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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.

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., Graphical abstract A new method of synthesis of insulin derivatives through sortase A (SrtA) ligation was reported. The insulin substrate (Ins-SA) can be site-selectively modified using sortase A in high efficiency.Image 1

The 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 non-opioid analgesic agents. We demonstrate that replacing disulfide loop I [Cys(I)-Cys(III)] with methylene thioacetal in the RgIA skeleton results in activity loss whereas substitution of loop II [Cys(II)-Cys(IV)] can be accommodated. The lead molecule, RgIA-5524, exhibits highly selective inhibition of α9α10 nAChRs with an IC(50) 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 disulfide surrogate in conotoxin-based, disulfide-rich peptide drugs.

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.

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

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.

Cucurbit[7]uril (CB[7]) is a supramolecular binding host for peptides and proteins with N-terminal Phe. However, the low occurrence of such peptides and proteins limits broader applications of this unique host-guest interaction. Here, we report a strategy to expand the scope of CB[7]-peptide interaction by site-specifically introducing N-terminal substitutions (e.g. benzyl groups) using reductive alkylation. N-terminal benzylated peptides have similar affinity to CB[7] as native peptides with N-terminal Phe and even stronger interactions can be achieved using better ligands. We further expanded this host-guest interaction to be stimuli responsive. By introducing benzyl carboxylate substituents, the CB[7]-peptide interaction shows pH-dependent binding. Furthermore, benzyl boronate substituents led to saccharide-dependent CB[7]-peptide interactions. We demonstrated that using this strategy to introduce stronger CB[7] binders to the N-terminus of human calcitonin (hCT) results in increased aggregation stability in the presence of CB[7]. This strategy to expand CB[7]-peptide interaction scope opens opportunities for future applications in peptides and proteins.

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.

The B-chain C-terminal region of insulin has been mutated or modified to achieve improved therapeutic efficacies. For ex-ample, all FDA-approved insulin analogs have altered C-terminal segments, which leads to improved pharmacokinetic prop-erties and provide significant clinical benefits on blood sugar regulation. Nonetheless, there is still no efficient method to synthesize insulin analogs with the altered C-terminal region. Herein, we report a facile synthesis using omniligase-1 to li-gate an insulin core with a peptide segment in high conversion. We further apply this ligation to M13 phage surface modifi-cations and demonstrate that the phage displayed insulin molecules can bind to insulin receptor ectodomain in an insulin-dependent manner. These results pave the way for building phage display insulin library for therapeutic selections and demonstrate the feasibility of using omniligase-1 to display other disulfide-rich peptides and proteins on phage.

The introduction of solid-phase peptide synthesis in the 1960’s 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.

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 a fully active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy and protein engineering to elucidate its interactions with the human insulin receptor ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of native insulin but also compromise therapeutic utility by delaying the onset action, suggesting approaches to develop improved therapeutic insulins. Curiously, a receptor conformation present in low abundance adopts a highly asymmetric structure that displays novel coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.

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. [Figure not available: see fulltext.]

Kim and colleagues show that OCA-B in T cells is essential for the generation of type 1 diabetes. OCA-B loss leaves the pancreatic lymph nodes largely undisturbed but associates autoreactive CD4+ T cells in the pancreas with anergy while deleting potentially autoreactive CD8+ T cells., The transcriptional coregulator OCA-B promotes expression of T cell target genes in cases of repeated antigen exposure, a necessary feature of autoimmunity. We hypothesized that T cell–specific OCA-B deletion and pharmacologic OCA-B inhibition would protect mice from autoimmune diabetes. We developed an Ocab conditional allele and backcrossed it onto a diabetes-prone NOD/ShiLtJ strain background. T cell–specific OCA-B loss protected mice from spontaneous disease. Protection was associated with large reductions in islet CD8+ T cell receptor specificities associated with diabetes pathogenesis. CD4+ clones associated with diabetes were present but associated with anergic phenotypes. The protective effect of OCA-B loss was recapitulated using autoantigen-specific NY8.3 mice but diminished in monoclonal models specific to artificial or neoantigens. Rationally designed membrane-penetrating OCA-B peptide inhibitors normalized glucose levels and reduced T cell infiltration and proinflammatory cytokine expression in newly diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition., Graphical Abstract

