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.
- Klíčová slova
- insulin, library, peptide, receptor, scaffold,
- MeSH
- inzulin metabolismus chemie MeSH
- knihovny malých molekul chemie farmakologie chemická syntéza MeSH
- lidé MeSH
- ligandy MeSH
- molekulární struktura MeSH
- peptidová knihovna MeSH
- peptidy chemie metabolismus chemická syntéza MeSH
- receptor inzulinu * metabolismus chemie MeSH
- techniky kombinatorické chemie * MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- inzulin MeSH
- knihovny malých molekul MeSH
- ligandy MeSH
- peptidová knihovna MeSH
- peptidy MeSH
- receptor inzulinu * MeSH
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.
- Klíčová slova
- antagonism, dicarba, disulfide mimetics, insulin mimetic peptide, insulin receptor, staple, triazole,
- MeSH
- disulfidy chemie MeSH
- inzulin * metabolismus MeSH
- peptidy chemie MeSH
- receptor inzulinu * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- disulfidy MeSH
- inzulin * MeSH
- peptidy MeSH
- receptor inzulinu * MeSH
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.
- MeSH
- insulinu podobný růstový faktor II * chemie MeSH
- kosti a kostní tkáň MeSH
- lidé MeSH
- peptidové fragmenty chemie MeSH
- peptidy MeSH
- vápník * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- insulinu podobný růstový faktor II * MeSH
- peptidové fragmenty MeSH
- peptidy MeSH
- preptin MeSH Prohlížeč
- vápník * MeSH
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.
- MeSH
- CD antigeny chemie metabolismus MeSH
- fosforylace MeSH
- inzulin analogy a deriváty metabolismus MeSH
- inzulinová rezistence MeSH
- kalorimetrie metody MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- protein - isoformy chemie metabolismus MeSH
- proteinové agregáty * MeSH
- receptor inzulinu chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- CD antigeny MeSH
- INSR protein, human MeSH Prohlížeč
- inzulin MeSH
- protein - isoformy MeSH
- proteinové agregáty * MeSH
- receptor inzulinu MeSH
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.
- Klíčová slova
- diketopiperazines, esters, peptides, protecting groups, solid-phase synthesis,
- MeSH
- estery chemická syntéza chemie MeSH
- imobilizované proteiny chemická syntéza MeSH
- indikátory a reagencie chemická syntéza chemie MeSH
- peptidy chemická syntéza MeSH
- techniky syntézy na pevné fázi metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- estery MeSH
- imobilizované proteiny MeSH
- indikátory a reagencie MeSH
- peptidy MeSH
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.
- Klíčová slova
- chiral anion exchanger, liquid chromatography, particle size distribution, pore volume effect, preferential sorption,
- Publikační typ
- časopisecké články MeSH
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.
- Klíčová slova
- Site 1, binding, insulin, insulin receptor, insulin-like growth factor (IGF), kinetics, protein design, structure-function,
- MeSH
- insulinu podobný růstový faktor I chemie genetika metabolismus MeSH
- inzulin agonisté metabolismus MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- receptor IGF typ 1 MeSH
- receptor inzulinu chemie genetika metabolismus MeSH
- receptory somatomedinů chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- IGF1R protein, human MeSH Prohlížeč
- insulinu podobný růstový faktor I MeSH
- inzulin MeSH
- receptor IGF typ 1 MeSH
- receptor inzulinu MeSH
- receptory somatomedinů MeSH
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.
- Klíčová slova
- N-protected amino acids, chiral anion exchange, chiral stationary phases, click-chemistry immobilization, enantiomer separation,
- MeSH
- chinovník chemie MeSH
- chromatografie iontoměničová MeSH
- molekulární struktura MeSH
- stereoizomerie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- arginin chemie MeSH
- buňky NIH 3T3 účinky léků MeSH
- cykloadiční reakce MeSH
- disulfidy chemie MeSH
- fibroblasty MeSH
- fosforylace MeSH
- insulinu podobný růstový faktor I analogy a deriváty chemická syntéza chemie metabolismus farmakologie MeSH
- lidé MeSH
- měď chemie MeSH
- methionin chemie MeSH
- myši MeSH
- preklinické hodnocení léčiv metody MeSH
- proteinové domény MeSH
- protoonkogenní proteiny c-akt metabolismus MeSH
- receptor IGF typ 1 metabolismus MeSH
- syntetická chemie okamžité shody MeSH
- techniky syntézy na pevné fázi MeSH
- triazoly chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- arginin MeSH
- disulfidy MeSH
- IGF1 protein, human MeSH Prohlížeč
- insulinu podobný růstový faktor I MeSH
- měď MeSH
- methionin MeSH
- protoonkogenní proteiny c-akt MeSH
- receptor IGF typ 1 MeSH
- triazoly MeSH
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: β-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.
- Klíčová slova
- alanine, azide elimination, azido amino acid, homoalanine, lysine, ornithine, synthesis,
- MeSH
- alkyny chemie MeSH
- aminokyseliny chemická syntéza MeSH
- azidy chemie MeSH
- ethylaminy chemie MeSH
- fluoreny chemická syntéza chemie MeSH
- močovina analogy a deriváty chemie MeSH
- peptidy chemická syntéza MeSH
- syntetická chemie okamžité shody metody MeSH
- triazoly chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate MeSH Prohlížeč
- alkyny MeSH
- aminokyseliny MeSH
- azidy MeSH
- ethylaminy MeSH
- fluoreny MeSH
- močovina MeSH
- N,N-diisopropylethylamine MeSH Prohlížeč
- N(alpha)-fluorenylmethyloxycarbonylamino acids MeSH Prohlížeč
- peptidy MeSH
- triazoly MeSH
