Modulation of the antagonistic properties of an insulin mimetic peptide by disulfide bridge modifications.

Insulin is a peptide responsible for regulating the metabolic homeostasis of the organism; it elicits its effects through binding to the transmembrane insulin receptor (IR). Insulin mimetics with agonistic or antagonistic effects toward the receptor are an exciting field of research and could find applications in treating diabetes or malignant diseases. We prepared five variants of a previously reported 20-amino acid insulin-mimicking peptide. These peptides differ from each other by the structure of the covalent bridge connecting positions 11 and 18. In addition to the peptide with a disulfide bridge, a derivative with a dicarba bridge and three derivatives with a 1,2,3-triazole differing from each other by the presence of sulfur or oxygen in their staples were prepared. The strongest binding to IR was exhibited by the peptide with a disulfide bridge. All other derivatives only weakly bound to IR, and a relationship between increasing bridge length and lower binding affinity can be inferred. Despite their nanomolar affinities, none of the prepared peptide mimetics was able to activate the insulin receptor even at high concentrations, but all mimetics were able to inhibit insulin-induced receptor activation. However, the receptor remained approximately 30% active even at the highest concentration of the agents; thus, the agents behave as partial antagonists. An interesting observation is that these mimetic peptides do not antagonize insulin action in proportion to their binding affinities. The compounds characterized in this study show that it is possible to modulate the functional properties of insulin receptor peptide ligands using disulfide mimetics.

Functional stapled fragments of human preptin of minimised length.

Preptin is a 34-amino-acid-long peptide derived from the E-domain of a precursor of insulin-like growth factor 2 (pro-IGF2) with bone-anabolic and insulin secretion amplifying properties. Here, we describe the synthesis, structures, and biological activities of six shortened analogues of human preptin. Eight- and nine-amino-acid-long peptide amides corresponding to the C-terminal part of human preptin were stabilised by two types of staples to induce a higher proportion of helicity in their secondary structure. We monitored the secondary structure of the stapled peptides using circular dichroism. The biological effect of the structural changes was determined afterwards by the ability of peptides to stimulate the release of intracellular calcium ions. We confirmed the previous observation that the stabilisation of the disordered conformation of human preptin has a deleterious effect on biological potency. However, surprisingly, one of our preptin analogues, a nonapeptide stabilised by olefin metathesis between positions 3 and 7 of the amino acid chain, had a similar ability to stimulate calcium ions' release to the full-length human preptin. Our findings could open up new ways to design new preptin analogues, which may have potential as drugs for the treatment of diabetes and osteoporosis.

A versatile insulin analog with high potency for both insulin and insulin-like growth factor 1 receptors: Structural implications for receptor binding.

Insulin and insulin-like growth factor 1 (IGF-1) are closely related hormones involved in the regulation of metabolism and growth. They elicit their functions through activation of tyrosine kinase-type receptors: insulin receptors (IR-A and IR-B) and IGF-1 receptor (IGF-1R). Despite similarity in primary and three-dimensional structures, insulin and IGF-1 bind the noncognate receptor with substantially reduced affinity. We prepared [d-HisB24, GlyB31, TyrB32]-insulin, which binds all three receptors with high affinity (251 or 338% binding affinity to IR-A respectively to IR-B relative to insulin and 12.4% binding affinity to IGF-1R relative to IGF-1). We prepared other modified insulins with the aim of explaining the versatility of [d-HisB24, GlyB31, TyrB32]-insulin. Through structural, activity, and kinetic studies of these insulin analogs, we concluded that the ability of [d-HisB24, GlyB31, TyrB32]-insulin to stimulate all three receptors is provided by structural changes caused by a reversed chirality at the B24 combined with the extension of the C terminus of the B chain by two extra residues. We assume that the structural changes allow the directing of the B chain C terminus to some extra interactions with the receptors. These unusual interactions lead to a decrease of dissociation rate from the IR and conversely enable easier association with IGF-1R. All of the structural changes were made at the hormones' Site 1, which is thought to interact with the Site 1 of the receptors. The results of the study suggest that merely modifications of Site 1 of the hormone are sufficient to change the receptor specificity of insulin.

Evaluation of silica from different vendors as the solid support of anion-exchange chiral stationary phases by means of preferential sorption and liquid chromatography.

