A cysteine-specific solubilizing tag strategy enables efficient chemical protein synthesis of difficult targets.

We developed a new cysteine-specific solubilizing tag strategy via a cysteine-conjugated succinimide. This solubilizing tag remains stable under common native chemical ligation conditions and can be efficiently removed with palladium-based catalysts. Utilizing this approach, we synthesized two proteins containing notably difficult peptide segments: interleukin-2 (IL-2) and insulin. This IL-2 chemical synthesis represents the simplest and most efficient approach to date, which is enabled by the cysteine-specific solubilizing tag to synthesize and ligate long peptide segments. Additionally, we synthesized a T8P insulin variant, previously identified in an infant with neonatal diabetes. We show that T8P insulin exhibits reduced bioactivity (a 30-fold decrease compared to standard insulin), potentially contributing to the onset of diabetes in these patients. In summary, our work provides an efficient tool to synthesize challenging proteins and opens new avenues for exploring research directions in understanding their biological functions.

Omniligase-1-Mediated Phage-Peptide Library Modification and Insulin Engineering.

Chemical and enzymatic modifications of peptide-displayed libraries have been successfully employed to expand the phage display library. However, the requirement of specific epitopes and scaffolds has limited the scope of protein engineering using phage display. In this study, we present a novel approach utilizing omniligase-1-mediated selective and specific ligation on the phage pIII protein, offering a high conversion rate and compatibility with commercially available phage libraries. We applied this method to perform high-throughput engineering of insulin analogues with randomized B chain C-terminal regions. Insulin analogues with different B chain C-terminal segments were selected and exhibited biological activity equivalent to that of human insulin. Molecular dynamics studies of insulin analogues revealed a novel interaction between the insulin B27 residue and insulin receptor L1 domain. In summary, our findings highlight the potential of omniligase-1-mediated phage display in the development and screening of disulfide-rich peptides and proteins. This approach holds promise for the creation of novel insulin analogues with enhanced therapeutic properties and exhibits potential for the development of other therapeutic compounds.

Serine-mediated hydrazone ligation displaying insulin-like peptides on M13 phage pIII.

Phage display has emerged as a tool for the discovery of therapeutic antibodies and proteins. However, the effective display and engineering of structurally complex proteins, such as insulin, pose significant challenges due to the sequence of insulin, which is composed of two peptide chains linked by three disulfide bonds. In this study, we developed a new approach for the display of insulin-like peptides on M13 phage pIII, employing N-terminal serine-mediated hydrazone ligation. The insulin-displaying phage retains the biological binding affinity of human insulin. To address the viability loss after ligation, we introduced a trypsin-cleavable spacer on pIII, enabling insulin-displayed phage library selection. This method offers a general pathway for the display of structurally complex proteins on pIII, enhancing the practicality of selecting chemically modified phage libraries and opening avenues for the engineering of new insulin analogs for the treatment of diabetes by using phage display.

Supramolecular approaches for insulin stabilization without prolonged duration of action.

Aggregation represents a significant challenge for the long-term formulation stability of insulin therapeutics. The supramolecular PEGylation of insulin with conjugates of cucurbit[7]uril and polyethylene glycol (CB[7]‒PEG) has been shown to stabilize insulin formulations by reducing aggregation propensity. Yet prolonged in vivo duration of action, arising from sustained complex formation in the subcutaneous depot, limits the application scope for meal-time insulin uses and could increase hypoglycemic risk several hours after a meal. Supramolecular affinity of CB[7] in binding the B1-Phe residue on insulin is central to supramolecular PEGylation using this approach. Accordingly, here we synthesized N-terminal acid-modified insulin analogs to reduce CB[7] interaction affinity at physiological pH and reduce the duration of action by decreasing the subcutaneous depot effect of the formulation. These insulin analogs show weak to no interaction with CB[7]‒PEG at physiological pH but demonstrate high formulation stability at reduced pH. Accordingly, N-terminal modified analogs have in vitro and in vivo bioactivity comparable to native insulin. Furthermore, in a rat model of diabetes, the acid-modified insulin formulated with CB[7]‒PEG offers a reduced duration of action compared to native insulin formulated with CB[7]‒PEG. This work extends the application of supramolecular PEGylation of insulin to achieve enhanced stability while reducing the risks arising from a subcutaneous depot effect prolonging in vivo duration of action.

