receptor autophosphorylation Dotaz Zobrazit nápovědu
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
- azidy chemická syntéza chemie MeSH
- inzulin analogy a deriváty chemie metabolismus MeSH
- knihovny malých molekul chemická syntéza chemie metabolismus farmakologie MeSH
- měď analýza MeSH
- molekulární struktura MeSH
- receptor inzulinu chemie metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- techniky kombinatorické chemie * MeSH
- techniky syntézy na pevné fázi MeSH
- vazba proteinů MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVE: The insulin/IGF superfamily is conserved across vertebrates and invertebrates. Our team has identified five viruses containing genes encoding viral insulin/IGF-1 like peptides (VILPs) closely resembling human insulin and IGF-1. This study aims to characterize the impact of Mandarin fish ranavirus (MFRV) and Lymphocystis disease virus-Sa (LCDV-Sa) VILPs on the insulin/IGF system for the first time. METHODS: We chemically synthesized single chain (sc, IGF-1 like) and double chain (dc, insulin like) forms of MFRV and LCDV-Sa VILPs. Using cell lines overexpressing either human insulin receptor isoform A (IR-A), isoform B (IR-B) or IGF-1 receptor (IGF1R), and AML12 murine hepatocytes, we characterized receptor binding, insulin/IGF signaling. We further characterized the VILPs' effects of proliferation and IGF1R and IR gene expression, and compared them to native ligands. Additionally, we performed insulin tolerance test in CB57BL/6 J mice to examine in vivo effects of VILPs on blood glucose levels. Finally, we employed cryo-electron microscopy (cryoEM) to analyze the structure of scMFRV-VILP in complex with the IGF1R ectodomain. RESULTS: VILPs can bind to human IR and IGF1R, stimulate receptor autophosphorylation and downstream signaling pathways. Notably, scMFRV-VILP exhibited a particularly strong affinity for IGF1R, with a mere 10-fold decrease compared to human IGF-1. At high concentrations, scMFRV-VILP selectively reduced IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation (Ras/MAPK pathway), while leaving Akt phosphorylation (PI3K/Akt pathway) unaffected, indicating a potential biased inhibitory function. Prolonged exposure to MFRV-VILP led to a significant decrease in IGF1R gene expression in IGF1R overexpressing cells and AML12 hepatocytes. Furthermore, insulin tolerance test revealed scMFRV-VILP's sustained glucose-lowering effect compared to insulin and IGF-1. Finally, cryo-EM analysis revealed that scMFRV-VILP engages with IGF1R in a manner closely resembling IGF-1 binding, resulting in a highly analogous structure. CONCLUSIONS: This study introduces MFRV and LCDV-Sa VILPs as novel members of the insulin/IGF superfamily. Particularly, scMFRV-VILP exhibits a biased inhibitory effect on IGF1R signaling at high concentrations, selectively inhibiting IGF-1 stimulated IGF1R autophosphorylation and Erk phosphorylation, without affecting Akt phosphorylation. In addition, MFRV-VILP specifically regulates IGF-1R gene expression and IGF1R protein levels without affecting IR. CryoEM analysis confirms that scMFRV-VILP' binding to IGF1R is mirroring the interaction pattern observed with IGF-1. These findings offer valuable insights into IGF1R action and inhibition, suggesting potential applications in development of IGF1R specific inhibitors and advancing long-lasting insulins.
- MeSH
- elektronová kryomikroskopie MeSH
- exprese genu MeSH
- fosfatidylinositol-3-kinasy metabolismus MeSH
- fosforylace MeSH
- insulinu podobný růstový faktor I * genetika metabolismus MeSH
- inzulin metabolismus MeSH
- lidé MeSH
- myši MeSH
- protein - isoformy metabolismus MeSH
- protoonkogenní proteiny c-akt metabolismus MeSH
- receptor IGF typ 1 * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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
- fosforylace MeSH
- insulinu podobný růstový faktor I chemie genetika MeSH
- insulinu podobný růstový faktor II chemie genetika MeSH
- inzulin chemie metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- molekulární evoluce MeSH
- mutace MeSH
- protein - isoformy MeSH
- receptor inzulinu chemie metabolismus MeSH
- receptory somatomedinů chemie genetika MeSH
- signální transdukce MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The constitutive androstane receptor (CAR) is a crucial transcriptional regulator of key xenobiotic-metabolizing enzymes such as cytochrome P450 CYP3A4, CYP2C9 and CYP2B6. The flavonoids chrysin, baicalein and galangin have been reported to activate CAR and interfere with EGFR signaling. Nevertheless, it is not known if these flavonoids are direct CAR ligands or indirect phenobarbital-like CAR activators via the inhibition of epidermal growth factor receptor (EGFR) signaling. We analyze the interactions of chrysin, galangin and baicalein and its glycoside baicalin with human CAR. We have employed and validated methods that can study direct interaction with the CAR ligand binding pocket. Secondly, we determined if the compounds affect human EGFR signaling and interact with EGFR. Employing a TR-FRET coactivator assay with recombinant CAR or CAR assembly assay, a consistent activation of CAR with flavonoids and phenobarbital was not observed. It was determined, however, that galangin, chrysin, and baicalein may slightly repress EGFR-Tyr1068 autophosphorylation after EGF treatment, phosphorylation of downstream transcription factor ELK1 and stimulate EGFP-CAR nuclear translocation in primary human hepatocytes. These data suggest that flavonoids chrysin, galangin and baicalein are indirect human CAR activators. This study also demonstrates new approach how to test the direct CAR interaction with its ligands.