Chromatographic performance of a chiral stationary phase is significantly influenced by the employed solid support. Properties of the most commonly used support, silica particles, such as size and size distribution, and pore size are of utmost importance for both superficially porous particles and fully porous particles. In this work, we have focused on evaluation of fully porous particles from three different vendors as solid supports for a brush-type chiral stationary phase based on 9-O-tert-butylcarbamoyl quinidine. We have prepared corresponding stationary phases under identical experimental conditions and determined the parameters of the modified silica by physisorption measurements and scanning electron microscopy. Enantiorecognition properties of the chiral stationary phases have been studied using preferential sorption experiments. The same material was slurry-packed into chromatographic columns and the chromatographic properties have been evaluated in liquid chromatography. We show that preferential sorption can provide valuable information about the influence of the pore size and total pore volume on the interaction of analytes of different size with the chirally-modified silica surface. The data can be used to understand differences observed in chromatographic evaluation of the chiral stationary phases. The combination of preferential sorption and liquid chromatography separation can provide detailed information on new chiral stationary phases.

Effect of different immobilization strategies on chiral recognition properties of Cinchona-based anion exchangers.

In the enantiomeric separation of highly polar compounds, a traditionally challenging task for high-performance liquid chromatography, ion-exchange chiral stationary phases have found the main field of application. In this contribution, we present a series of novel anion-exchange-type chiral stationary phases for enantiomer separation of protected amino phosphonates and N-protected amino acids. Two of the prepared selectors possessed a double and triple bond within a single molecule. Thus, they were immobilized onto silica support employing either a thiol-ene (radical) or an azide-yne (copper(I)-catalyzed) click reaction. We evaluated the selectivity and the effect of immobilization proceeding either by the double bond of the Cinchona alkaloid or a triple bond of the carbamoyl moiety on the chromatographic performance of the chiral stationary phases using analytes with protecting groups of different size, flexibility, and π-acidity. The previously observed preference toward protecting groups possessing π-acidic units, which is a typical feature of Cinchona-based chiral stationary phases, was preserved. In addition, increasing the bulkiness of the selectors' carbamoyl units leads to significantly reduced retention times, while very high selectivity toward the tested analytes is retained.

Optimized syntheses of Fmoc azido amino acids for the preparation of azidopeptides.

The rise of CuI-catalyzed click chemistry has initiated an increased demand for azido and alkyne derivatives of amino acid as precursors for the synthesis of clicked peptides. However, the use of azido and alkyne amino acids in peptide chemistry is complicated by their high cost. For this reason, we investigated the possibility of the in-house preparation of a set of five Fmoc azido amino acids: ß-azido l-alanine and d-alanine, γ-azido l-homoalanine, δ-azido l-ornithine and ω-azido l-lysine. We investigated several reaction pathways described in the literature, suggested several improvements and proposed several alternative routes for the synthesis of these compounds in high purity. Here, we demonstrate that multigram quantities of these Fmoc azido amino acids can be prepared within a week or two and at user-friendly costs. We also incorporated these azido amino acids into several model tripeptides, and we observed the formation of a new elimination product of the azido moiety upon conditions of prolonged couplings with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/DIPEA. We hope that our detailed synthetic protocols will inspire some peptide chemists to prepare these Fmoc azido acids in their laboratories and will assist them in avoiding the too extensive costs of azidopeptide syntheses. Experimental procedures and/or analytical data for compounds 3-5, 20, 25, 26, 30 and 43-47 are provided in the supporting information. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

A CuAAC-Hydrazone-CuAAC Trifunctional Scaffold for the Solid-Phase Synthesis of Trimodal Compounds: Possibilities and Limitations.

We present a trifunctional scaffold designed for the solid-phase synthesis of trimodal compounds. This scaffold holds two alkyne arms in a free and TIPS-protected form for consecutive CuAAC (copper(I)-catalyzed azide-alkyne cycloaddition), one Fmoc-protected hydrazide arm for reaction with aldehydes, and one carboxylic acid arm with CF2 groups for attachment to the resin and (19)F-NMR quantification. This scaffold was attached to a resin and derivatized with model azides and aliphatic, electron-rich or electron-poor aromatic aldehydes. We identified several limitations of the scaffold caused by the instability of hydrazones in acidic conditions, in the presence of copper during CuAAC, and when copper accumulated in the resin. We successfully overcame these drawbacks by optimizing synthetic conditions for the derivatization of the scaffold with aromatic aldehydes. Overall, the new trifunctional scaffold combines CuAAC and hydrazone chemistries, offering a broader chemical space for the development of bioactive compounds.

Mono-N-acyl-2,6-diaminopimelic acid derivatives: analysis by electromigration and spectroscopic methods and examination of enzyme inhibitory activity.