Antagonistic Insulin Derivative Suppresses Insulin-Induced Hypoglycemia.

Insulin derivatives provide new functions that are distinctive from native insulin. We investigated insulin modifications on the C-terminal A chain with insulin receptor (IR) peptide binders and presented a full and potent IR antagonist. We prepared insulin precursors featuring a sortase A (SrtA) recognition sequence, LPETGG, at the C-terminal A chain and used a SrtA-mediated ligation method to synthesize insulin derivatives. The insulin precursor exhibits full IR agonism potency, similar to native human insulin. We explored derivatives with linear IR binding peptides attached to the insulin C-terminal A chain. One insulin derivative with an IR binder (Ins-AC-S2) can fully antagonize IR activation by insulin, as confirmed by cell-based assays. This IR antagonist suppresses insulin-induced hypoglycemia in a streptozotocin-induced diabetic rat model. This study provides a new direction toward insulin antagonist development.

Supramolecular Protein Stabilization with Zwitterionic Polypeptide-Cucurbit[7]uril Conjugates.

Protein aggregation is an obstacle for the development of new biopharmaceuticals, presenting challenges in shipping and storage of vital therapies. Though a variety of materials and methods have been explored, the need remains for a simple material that is biodegradable, nontoxic, and highly efficient at stabilizing protein therapeutics. In this work, we investigated zwitterionic polypeptides prepared using a rapid and scalable polymerization technique and conjugated to a supramolecular macrocycle host, cucurbit[7]uril, for the ability to inhibit aggregation of model protein therapeutics insulin and calcitonin. The polypeptides are based on the natural amino acid methionine, and zwitterion sulfonium modifications were compared to analogous cationic and neutral structures. Each polymer was end-modified with a single cucurbit[7]uril macrocycle to afford supramolecular recognition and binding to terminal aromatic amino acids on proteins. Only conjugates prepared from zwitterionic structures of sufficient chain lengths were efficient inhibitors of insulin aggregation and could also inhibit aggregation of calcitonin. This polypeptide exhibited no cytotoxicity in human cells even at concentrations that were five-fold of the intended therapeutic regime. We explored treatment of the zwitterionic polypeptides with a panel of natural proteases and found steady biodegradation as expected, supporting eventual clearance when used as a protein formulation additive.

Modifying insulin to improve performance.

A platform to enable precise modifications of insulin could improve drug functionality.

Improved Handling of Peptide Segments Using Side Chain-Based "Helping Hand" Solubilizing Tools.

Maintaining high, or even sufficient, solubility of every peptide segment in chemical protein synthesis (CPS) remains a critical challenge; insolubility of just a single peptide segment can thwart a total synthesis venture. Multiple approaches have been used to address this challenge, most commonly by employing a chemical tool to temporarily improve peptide solubility. In this chapter, we discuss chemical tools for introducing semipermanent solubilizing sequences (termed helping hands) at the side chains of Lys and Glu residues. We describe the synthesis, incorporation by Fmoc-SPPS, and cleavage conditions for utilizing these two tools. For Lys sites, we discuss the Fmoc-Ddap-OH dimedone-based linker, which is achiral, synthesized in one step, can be introduced directly at primary amines, and is removed using hydroxylamine (or hydrazine). For Glu sites, we detail the new Fmoc-SPPS building block, Fmoc-Glu(AlHx)-OH, which can be prepared in an efficient process over two purifications. Solubilizing sequences are introduced directly on-resin and later cleaved with palladium-catalyzed transfer under aqueous conditions to restore a native Glu side chain. These two chemical tools are straightforward to prepare and implement, and we anticipate continued usage in "difficult" peptide segments following the protocols described herein.

Unconventional insulins from predators and pathogens.

