Insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively) are protein hormones involved not only in normal growth and development but also in life span regulation and cancer. They exert their functions mainly through the IGF-1R or by binding to isoform A of the insulin receptor (IR-A). The development of IGF-1 and IGF-2 antagonists is of great clinical interest. Mutations of A4 and A8 sites of human insulin lead to disproportionate effects on hormone IR binding and activation. Here, we systematically modified IGF-1 sites 45, 46, and 49 and IGF-2 sites 45 and 48, which correspond, or are close, to insulin sites A4 and A8. The IGF-1R and IR-A binding and autophosphorylation potencies of these analogues were characterized. They retained the main IGF-1R-related properties, but the hormones with His49 in IGF-1 and His48 in IGF-2 showed significantly higher affinities for IR-A and for IR-B, being the strongest IGF-1- and IGF-2-like binders of these receptors ever reported. All analogues activated IR-A and IGF-1R without major discrepancies in their binding affinities. This study revealed that IR-A and IGF-1R contain specific sites, likely parts of their so-called sites 2', which can interact differently with specifically modified IGF analogues. Moreover, a clear importance of IGF-2 site 44 for effective hormone folding was also observed. These findings may facilitate novel and rational engineering of new hormone analogues for IR-A and IGF-1R studies and for potential medical applications.

• MeSH
• Phosphorylation MeSH
• Insulin-Like Growth Factor I chemistry   genetics   MeSH
• Insulin-Like Growth Factor II chemistry   genetics   MeSH
• Insulin chemistry   metabolism   MeSH
• Humans MeSH
• Ligands MeSH
• Evolution, Molecular MeSH
• Mutation MeSH
• Protein Isoforms MeSH
• Receptor, Insulin chemistry   metabolism   MeSH
• Receptors, Somatomedin chemistry   genetics   MeSH
• Signal Transduction MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Arginine chemistry   MeSH
• NIH 3T3 Cells drug effects   MeSH
• Click Chemistry MeSH
• Cycloaddition Reaction MeSH
• Disulfides chemistry   MeSH
• Fibroblasts MeSH
• Phosphorylation MeSH
• Insulin-Like Growth Factor I analogs & derivatives   chemical synthesis   chemistry   metabolism   pharmacology   MeSH
• Humans MeSH
• Copper chemistry   MeSH
• Methionine chemistry   MeSH
• Mice MeSH
• Drug Evaluation, Preclinical methods   MeSH
• Protein Domains MeSH
• Proto-Oncogene Proteins c-akt metabolism   MeSH
• Receptor, IGF Type 1 metabolism   MeSH
• Solid-Phase Synthesis Techniques MeSH
• Triazoles chemistry   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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.

• MeSH
• Alkynes chemistry   MeSH
• Amino Acids chemical synthesis   MeSH
• Azides chemistry   MeSH
• Click Chemistry methods   MeSH
• Ethylamines chemistry   MeSH
• Fluorenes chemical synthesis   chemistry   MeSH
• Urea analogs & derivatives   chemistry   MeSH
• Peptides chemical synthesis   MeSH
• Triazoles chemistry   MeSH
• Publication type
• Journal Article MeSH

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.

• MeSH
• Azides chemical synthesis   chemistry   MeSH
• Insulin analogs & derivatives   chemistry   metabolism   MeSH
• Small Molecule Libraries chemical synthesis   chemistry   metabolism   pharmacology   MeSH
• Copper analysis   MeSH
• Molecular Structure MeSH
• Receptor, Insulin chemistry   metabolism   MeSH
• Reproducibility of Results MeSH
• Combinatorial Chemistry Techniques * MeSH
• Solid-Phase Synthesis Techniques MeSH
• Protein Binding MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Publication type
• Journal Article MeSH

Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains.

