methodology. Prices were expressed as Median Price Ratios (MPRs) to Australian Pharmaceutical Benefit Scheme (PBS) prices. In addition, we investigated the price components of seven selected insulin products as a case study before and after the Online Centralized Procurement Policy for Hospital Drugs in May, 2018. Affordability was presented as the number of daily wages of the lowest paid unskilled government worker (LPGW) required to purchase 1000IU of insulin based on the average courses of treatment, approximately 30 days' treatment. The availability of insulin products was very high in secondary hospitals and tertiary hospitals both in 2016 and 2018, but in community hospitals was very low. In 2018, the availability of prandial insulin products showed fluctuation compared to 2016. The availability of pre-mixed human insulin products was over 95% overall, and also very high (80%) in community hospitals in 2018. The prices of insulin products were much lower than PBS prices of Australian in this study, with the MPRs less than 1 (0.32 to 0.71 in 2016 vs. 0.30 to 0.68 in 2018) for all insulin types. But insulin products in Nanjing in 2016 and 2018 were considered unaffordable, because the number of daily wages of the LPGW needed to purchase for the 30 days treatment of insulin products ranged from 2.26 to 8.49 in 2016 and 1.88 to 7.09 in 2018. The manufacturers' selling price contributed the main part (74.15% to 77.70% before and 74.86% to 91.51% after the implementation of the bidding policy) of the price components of target insulin brands. The availability of insulin products was high in secondary hospitals and tertiary hospitals, but lower in community hospitals. However, the affordability in community hospitals was better than other hospitals, but the insulin products were still unaffordable for patients on low incomes. Further improvements of the availability accessibility and affordability of medicines in advancing health insurance policies and lowering drug prices should be put forward.

The Chemical Synthesis of Insulin: An Enduring Challenge.

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.

[Studies on Functional Molecules Based on Peptide Chemistry].

Studies on functional molecules starting from syntheses of cysteine-containing peptides and protein are described. Starting from evaluation of a cysteine specific side-reaction, a specific reaction for disulfide-bond formation was developed. The reaction made it possible to independently construct a disulfide bridge without effecting the existing disulfide bonds, which resulted in a unique approach for the synthesis of human insulin by site-specific disulfide bond formation. In a series of studies on sulfur-containing amino acids, another cysteine related un-natural amino acid, α-methyl cysteine, was used for the total syntheses of natural products containing a unique thiazorine/thiazole ring system. Chloroimidazolidium coupling reagent developed by us was effective for the successive couplings of the α-methyl cysteine residues. Based on these synthetic studies, design and evaluation of protease inhibitors were then studied, since a stereo-specific synthesis of the key structure is crucial to make the inhibitor an effective functional molecule in the interactions with its target protease. As the target proteases, ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) and chymotrypsin-like protease of severe acute respiratory syndrome (SARS 3CL protease) were selected: the former is a crucial enzyme for amyloid ß production and the latter is an essential enzyme for the re-construction of SARS corona virus in host cells. Structure optimization procedure of the respective inhibitors are described based on X-ray crystal structure analyses of the inhibitor-protease complex.

Nanoparticles integrating natural and synthetic polymers for in vivo insulin delivery.

The aims of the current study were to develop insulin-loaded nanoparticles comprised of various polymers at different compositions, and to evaluate their ability to lower blood glucose levels in diabetic rats following subcutaneous and oral administrations. Several combinations of natural and synthetic polymers have been utilized for preparation of nanoparticles including, chitosan, alginate, albumin and Pluronic. Nanosized (170 nm-800 nm) spherical particles of high encapsulation efficiency (15-52%) have been prepared. Composition and ratios between the integrated polymers played a pivotal role in determining size, zeta potential, and in vivo hypoglycemic activity of particles. After subcutaneous and oral administration in diabetic rats, some of the insulin-loaded nanoparticles were able to induce much higher hypoglycemic effect as compared to the unloaded free insulin. For instance, subcutaneous injection of nanoparticles comprised of chitosan combined with sodium tripolyphosphate, Pluronic or alginate/calcium chloride, resulted in comparable hypoglycemic effects to free insulin, at two-fold lower dose. Nanoparticles were well-tolerated after oral administration in rats, as evidenced by by measuring levels of alanine aminotransferase, aspartate aminotransferases, albumin, creatinine and urea. This study indicates that characteristics and delivery efficiency of nanomaterials can be controlled via utilizing several natural/synthetic polymers and by fine-tuning of combination ratio between polymers.

Functionality of receptor targeted zinc-insulin quantum clusters in skin tissue augmentation and bioimaging.