- MeSH
- buněčné linie MeSH
- cytochrom P450 CYP2B6 metabolismus MeSH
- erbB receptory účinky léků MeSH
- fenobarbital farmakologie MeSH
- flavanony farmakologie MeSH
- flavonoidy farmakologie MeSH
- hepatocyty účinky léků metabolismus MeSH
- lidé MeSH
- protein Elk-1 s doménou ets účinky léků genetika MeSH
- receptory cytoplazmatické a nukleární agonisté MeSH
- transport proteinů účinky léků MeSH
- vazebná místa účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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 savčí cytologie metabolismus MeSH
- fibroblasty cytologie metabolismus MeSH
- fosforylace MeSH
- hypoglykemika metabolismus MeSH
- insulinu podobný růstový faktor I metabolismus MeSH
- insulinu podobný růstový faktor II metabolismus MeSH
- inzulin metabolismus MeSH
- konformace proteinů MeSH
- kultivované buňky MeSH
- lidé MeSH
- lymfocyty cytologie metabolismus MeSH
- molekulární sondy metabolismus MeSH
- myši knockoutované MeSH
- myši MeSH
- receptor IGF typ 1 metabolismus MeSH
- receptor inzulinu metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Inhibition of protein kinases is a validated concept for pharmacological intervention in cancers. Many kinase inhibitors have been approved for clinical use, but their practical application is often limited. Here, we describe a collection of 23 novel 2,6,9-trisubstituted purine derivatives with nanomolar inhibitory activities against PDGFRα, a receptor tyrosine kinase often found constitutively activated in various tumours. The compounds demonstrated strong and selective cytotoxicity in the human eosinophilic leukemia cell line EOL-1, whereas several other cell lines were substantially less sensitive. The cytotoxicity in EOL-1, which is known to express the FIP1L1-PDGFRA fusion gene encoding an oncogenic kinase, correlated significantly with PDGFRα inhibition. EOL-1 cells treated with the compounds also exhibited dose-dependent inhibition of PDGFRα autophosphorylation and suppression of its downstream signaling pathways with concomitant G1 phase arrest, confirming the proposed mechanism of action. Our results show that substituted purines can be used as platforms for preparing tyrosine kinase inhibitors with specific activity towards eosinophilic leukemia.
- MeSH
- antitumorózní látky chemická syntéza chemie farmakologie MeSH
- inhibitory proteinkinas chemická syntéza chemie farmakologie MeSH
- léky antitumorózní - screeningové testy MeSH
- lidé MeSH
- molekulární struktura MeSH
- nádorové buňky kultivované MeSH
- proliferace buněk účinky léků MeSH
- puriny chemická syntéza chemie farmakologie MeSH
- růstový faktor odvozený z trombocytů - receptor alfa antagonisté a inhibitory genetika metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Receptor tyrosine kinase PDGFRα is often constitutively activated in various tumours and is regarded as a drug target. Here, we present a collection of 2,6,9-trisubstituted purines with nanomolar potency against PDGFRα and strong and selective cytotoxicity in the human eosinophilic leukaemia cell line EOL-1 that expresses the FIP1L1-PDGFRA oncogene. In treated EOL-1 cells, the example compound 14q inhibited the autophosphorylation of PDGFRα and the phosphorylation of STAT3 and ERK1/2. Interestingly, we observed pronounced and even increased effects of 14q on PDGFRα and some of its downstream signalling pathways after drug washout. In accordance with suppressed PDGFRα signalling, treated cells were arrested in the G1 phase of the cell cycle and eventually underwent apoptosis. Our results show that substituted purines can be used as specific modulators of eosinophilic leukaemia.