Insulin and its related peptides are found throughout the animal kingdom, in which they serve diverse functions. This includes regulation of glucose homeostasis, neuronal development and cognition. The surprising recent discovery that venomous snails evolved specialized insulins to capture fish demonstrated the nefarious use of this hormone in nature. Because of their streamlined role in predation, these repurposed insulins exhibit unique characteristics that have unraveled new aspects of the chemical ecology and structural biology of this important hormone. Recently, insulins were also reported in other venomous predators and pathogenic viruses, demonstrating the broader use of insulin by one organism to manipulate the physiology of another. In this Review, we provide an overview of the discovery and biomedical application of repurposed insulins and other hormones found in nature and highlight several unique insights gained from these unusual compounds.

Synthesis and Characterization of Phenylboronic Acid-Modified Insulin With Glucose-Dependent Solubility.

Glucose-responsive insulin represents a promising approach to regulate blood glucose levels. We previously showed that attaching two fluorophenylboronic acid (FPBA) residues to the C-terminal B chain of insulin glargine led to glucose-dependent solubility. Herein, we demonstrated that relocating FPBA from B chain to A chain increased the baseline solubility without affecting its potency. Furthermore, increasing the number of FPBA groups led to increased glucose-dependent solubility.

Symmetric and asymmetric receptor conformation continuum induced by a new insulin.

Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes and biochemical properties. Here, we report an active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy (cryo-EM) and protein engineering to elucidate its interactions with the human insulin receptor (IR) ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of human insulin but also compromise therapeutic utility by delaying dissolution from the site of subcutaneous injection. This finding suggests approaches to developing improved therapeutic insulins. Curiously, the receptor displays a continuum of conformations from the symmetric state to a highly asymmetric low-abundance structure that displays coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.

Facile synthesis of insulin fusion derivatives through sortase A ligation.

Insulin derivatives such as insulin detemir and insulin degludec are U.S. Food and Drug Administration (FDA)-approved long-acting insulin currently used by millions of people with diabetes. These derivatives are modified in C-terminal B29 lysine to retain insulin bioactivity. New and efficient methods for facile synthesis of insulin derivatives may lead to new discovery of therapeutic insulin. Herein, we report a new method using sortase A (SrtA)-mediated ligation for the synthesis of insulin derivatives with high efficiency and functional group tolerance in the C-terminal B chain. This new insulin molecule (Ins-SA) with an SrtA-recognizing motif can be conjugated to diverse groups with N-terminal oligoglycines to generate new insulin derivatives. We further demonstrated that a new insulin derivative synthesized by this SrtA-mediated ligation shows strong cellular and in vivo bioactivity. This enzymatic method can therefore be used for future insulin design and development.

Discovery of Methylene Thioacetal-Incorporated α-RgIA Analogues as Potent and Stable Antagonists of the Human α9α10 Nicotinic Acetylcholine Receptor for the Treatment of Neuropathic Pain.

α9-Containing nicotinic acetylcholine receptors (nAChRs) are key targets for the treatment of neuropathic pain. α-Conotoxin RgIA4 is a peptide antagonist of human α9α10 nAChRs with high selectivity. However, structural rearrangement reveals a potential liability for clinical applications. We herein report our designer RgIA analogues stabilized by methylene thioacetal as nonopioid analgesic agents. We demonstrate that replacing disulfide loop I [CysI-CysIII] with methylene thioacetal in the RgIA skeleton results in activity loss, whereas substitution of loop II [CysII-CysIV] can be accommodated. The lead molecule, RgIA-5524, exhibits highly selective inhibition of α9α10 nAChRs with an IC50 of 0.9 nM and much reduced degradation in human serum. In vivo studies showed that RgIA-5524 relieves chemotherapy-induced neuropathic pain in wild type but not α9 knockout mouse models, demonstrating that α9-containing nAChRs are necessary for the therapeutic effects. This work highlights the application of methylene thioacetal as a disulfide surrogate in conotoxin-based, disulfide-rich peptide drugs.