Quantum clusters with target specificity are suitable for tissue-specific imaging. In the present work, amorphous zinc insulin quantum clusters (IZnQCs) had been synthesised to promote and monitor wound recovery. Easy synthesis, biocompatibility, stability, enhanced quantum yield, and solubility made the cluster suitable for preclinical/clinical exploration. Zn2+ is known for its binding to insulin hexamer. Here we report the reformation of the structure in a quantum cluster form in the presence of Zn2+. The formation of IZnQCs was confirmed by the change in zeta potential from -25.6 mV to -17.9 mV and also the formation of protein metal interaction was confirmed in FTIR bands at 450, 480, and 613 cm-1 for Zn-O, Zn-N, and Zn-S, respectively. HRTEM-EDS and SAED data analysis showed an amorphous nature of the cluster. The binding of IZnQCs to the cells has been confirmed using confocal microscopy. IZnQCs showed a synergistic effect in wound recovery than insulin or Zn2+ alone. Further due to high fluorescence this recovery process can be monitored under an appropriate setup. Wound healing promotional activity, target specificity, and fluorescence properties make the IZnQCs ideal to use for bioimaging along with promoting and monitoring of wound recovery agent.

Multipodal insulin mimetics built on adamantane or proline scaffolds.

Multi-orthogonal molecular scaffolds can be applied as core structures of bioactive compounds. Here, we prepared four tri-orthogonal scaffolds based on adamantane or proline skeletons. The scaffolds were used for the solid-phase synthesis of model insulin mimetics bearing two different peptides on the scaffolds. We found that adamantane-derived compounds bind to the insulin receptor more effectively (Kd value of 0.5 µM) than proline-derived compounds (Kd values of 15-38 µM) bearing the same peptides. Molecular dynamics simulations suggest that spacers between peptides and central scaffolds can provide greater flexibility that can contribute to increased binding affinity. Molecular modeling showed possible binding modes of mimetics to the insulin receptor. Our data show that the structure of the central scaffold and flexibility of attached peptides in this type of compound are important and that different scaffolds should be considered when designing peptide hormone mimetics.

Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly.

Ensembles of protein aggregates are characterized by a nano- and micro-scale heterogeneity of the species. This diversity translates into a variety of effects that protein aggregates may have in biological systems, both in connection to neurodegenerative diseases and immunogenic risk of protein drug products. Moreover, this naturally occurring variety offers unique opportunities in the field of protein-based biomaterials. In the above-mentioned fields, the isolation and structural analysis of the different amyloid types within the same ensemble remain a priority, still representing a significant experimental challenge. Here we address such complexity in the case of insulin for its relevance as biopharmaceutical and its involvement in insulin-derived amyloidosis. By combining Fourier Transform Infrared Microscopy (micro-FTIR) and fluorescence lifetime imaging microscopy (FLIM) we show the occurrence, within the same ensemble of insulin protein aggregates, of a variable ß-structure architecture and content not only dependent on the species analyzed (spherulites or fibrils), but also on the position within a single spherulite at submicron scale. We unambiguously reveal that the surface of the spherulites are characterized by ß-structures with an enhanced H-bond coupling compared to the core. This information, inaccessible via bulk methods, allows us to relate the aggregate structure at molecular level to the overall morphology of the aggregates. Our findings robustly solve the problem of probing the ensemble and single particle heterogeneity of amyloid samples. Furthermore, we offer a unique, scalable and ready-to-use screening methodology for in-depth characterization of self-assembled structures, being this translatable to material sciences, drug quality control and clinical imaging of amyloid-affected tissues.

Role of Proinsulin Self-Association in Mutant INS Gene-Induced Diabetes of Youth.

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin (PI) in the endoplasmic reticulum (ER) drive the molecular pathogenesis of mutant INS gene-induced diabetes of youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, PI-WT dimerizes in the ER. Here, we suggest that the normal PI-PI contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that PI B-chain tyrosine-16 (Tyr-B16), which is a key residue in normal PI dimerization, helps confer dominant-negative behavior of MIDY mutant PI-C(A7)Y. Substitutions of Tyr-B16 with either Ala, Asp, or Pro in PI-C(A7)Y decrease the abnormal interactions between the MIDY mutant and PI-WT, rescuing PI-WT export, limiting ER stress, and increasing insulin production in ß-cells and human islets. This study reveals the first evidence indicating that noncovalent PI-PI contact initiates dominant-negative behavior of misfolded PI, pointing to a novel therapeutic target to enhance PI-WT export and increase insulin production.

Glucose-Responsive Insulin Through Bioconjugation Approaches.

Although insulin analogs have markedly improved glycemic control for people with diabetes, glycemic excursions still cause major health problems and complications. In particular, the narrow therapeutic window of current insulin therapy makes it extremely difficult to maintain normoglycemia without risking severe hypoglycemia. Currently, there are no FDA-approved insulin therapeutics whose bioactivity is regulated by blood glucose levels. This review discusses recent progress on developing glucose-responsive insulin (GRI) bioconjugates without the need of exogenous matrices. Through this approach, tremendous efforts have been made over the years to demonstrate the promise of better glycemic control and reduced risk of hypoglycemia. Last, we discuss future directions of GRI development with a goal to maximize the glucose responsiveness.