- MeSH
- antitumorózní látky chemická syntéza chemie farmakologie MeSH
- apoptóza účinky léků MeSH
- buněčný cyklus účinky léků MeSH
- fosforylace účinky léků MeSH
- hypereozinofilní syndrom farmakoterapie metabolismus patologie MeSH
- inhibitory proteinkinas chemická syntéza chemie farmakologie MeSH
- léky antitumorózní - screeningové testy MeSH
- lidé MeSH
- molekulární struktura MeSH
- nádorové buňky kultivované MeSH
- proliferace buněk účinky léků MeSH
- puriny chemická syntéza chemie farmakologie MeSH
- růstový faktor odvozený z trombocytů - receptor alfa antagonisté a inhibitory genetika metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The multistep phosphorelay (MSP) is a central signaling pathway in plants integrating a wide spectrum of hormonal and environmental inputs and controlling numerous developmental adaptations. For the thorough comprehension of the molecular mechanisms underlying the MSP-mediated signal recognition and transduction, the detailed structural characterization of individual members of the pathway is critical. In this review we describe and discuss the recently known crystal and nuclear magnetic resonance structures of proteins acting in MSP signaling in higher plants, focusing particularly on cytokinin and ethylene signaling in Arabidopsis thaliana. We discuss the range of functional aspects of available structural information including determination of ligand specificity, activation of the receptor via its autophosphorylation, and downstream signal transduction through the phosphorelay. We compare the plant structures with their bacterial counterparts and show that although the overall similarity is high, the differences in structural details are frequent and functionally important. Finally, we discuss emerging knowledge on molecular recognition mechanisms in the MSP, and mention the latest findings regarding structural determinants of signaling specificity in the Arabidopsis MSP that could serve as a general model of this pathway in all higher plants.
Ceritinib je nový inhibitor kinázy anaplastického lymfomu (anaplastic lymphoma kinase, ALK) určený pro pacienty s ALK pozitivním nemalobuněčným karcinomem plic, kteří již byli léčeni crizotinibem. Ceritinib inhibuje autofosforylaci ALK, čímž je snížena produkce signálních proteinů a proliferace závislá na buněčné populaci ALK. Účinek ceritinibu byl ověřen dvěma klinickými studiemi fáze II, kde dosáhl celkové odpovědi 37,1 % (95% CI: 29,1–45,7) a mediánu doby bez progrese 5,7 měsíce (95% CI: 5,3–7,4). Nejčastějšími nežádoucími účinky jsou gastrointestinální toxicita, únava, nechutenství, vyšší sérová koncentrace kreatininu, vyrážka a anemie.
Ceritinib is indicated for the treatment of patients with anaplastic lymphoma kinase (ALK) positive advanced non small cell lung cancer previously treated with crizotinib. Ceritinib inhibits autophosphorylation of ALK, ALK mediated phosphorylation of downstream signalling proteins, and proliferation of ALK dependent cancer cells both in vitro and in vivo. Ceritinib was evaluated in two phase II single arm studies in patients with ALK positive non small cell lung cancer previously treated with an ALK inhibitor. Overall response rate with ceritinib was 37.1% (95% CI 29.1–45.7) and median progression free survival was 5.7 months (95% CI 5.3–7.4). The most commonly reported adverse effects were gastrointestinal disorders, fatigue, anorexia, raised blood creatinine, rash, and anaemia.
- Klíčová slova
- studie ASCEND-2, studie ASCEND-3,
- MeSH
- analýza přežití MeSH
- chemorezistence MeSH
- klinické zkoušky, fáze II jako téma MeSH
- lidé MeSH
- multicentrické studie jako téma MeSH
- mutace MeSH
- nádory plic farmakoterapie genetika MeSH
- nemalobuněčný karcinom plic * farmakoterapie genetika MeSH
- přežití po terapii bez příznaků nemoci MeSH
- pyrimidiny farmakologie škodlivé účinky terapeutické užití MeSH
- sulfony farmakologie škodlivé účinky terapeutické užití MeSH
- tyrosinkinasové receptory * antagonisté a inhibitory farmakologie škodlivé účinky terapeutické užití MeSH
- Check Tag
- lidé MeSH
Streptococcus pneumoniae carries a single Ser/Thr protein kinase gene stkP in its genome. Biochemical studies performed with recombinant StkP have revealed that this protein is a functional membrane-linked eukaryotic-type Ser/Thr protein kinase. Here, we demonstrate that the deletion of its extracellular domain negatively affects the stability of a core kinase domain. In contrast, the membrane anchored kinase domain and the full-length form of StkP were stable and capable of autophosphorylation. Furthermore, evidence is presented that StkP forms dimers through its transmembrane and extracellular domains.