The transcriptional coregulator OCA-B promotes expression of T cell target genes in cases of repeated antigen exposure, a necessary feature of autoimmunity. We hypothesized that T cell-specific OCA-B deletion and pharmacologic OCA-B inhibition would protect mice from autoimmune diabetes. We developed an Ocab conditional allele and backcrossed it onto a diabetes-prone NOD/ShiLtJ strain background. T cell-specific OCA-B loss protected mice from spontaneous disease. Protection was associated with large reductions in islet CD8+ T cell receptor specificities associated with diabetes pathogenesis. CD4+ clones associated with diabetes were present, but associated with anergic phenotypes. The protective effect of OCA-B loss was recapitulated using autoantigen-specific NY8.3 mice, but diminished in monoclonal models specific to artificial or neoantigens. Rationally-designed membrane-penetrating OCA-B peptide inhibitors normalized glucose levels, and reduced T cell infiltration and proinflammatory cytokine expression in newly-diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition.~40-word summary statement for the online JEM table of contents and alertsKim and colleagues show that OCA-B in T cells is essential for the generation of type-1 diabetes. OCA-B loss leaves the pancreatic lymph nodes largely undisturbed, but associates autoreactive CD4+ T cells in the pancreas with anergy while deleting potentially autoreactive CD8+ T cells.SummaryKim et al. show that loss or inhibition of OCA-B in T cells protects mice from type-1 diabetes.

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 novel direction for therapeutic insulin development.

We report an efficient, highly selective modification on the N-terminal amines of peptides and proteins using aldehyde derivatives via reductive alkylation. After modification of a library of unprotected peptides XYSKEASAL (X varies over 20 natural amino acids) by benzaldehyde at room temperature, pH 6.1 resulted in excellent N-terminal selectivity (α-amino/e-amino: >99 : 1) and high reaction conversion for 19 out of the 20 peptides. Under similar conditions, highly selective N-terminal modifications were achieved with a variety of aldehydes. Furthermore, N-termini of native peptides and proteins could be selectively modified under the same conditions to introduce bioorthogonal functional groups. Using human insulin as an example, we further demonstrated that preserving the positive charge in the N-terminus using reductive alkylation instead of acylation leads to a 5-fold increase in bioactivity. In summary, our reported method provides a universal strategy for site-selective N-terminal functionalization in native peptides and proteins.

A novel four-disulfide insulin analog was designed with retained bioactivity and increased fibrillation stability., 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.

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.

The fish-hunting marine cone snail Conus geographus uses a specialized venom insulin to induce hypoglycemic shock in its prey. We recently showed that this venom insulin, Con-Ins G1, has unique characteristics relevant to the design of new insulin therapeutics. Here, we show that fish-hunting cone snails provide a rich source of minimized ligands of the vertebrate insulin receptor. Insulins from C. geographus, Conus tulipa and Conus kinoshitai exhibit diverse sequences, yet all bind to and activate the human insulin receptor. Molecular dynamics reveal unique modes of action that are distinct from any other insulins known in nature. When tested in zebrafish and mice, venom insulins significantly lower blood glucose in the streptozotocin-induced model of diabetes. Our findings suggest that cone snails have evolved diverse strategies to activate the vertebrate insulin receptor and provide unique insight into the design of novel drugs for the treatment of diabetes.

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.

Insulins in the venom of certain fish-hunting cone snails facilitate prey capture by rapidly inducing hypoglycemic shock. One such insulin, Conus geographus G1 (Con-Ins G1), is the smallest known insulin found in nature and lacks the C-terminal segment of the B chain that, in human insulin, mediates engagement of the insulin receptor and assembly of the hormone's hexameric storage form. Removal of this segment (residues B23-B30) in human insulin results in substantial loss of receptor affinity. Here, we found that Con-Ins G1 is monomeric, strongly binds the human insulin receptor and activates receptor signaling. Con-Ins G1 thus is a naturally occurring B-chain-minimized mimetic of human insulin. Our crystal structure of Con-Ins G1 reveals a tertiary structure highly similar to that of human insulin and indicates how Con-Ins G1's lack of an equivalent to the key receptor-engaging residue PheB24 is mitigated. These findings may facilitate efforts to design ultrarapid-acting therapeutic insulins.

Since its discovery and isolation, exogenous insulin has dramatically changed the outlook for patients with diabetes. However, even when patients strictly follow an insulin regimen, serious complications can result as patients experience both hyperglycemic and hypoglycemic states. Several chemically or genetically modified insulins have been developed that tune the pharmacokinetics of insulin activity for personalized therapy. Here, we demonstrate a strategy for the chemical modification of insulin intended to promote both long-lasting and glucose-responsive activity through the incorporation of an aliphatic domain to facilitate hydrophobic interactions, as well as a phenylboronic acid for glucose sensing. These synthetic insulin derivatives enable rapid reversal of blood glucose in a diabetic mouse model following glucose challenge, with some derivatives responding to repeated glucose challenges over a 13-h period. The best-performing insulin derivative provides glucose control that is superior to native insulin, with responsiveness to glucose challenge improved over a clinically used long-acting insulin derivative. Moreover, continuous glucose monitoring reveals responsiveness matching that of a healthy pancreas. This synthetic approach to insulin modification could afford both long-term and glucose-mediated insulin activity, thereby reducing the number of administrations and improving the fidelity of glycemic control for insulin therapy. The described work is to our knowledge the first demonstration of a glucose-binding modified insulin molecule with glucose-responsive activity verified in vivo.