Total Chemical Synthesis of a Nonfibrillating Human Glycoinsulin.

Glycosylation is an accepted strategy to improve the therapeutic value of peptide and protein drugs. Insulin and its analogues are life-saving drugs for all type I and 30% of type II diabetic patients. However, they can readily form fibrils which is a significant problem especially for their use in insulin pumps. Because of the solubilizing and hydration effects of sugars, it was thought that glycosylation of insulin could inhibit fibril formation and lead to a more stable formulation. Since enzymatic glycosylation results in heterogeneous products, we developed a novel chemical strategy to produce a homogeneous glycoinsulin (disialo-glycoinsulin) in excellent yield (∼60%). It showed a near-native binding affinity for insulin receptors A and B in vitro and high glucose-lowering effects in vivo, irrespective of the route of administration (s.c. vs i.p.). The glycoinsulin retained insulin-like helical structure and exhibited improved stability in human serum. Importantly, our disialo-glycoinsulin analogue does not form fibrils at both high concentration and temperature. Therefore, it is an excellent candidate for clinical use in insulin pumps.

Mutations at hypothetical binding site 2 in insulin and insulin-like growth factors 1 and 2 result in receptor- and hormone-specific responses.

Information on how insulin and insulin-like growth factors 1 and 2 (IGF-1 and -2) activate insulin receptors (IR-A and -B) and the IGF-1 receptor (IGF-1R) is crucial for understanding the difference in the biological activities of these peptide hormones. Cryo-EM studies have revealed that insulin uses its binding sites 1 and 2 to interact with IR-A and have identified several critical residues in binding site 2. However, mutagenesis studies suggest that Ile-A10, Ser-A12, Leu-A13, and Glu-A17 also belong to insulin's site 2. Here, to resolve this discrepancy, we mutated these insulin residues and the equivalent residues in IGFs. Our findings revealed that equivalent mutations in the hormones can result in differential biological effects and that these effects can be receptor-specific. We noted that the insulin positions A10 and A17 are important for its binding to IR-A and IR-B and IGF-1R and that A13 is important only for IR-A and IR-B binding. The IGF-1/IGF-2 positions 51/50 and 54/53 did not appear to play critical roles in receptor binding, but mutations at IGF-1 position 58 and IGF-2 position 57 affected the binding. We propose that IGF-1 Glu-58 interacts with IGF-1R Arg-704 and belongs to IGF-1 site 1, a finding supported by the NMR structure of the less active Asp-58-IGF-1 variant. Computational analyses indicated that the aforementioned mutations can affect internal insulin dynamics and inhibit adoption of a receptor-bound conformation, important for binding to receptor site 1. We provide a molecular model and alternative hypotheses for how the mutated insulin residues affect activity.

A Disulfide Scan of Insulin by [3 + 1] Methodology Exhibits Site-Specific Influence on Bioactivity.

Insulin is the principal hormone involved in the regulation of metabolism and has served a seminal role in the treatment of diabetes. Building upon advances in insulin synthetic methodology, we have developed a straightforward route to novel insulins containing a fourth disulfide bond in a [3 + 1] fashion establishing the first disulfide scan of the hormone. All the targeted analogs accommodated the constraint to demonstrate an unexpected conformational flexibility of native insulin. The bioactivity was established for the constrained (4-DS) and unconstrained (3-DS) analogs by in vitro methods, and extended to in vivo study for select peptides. We also identified residue B10 as a preferred anchor to introduce a tether that would regulate insulin bioactivity. We believe that the described [3 + 1] methodology might constitute the preferred approach for performing similar disulfide scanning in peptides that contain multiple disulfides.

Synthesis of hydrophobic insulin-based peptides using a helping hand strategy.

The introduction of solid-phase peptide synthesis in the 1960s improved the chemical synthesis of both the A- and B-chains of insulin and insulin analogs. However, the subsequent elaboration of the synthetic peptides to generate active hormones continues to be difficult and complex due in part to the hydrophobicity of the A-chain. Over the past decade, several groups have developed different methods to enhance A-chain solubility. Two of the most popular methods are use of isoacyl dipeptides, and the attachment of an A-chain C-terminal pentalysine tag with a base-labile 4-hydroxymethylbenzoic acid linker. These methods have proven effective but can be limited in scope depending on the peptide sequence of a specific insulin. Herein we describe an auxiliary approach to enhance the solubility of insulin-based peptides by incorporating a tri-lysine tag attached to a cleavable Fmoc-Ddae-OH linker. Incorporation of this linker, or "helping hand", on the N-terminus greatly improved the solubility of chicken insulin A-chain, which is analogous to human insulin, and allowed for coupling of the insulin A- and B-chain via directed disulfide bond formation. After formation of the insulin heterodimer, the linker and tag could be easily removed using a hydrazine buffer (pH 7.5) to obtain an overall 12.6% yield based on A-chain. This strategy offers an efficient method to enhance the solubility of hydrophobic insulin-based peptides as well as other traditionally difficult peptides.