Thirteen mono-N-acyl derivatives of 2,6-diaminopimelic acid (DAP)-new potential inhibitors of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18)-were analyzed and characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and two capillary electromigration methods: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Structural features of DAP derivatives were characterized by IR and NMR spectroscopies, whereas CZE and MEKC were applied to evaluate their purity and to investigate their electromigration properties. Effective electrophoretic mobilities of these compounds were determined by CZE in acidic and alkaline background electrolytes (BGEs) and by MEKC in acidic and alkaline BGEs containing a pseudostationary phase of anionic detergent sodium dodecyl sulfate (SDS) or cationic detergent cetyltrimethylammonium bromide (CTAB). The best separation of DAP derivatives, including diastereomers of some of them, was achieved by MEKC in an acidic BGE (500 mM acetic acid [pH 2.54] and 60mM SDS). All DAP derivatives were examined for their ability to inhibit catalytic activity of DapE from Haemophilus influenzae (HiDapE) and ArgE from Escherichia coli (EcArgE). None of these DAP derivatives worked as an effective inhibitor of HiDapE, but one derivative-N-fumaryl, Me-ester-DAP-was found to be a moderate inhibitor of EcArgE, thereby providing a promising lead structure for further studies on ArgE inhibitors.

Combinatorial Libraries of Bipodal Binders of the Insulin Receptor.

The binding process of insulin to its transmembrane receptor entails a sophisticated interplay between two proteins, each possessing two binding sites. Given the difficulties associated with the use of insulin in the treatment of diabetes, despite its remarkable efficacy, there is interest in smaller and more stable compounds than the native hormone that would effectively activate the receptor. Our study adopts a strategy focused on synthesizing extensive combinatorial libraries of bipodal compounds consisting of two distinct peptides linked to a molecular scaffold. These constructs, evaluated in a resin bead-bound format, were designed to assess their binding to the insulin receptor. Despite notable nonspecific binding, our approach successfully generated and tested millions of compounds. Rigorous evaluations via flow cytometry and specific antibodies revealed peptide sequences with specific interactions at either receptor binding Site 1 or 2. Notably, these sequences bear similarity to peptides discovered through phage display by other researchers. This convergence of chemical and biological methods underscores nature's beauty, revealing general principles in peptide binding to the insulin receptor. Overall, our study deepens the understanding of molecular interactions in ligand binding to the insulin receptor, highlighting the challenges of targeting large proteins with small synthetic peptides.

Unusual activity pattern of leucine aminopeptidase inhibitors based on phosphorus containing derivatives of methionine and norleucine.

Ligands containing bulky aliphatic P1 residues exhibit a high affinity towards cytosolic leucine aminopeptidase, a bizinc protease of biomedical significance. According to this specificity, a series of phosphonic and phosphinic compounds have been put forward as novel putative inhibitors of the enzyme. These phosphonic and phosphinic compounds were derivatives of methionine and norleucine as both single amino acids and dipeptides. The designed inhibitors were synthesised and tested towards the peptidase isolated from porcine kidneys using an improved separation procedure affording superior homogeneity. Unexpectedly, organophosphorus derivatives of methionine and norleucine exhibited moderate activity with K(i) values in the micromolar range.

Insulin Analogues with Altered Insulin Receptor Isoform Binding Specificities and Enhanced Aggregation Stabilities.

Insulin is a lifesaver for millions of diabetic patients. There is a need for new insulin analogues with more physiological profiles and analogues that will be thermally more stable than human insulin. Here, we describe the chemical engineering of 48 insulin analogues that were designed to have changed binding specificities toward isoforms A and B of the insulin receptor (IR-A and IR-B). We systematically modified insulin at the C-terminus of the B-chain, at the N-terminus of the A-chain, and at A14 and A18 positions. We discovered an insulin analogue that has Cα-carboxyamidated Glu at B31 and Ala at B29 and that has a more than 3-fold-enhanced binding specificity in favor of the "metabolic" IR-B isoform. The analogue is more resistant to the formation of insulin fibrils at 37 °C and is also more efficient in mice than human insulin. Therefore, [AlaB29,GluB31,amideB31]-insulin may be interesting for further clinical evaluation.

Synthesis of α-carboxyphosphinopeptides derived from norleucine.

In the present study, we describe in detail the synthesis of a relatively rare class of phosphorus compounds, α-carboxyphosphinopeptides. We prepared several norleucine-derived α-carboxyphosphinic pseudopeptides of the general formula Nle-Ψ[PO(OH)]-Gly. These compounds could have important applications as transition state-mimicking inhibitors for methionine or leucine aminopeptidases or other enzymes. For the preparation of the key α-carboxyphosphinate protected precursors, we investigated, compared and improved two different synthetic methods described in literature: the Arbuzov reaction of a silylated N-protected phosphinic acid with a bromoacetate ester and the nucleophilic addition of a mixed O-methyl S-phenyl N-protected phosphonic acid or a methyl N-protected phosphonochloridate with tert-butyl lithioacetate. We also prepared two N-Fmoc protected synthons, Fmoc-Nle-Ψ[PO(OH)]-Gly-COOH and Fmoc-Nle-Ψ[PO(OAd)]-Gly-COOH, and demonstrated that these precursors are suitable building blocks for the solid-phase synthesis of α-carboxyphosphinopeptides.