The application of thiol-yne/thiol-ene reactions to synthesize mono- and bicyclic-stapled peptides and proteins is reported. First, a thiol-ene-based peptide-stapling method in aqueous conditions was developed. This method enabled the efficient stapling of recombinantly expressed coil-coiled proteins. The resulting stapled protein demonstrated higher stability in its secondary structure than the unstapled version. Furthermore, a thiol-yne coupling was performed by using an α,ω-diyne to react with two cysteine residues to synthesize a stapled peptide with two vinyl sulfide groups. The stapled peptide could further react with another biscysteine peptide to yield a bicyclic stapled peptide with enhanced properties. For example, the cell permeability of a stapled peptide was further increased by appending an oligoarginine cell-penetrating peptide. The robustness and versatility of thiol-yne/thiol-ene reactions that can be applied to both synthetic and expressed peptides and proteins were demonstrated.

Cytokine-induced beta-cell apoptosis is important to the etiology of type-1 diabetes. Although previous reports have shown that general inhibitors of histone deacetylase (HDAC) activity, such as suberoylanilide hydroxamic acid and trichostatin A, can partially prevent beta-cell death, they do not fully restore beta-cell function. To understand HDAC isoform selectivity in beta cells, we measured the cellular effects of eleven structurally diverse HDAC inhibitors on cytokine-induced apoptosis in the rat INS-1E cell line. All eleven compounds restored ATP levels and reduced nitrite secretion. However, caspase-3 activity was reduced only by MS-275 and CI-994, both of which target HDAC1, 2, and 3. Importantly, both MS-275 and genetic knock-down of Hdac3 alone were sufficient to restore glucose-stimulated insulin secretion in the presence of cytokines. These results suggest that HDAC3-selective inhibitors may be effective in preventing cytokine-induced beta-cell apoptosis.

The synthesis of a stereochemically diverse library of medium-sized rings accessible via a 'build/couple/pair' strategy is described. Key aspects of the synthesis include S(N)Ar cycloetherification of a linear amine template to afford eight stereoisomeric 8-membered lactams and subsequent solid-phase diversification of these scaffolds to yield a 6488-membered library. Screening of this compound collection in a cell-based assay for the suppression of cytokine-induced beta-cell apoptosis resulted in the identification of a small-molecule suppressor capable of restoring glucose-stimulated insulin secretion in a rat beta-cell line. The presence of all stereoisomers in the screening collection enabled preliminary determination of the structural and stereochemical requirements for cellular activity, while efficient follow-up chemistry afforded BRD-0476 (probe ML187), which had an approximately three-fold increase in activity. These results demonstrate the utility of diversity-oriented synthesis to probe discovery using cell-based screening, and the importance of including stereochemical diversity in screening collections for the development of stereo/structure-activity relationships.

Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic β-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote β-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat β cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation.

We report the discovery of a peptide stapling and macrocyclization method using thiol-ene reactions between two cysteine residues and an α,ω-diene in high yields. This new approach enabled us to selectively modify cysteine residues in native, unprotected peptides with a variety of stapling modifications for helix stabilization or general macrocyclization. We synthesized stapled Axin mimetic analogues and demonstrated increased alpha helicity upon peptide stapling. We then synthesized stapled p53 mimetic analogues using pure hydrocarbon linkers and demonstrated their abilities to block the p53-MDM2 interaction and selectively kill p53 wild-type colorectal carcinoma HCT-116 cells but not p53 null cells. In summary, we demonstrated a robust and versatile peptide stapling method that could be potentially applied to both synthetic and expressed peptides.

Pancreatic beta-cell apoptosis is a critical event during the development of type-1 diabetes. The identification of small molecules capable of preventing cytokine-induced apoptosis could lead to avenues for therapeutic intervention. We developed a set of phenotypic cell-based assays designed to identify such small-molecule suppressors. Rat INS-1E cells were simultaneously treated with a cocktail of inflammatory cytokines and a collection of 2,240 diverse small molecules and screened using an assay for cellular ATP levels. Forty-nine top-scoring compounds included glucocorticoids, several pyrazole derivatives, and known inhibitors of glycogen synthase kinase-3beta. Two compounds were able to increase cellular ATP levels, reduce caspase-3 activity and nitrite production, and increase glucose-stimulated insulin secretion in the presence of cytokines. These results indicate that small molecules identified by this screening approach may protect beta cells from autoimmune attack and may be good candidates for therapeutic intervention in early stages of type-1 diabetes.