Formulary Considerations for Insulins Approved Through the 505(b)(2) "Follow-on" Pathway.

OBJECTIVE: To summarize formulary-relevant issues for follow-on insulins approved through the Food and Drug Administration (FDA) 505(b)(2) approval pathway (Basaglar and Admelog). DATA SOURCES: A search of the MEDLINE database was performed for articles pertaining to clinical and formulary considerations for follow-on insulin products through July 2018. STUDY SELECTION AND DATA EXTRACTION: All clinical trials used in the 505(b)(2) approval process for follow-on insulin glargine and insulin lispro products were included and summarized. DATA SYNTHESIS: Follow-on insulin glargine and insulin lispro products have been recently approved as the first lower-cost alternatives to innovator insulin products. The follow-on insulins were approved via the 505(b)(2) pathway, making them neither generics nor biosimilars. Current data do not suggest any clinically relevant differences between the follow-on insulins and their respective innovator products. Clinicians should be aware that follow-on insulins will be reclassified as biologic products in the year 2020. Relevance to Patient Care and Clinical Practice: This article provides information about currently available follow-on insulin products that were approved through the 505(b)(2) pathway, including product characteristics and efficacy and safety data. These products will likely be considered for both clinical use and formulary placement because of their potentially lower cost compared with innovator products. CONCLUSIONS: Follow-on insulin products approved through the 505(b)(2) pathway are supported by robust efficacy and safety data. As new follow-on insulins are approved and the regulatory change that will occur with these products in 2020 approaches, formulary decisions and clinical policies (eg, substitution) will continue to be revisited.

Structurally Constrained Insulin Analogs by Directed Stepwise Crosslinking.

BACKGROUND: Research has been directed at the optimization of insulin for medicinal purposes. An insulin analog that could be reversibly activated might provide more precise pharmacokinetic control and broaden the inherent therapeutic index of the hormone. The prospect of using intramolecular structural constraint to reversibly inactive insulin might constitute the first step to achieving such an optimized analog. Chemically crosslinked insulin analogs have been reported where two amines are covalently linked by reaction with symmetrical bifunctional active esters. There is little selectivity in this synthetic approach to molecular constraint with multiple derivatives being formed. OBJECTIVE: To systematically evaluate the synthesis of covalently crosslinked insulin analogs by asymmetric methods and the biological consequences. METHOD: We report synthesis of amine crosslinked insulin analogs via a two-step procedure. The stepwise approach was initiated by amide bond formation and followed by second site alkylation to produce site-specific, cross-linked insulin analogs. RESULTS: A set of unique insulin analogs crosslinked at the two of the three native amines were synthesized. They were chemical characterized and assessed by in vitro bioanalysis to result in a significant and reasonably consistent reduction in biological potency. CONCLUSION: We achieved an unambiguous two-step synthesis of several crosslinked insulin analogs differing in location of the chemical tether. Bioanalysis demonstrated the ability of the molecular constraint to reduce bioactivity. The results set the stage for in vivo assessment of whether such a reduction in potency can be used pharmacologically to establish a constrained hormone upon which reversible tethering might be subsequently introduced.

High-Yield Synthesis of Human Insulin-Like Peptide 5 Employing a Nonconventional Strategy.

A simplified route to synthesis of INSL5 is reported, where the elimination of intermediate purification steps and nonconventional disulfide pairing results in final yields that are an order of magnitude higher than in previously reported stepwise syntheses. The intramolecular disulfide of A-chain was produced by a thiol displacement of StBu-protected cysteine, and was followed by an A-B chain disulfide formation in dimethylsulfoxide (DMSO). The final disulfide was formed by deprotection of StBu-cysteines in hydrofluoric acid (HF) at room temperature, which is a historical approach infrequently employed today, followed by oxidation using 2,2-dithiobis(5-nitropyridine) (DTNP) in acidic aqueous buffer. Throughout the synthesis, an isoacyl surrogate to a midsequence native amide bond was utilized to enhance solubility of the intermediate compounds.

Lessons for modern insulin development.

There have been many advances in insulin with a realistic possibility of mimicking nature to improve insulin replacement, with a view to achieving improved metabolic control. Lessons can be learnt from the evolution of insulin, insulin development, and new advances in technology. This may lead to fewer side effects of therapy resulting in a lower risk of hypoglycaemia and less weight gain, which could in turn could reduce long-term complications for people with diabetes.