Author Correction: A structurally minimized yet fully active insulin based on cone-snail venom insulin principles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development of Conformationally Constrained α-RgIA Analogues as Stable Peptide Antagonists of Human α9α10 Nicotinic Acetylcholine Receptors.

Non-opioid therapeutics for the treatment of neuropathic pain are urgently needed to address the ongoing opioid crisis. Peptides from cone snail venoms have served as invaluable molecules to target key pain-related receptors but can suffer from unfavorable physicochemical properties, which limit their therapeutic potential. In this work, we developed conformationally constrained α-RgIA analogues with high potency, receptor selectivity, and enhanced human serum stability to target the human α9α10 nicotinic acetylcholine receptor. The key lactam linkage introduced in α-RgIA fixed the favored globular conformation and suppressed disulfide scrambling. The NMR structure of the macrocyclic peptide overlays well with that of α-RgIA4, demonstrating that the cyclization does not perturb the overall conformation of backbone and key side-chain residues. Finally, a molecular docking model was used to rationalize the selective binding between a macrocyclic analogue and the α9α10 nicotinic acetylcholine receptor. These conformationally constrained antagonists are therefore promising candidates for antinociceptive therapeutic intervention.

A structurally minimized yet fully active insulin based on cone-snail venom insulin principles.

Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone-snail venom with moderate human insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins-a human des-octapeptide insulin analog-as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24-PheB25-TyrB26 aromatic triplet and opens a new direction for therapeutic insulin development.

Novel four-disulfide insulin analog with high aggregation stability and potency.

Although insulin was first purified and used therapeutically almost a century ago, there is still a need to improve therapeutic efficacy and patient convenience. A key challenge is the requirement for refrigeration to avoid inactivation of insulin by aggregation/fibrillation. Here, in an effort to mitigate this problem, we introduced a 4th disulfide bond between a C-terminal extended insulin A chain and residues near the C-terminus of the B chain. Insulin activity was retained by an analog with an additional disulfide bond between residues A22 and B22, while other linkages tested resulted in much reduced potency. Furthermore, the A22-B22 analog maintains the native insulin tertiary structure as demonstrated by X-ray crystal structure determination. We further demonstrate that this four-disulfide analog has similar in vivo potency in mice compared to native insulin and demonstrates higher aggregation stability. In conclusion, we have discovered a novel four-disulfide insulin analog with high aggregation stability and potency.

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.

Synthesis and Characterization of an A6-A11 Methylene Thioacetal Human Insulin Analogue with Enhanced Stability.

Insulin has been a life-saving drug for millions of people with diabetes. However, several challenges exist which limit therapeutic benefits and reduce patient convenience. One key challenge is the fibrillation propensity, which necessitates refrigeration for storage. To address this limitation, we chemically synthesized and evaluated a methylene thioacetal human insulin analogue (SCS-Ins). The synthesized SCS-Ins showed enhanced serum stability and aggregation resistance while retaining bioactivity compared with native insulin.

Display of Single-Chain Insulin-like Peptides on a Yeast Surface.

Insulin and insulin-like peptides play a pivotal role in a wide variety of cellular and physiological events, including energy storage, proliferation, aging, and differentiation. Variants of insulin and insulin-like peptides may therefore be probes for studying the insulin signaling pathway and therapeutic candidates for treating metabolic diseases. Here, we report a method for genetically displaying single-chain insulin-like peptides on the surface of Saccharomyces cerevisiae strain DY1632. Using a previously reported single-chain insulin analogue, SCI-57, as a model, we demonstrate that nearly 70% of yeast binds to insulin receptor (IR), suggesting that SCI-57 is folded correctly and maintains its IR binding property. Furthermore, the interaction between displayed SCI-57 and IR can be weakened using increasing concentrations of native insulin as a soluble competitor, suggesting that the interaction is insulin-dependent. We further applied this methodology to three other single-chain insulin analogues with various lengths and confirmed their interactions with IR. In summary, we successfully displayed a number of insulin-like peptides on a yeast surface and demonstrated insulin-dependent interactions with IR. This method may, therefore, be used for construction of libraries of insulin-like peptides to select for chemical probes or therapeutic molecules.