• MeSH
• Antigens, CD chemistry   genetics   metabolism   MeSH
• Insulin-Like Growth Factor II chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Protein Isoforms chemistry   genetics   metabolism   MeSH
• Protein Domains MeSH
• Receptor, IGF Type 1 chemistry   genetics   metabolism   MeSH
• Receptor, Insulin chemistry   genetics   metabolism   MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Amino Acid Substitution MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Insulin, insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively), and their receptors (IR and IGF-1R) are the key elements of a complex hormonal system that is essential for the development and functioning of humans. The C and D domains of IGFs (absent in insulin) likely play important roles in the differential binding of IGF-1 and -2 to IGF-1R and to the isoforms of IR (IR-A and IR-B) and specific activation of these receptors. Here, we attempted to probe the impact of IGF-1 and IGF-2 D domains (DI and DII, respectively) and the IGF-2 C domain (CII) on the receptor specificity of these hormones. For this, we made two types of insulin hybrid analogues: (i) with the C-terminus of the insulin A chain extended by the amino acids from the DI and DII domains and (ii) with the C-terminus of the insulin B chain extended by some amino acids derived from the CII domain. The receptor binding affinities of these analogues and their receptor autophosphorylation potentials were characterized. Our results indicate that the DI domain has a more negative impact than the DII domain does on binding to IR, and that the DI domain Pro-Leu-Lys residues are important factors for a different IR-A versus IR-B binding affinity of IGF-1. We also showed that the additions of amino acids that partially "mimic" the CII domain, to the C-terminus of the insulin B chain, change the binding and autophosphorylation specificity of insulin in favor of the "metabolic" IR-B isoform. This opens new venues for rational enhancement of insulin IR-B specificity by modifications beyond the C-terminus of its B chain.

• MeSH
• Embryo, Mammalian cytology   metabolism   MeSH
• Fibroblasts cytology   metabolism   MeSH
• Phosphorylation MeSH
• Hypoglycemic Agents metabolism   MeSH
• Insulin-Like Growth Factor I metabolism   MeSH
• Insulin-Like Growth Factor II metabolism   MeSH
• Insulin metabolism   MeSH
• Protein Conformation MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Lymphocytes cytology   metabolism   MeSH
• Molecular Probes metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Receptor, IGF Type 1 metabolism   MeSH
• Receptor, Insulin metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone's B-chain C-terminus for its IR-B specificity.

• MeSH
• Alkynes chemistry   MeSH
• Azides chemistry   MeSH
• Cycloaddition Reaction MeSH
• Insulin chemistry   metabolism   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Models, Molecular MeSH
• Protein Isoforms MeSH
• Receptor, IGF Type 1 chemistry   metabolism   MeSH
• Receptor, Insulin chemistry   metabolism   MeSH
• Protein Stability MeSH
• Protein Binding MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin-insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral φ and ψ main-chain angles. α-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1-B8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the "classical" T-state and that a substantial flexibility of the B1-B8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin-IR interaction.

• MeSH
• Circular Dichroism MeSH
• Insulin analogs & derivatives   chemistry   MeSH
• Crystallography, X-Ray MeSH
• Aminoisobutyric Acids chemistry   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The structural characterization of the insulin-insulin receptor (IR) interaction still lacks the conformation of the crucial B21-B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms.

• MeSH
• Phenylalanine MeSH
• Fibroblasts metabolism   MeSH
• Insulin analogs & derivatives   chemistry   genetics   metabolism   MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Lymphocytes metabolism   MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Rats, Wistar MeSH
• Receptor, Insulin chemistry   metabolism   MeSH
• Amino Acid Substitution MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Analýza proteomu zhoubných nádorů prsu. Zjišťování nových faktorů efektivity léčby a selhání léčby s cílem zlepšit dosavadní léčebné výsledky. Vztah nových faktorů a stávajících prognostických a prediktivních faktorů. Kultivace nádorů in vitro. Data získaná v rámci projektu budou zpracována metodou explorační analýzy dat GUHA. Tato metoda umožňuje automatickou tvorbu a testování hypotéz o skutečnostech podporovaných daty.; Analysis of the proteome of malignant breast tumors. Determination of novel prognostic and predictive factors.The validity of the classical prognostic factors versus molecular prognostic factors will be determined. Statistical analysis of breast cancer patients using GUHA method. General Unary Hypotheses Automaton method of exploration data analysis.

• MeSH
• Disease Management MeSH
• Biomarkers, Tumor analysis   MeSH
• Breast Neoplasms therapy   MeSH
• Disease-Free Survival MeSH
• Prognosis MeSH
• Proteome analysis   MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• gynekologie a porodnictví
• onkologie
• NML Publication type
• závěrečné zprávy o řešení grantu IGA MZ ČR