Acid-Stable Ester Linkers for the Solid-Phase Synthesis of Immobilized Peptides.

A series of N-terminally Fmoc-protected linkers of the general formula Fmoc-X-CO-O-Y-COOH have been prepared, where X is -NH-CH2 -CH2 - or -p-(aminomethyl)phenyl- and Y is -(CH2 )n - (n is 1 or 4) or -p-(methyl)phenyl-. These linkers can easily be covalently attached via their C-terminal carboxyl group to a resin bearing a free amino group. After cleavage of the N-terminal Fmoc group, the linkers can be extended by standard solid-phase peptide synthesis techniques. These ester linkers are acid-stable and resistant to the base-mediated diketopiperazine formation that often occurs during the synthesis of ester-bound peptides; they are stable at neutral pH in aqueous buffers for days but can be effectively cleaved with 0.1 m NaOH or aq. ammonia within minutes or hours, respectively. These properties make these ester handles well suited for use as linkers for the solid-phase peptide synthesis of immobilized peptides when the stable on-resin immobilization of the peptides and the testing of their biological properties in aqueous buffers at neutral pH are necessary.

Insulin-like Growth Factor 1 Analogs Clicked in the C Domain: Chemical Synthesis and Biological Activities.

Human insulin-like growth factor 1 (IGF-1) is a 70 amino acid protein hormone, with key impact on growth, development, and lifespan. The physiological and clinical importance of IGF-1 prompted challenging chemical and biological trials toward the development of its analogs as molecular tools for the IGF-1 receptor (IGF1-R) studies and as new therapeutics. Here, we report a new method for the total chemical synthesis of IGF-1 analogs, which entails the solid-phase synthesis of two IGF-1 precursor chains that is followed by the CuI-catalyzed azide-alkyne cycloaddition ligation and by biomimetic formation of a native pattern of disulfides. The connection of the two IGF-1 precursor chains by the triazole-containing moieties, and variation of its neighboring sequences (Arg36 and Arg37), was tolerated in IGF-1R binding and its activation. These new synthetic IGF-1 analogs are unique examples of disulfide bonds' rich proteins with intra main-chain triazole links. The methodology reported here also presents a convenient synthetic platform for the design and production of new analogs of this important human hormone with non-standard protein modifications.

Electroreduction of aromatic oximes: diprotonation, adsorption, imine formation, and substituent effects.

Aromatic oximes are reduced in aqueous solution in a four-electron process. The reducible species in the pH range 5-8 is a diprotonated form of the oxime. This species is generated in the course of electrolysis in the vicinity of the electrode surface from the adsorbed neutral form of the oxime. The reduction is initiated by a cleavage of the N-O bond. The diprotonation facilitates the reduction process by the preformation of OH2+ as a good leaving group and by a positive charge on the azomethine nitrogen. Diprotonation has been proven based on shapes of i = f(pH) plots, by observed shifts of half-wave potentials with pH and by comparison with the reduction of nitrones. Some observed deviations from theoretical i = f(pH) plots were attributed to the role of adsorption on the rate of protonation. Adsorption is also responsible for dips on some of the i-E curves. Adsorption plays a role at concentrations as low as 1 x 10(-5) M, when the electrode surface is still not fully covered. This indicates that catalyzed protonation occurs on islets of adsorbed materials. At pH 2-5 the studied oximes in the vicinity of the electrode are predominately present in a protonated form, which is less strongly adsorbed. In this pH range the protonation takes place in a homogeneous reaction layer of the electrode. It yields a monoprotonated form, which is reduced. The separation of two two-electron waves observed for some oximes in acidic media serves as an experimental proof of the formation of imines as reduction intermediates. This separation is caused by the differences in pKa values of protonated forms of oximes and imines. The effects of substituents in the para position on the benzene ring are characterized by correlation with the Hammett substituent constant sigmax. This has been proven at pH 1.5 for substituted benzaldehyde oximes and at pH 5.0 for substituted acetophenone oximes.

Reactivation potency of new group of acetylcholinesterase reactivators and their comparison with currently available oximes.

In this work, in vitro potency of novel serie of monoquaternary pyridinium oximes to reactivate cyclosarin-inhibited acetylcholinesterase (AChE) was tested. Currently available oximes (pralidoxime, obidoxime, trimedoxime, HI-6 and BI-6) were used as oximes for comparison. As resulted, none of tested new reactivators was able to reactivate AChE inhibited by cyclosarin. Also pralidoxime, obidoxime and trimedoxime did not reach good reactivation results. Only oximes HI-6 and BI-6 achieved sufficient reactivation potency. From obtained results, it can be deduced, that only reactivators with oxime group in position two are able to reactivate cyclosarin-inhibited AChE.

Synthesis and Evaluation of a Library of Trifunctional Scaffold-Derived Compounds as Modulators of the Insulin Receptor.

We designed a combinatorial library of trifunctional scaffold-derived compounds, which were derivatized with 30 different in-house-made azides. The compounds were proposed to mimic insulin receptor (IR)-binding epitopes in the insulin molecule and bind to and activate this receptor. This work has enabled us to test our synthetic and biological methodology and to prove its robustness and reliability for the solid-phase synthesis and testing of combinatorial libraries of the trifunctional scaffold-derived compounds. Our effort resulted in the discovery of two compounds, which were able to weakly induce the autophosphorylation of IR and weakly bind to this receptor at a 0.1 mM concentration. Despite these modest biological results, which well document the well-known difficulty in modulating protein-protein interactions, this study represents a unique example of targeting the IR with a set of nonpeptide compounds that were specifically designed and synthesized for this purpose. We believe that this work can open new perspectives for the development of next-generation insulin mimetics based on the scaffold structure.

The development of a new class of inhibitors for betaine-homocysteine S-methyltransferase.

Betaine-homocysteine S-methyltransferase (BHMT) is an important zinc-dependent methyltransferase that uses betaine as the methyl donor for the remethylation of homocysteine to form methionine. In the liver, BHMT performs to half of the homocysteine remethylation. In this study, we systematically investigated the tolerance of the enzyme for modifications at the "homocysteine" part of the previously reported potent inhibitor (R,S)-5-(3-amino-3-carboxy-propylsulfanyl)-pentanoic acid (1). In the new compounds, which are S-alkylated homocysteine derivatives, we replaced the carboxylic group in the "homocysteine" part of inhibitor 1 with different isosteric moieties (tetrazole and oxadiazolone); we suppressed the carboxylic negative charge by amidations; we enhanced acidity by replacing the carboxylate with phosphonic or phosphinic acids; and we introduced pyrrolidine steric constraints. Some of these compounds display high affinity toward human BHMT and may be useful for further pharmacological studies of this enzyme. Although none of the new compounds were more potent inhibitors than the reference inhibitor 1, this study helped to completely define the structural requirements of the active site of BHMT and revealed the remarkable selectivity of the enzyme for homocysteine.

S1 pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors.

Methionyl aminopeptidases (MetAPs) are metallo-dependent proteases responsible for removing of N-terminal methionine residue of peptides and proteins during protein maturation and activation. In this report we use a comprehensive strategy to screen the substrate specificity of three methionyl aminopeptidases: Homo sapiens MetAP-1, Homo sapiens MetAP-2 and Escherichia coli MetAP-1. By utilizing a 65-membered fluorogenic substrate library consisting of natural and unnatural amino acids we established detailed substrate preferences of each enzyme in the S1 pocket. Our results show that this pocket is highly conserved for all investigated MetAPs, very stringent for methionine, and that several unnatural amino acids with methionine-like characteristics were also well hydrolyzed by MetAPs. The substrate-derived results were verified using several phosphonate and phosphinate-based inhibitors.

Potency of novel oximes to reactivate sarin inhibited human cholinesterases.

Class of monoquaternary pyridinium oximes was in vitro tested as potential reactivators of acetylcholinesterase (AChE; EC 3.1.1.7) inhibited by nerve agent sarin. Human brain homogenate was used as an appropriate source of cholinesterases. Reactivation potency of novel oximes was compared with currently available reactivators - pralidoxime, obidoxime, and HI-6. According to the obtained results, only five reactivators were able to satisfactorily renew cholinesterase potency (pralidoxime, obidoxime, HI-6, 4-PAM, and K119). Unfortunately, none of the novel tested reactivators surpassed the reactivation potency of the currently most promising reactivator, HI-6. This study shows that monoquaternary reactivators are unable to reactivate nerve agent-inhibited AChE. Due to this, in future, only bisquaternary compounds derived from HI-6 or obidoxime should be designed as new potential cholinesterase reactivators.