Casein kinases 1 (CK1) are key signaling molecules that have emerged recently as attractive therapeutic targets in particular for the treatment of hematological malignancies. Herein, we report the identification of a new class of potent and highly selective inhibitors of CK1α, δ and ϵ. Based on their optimal in vitro and in vivo profiles and their exclusive selectivity, MU1250, MU1500 and MU1742 were selected as quality chemical probes for those CK1 isoforms. At proper concentrations, MU1250 and MU1500 allow for specific targeting of CK1δ or dual inhibition of CK1δ/ϵ in cells. The compound MU1742 also efficiently inhibits CK1α and, to our knowledge, represents the first potent and highly selective inhibitor of this enzyme. In addition, we demonstrate that the central 1H-pyrrolo[2,3-b]pyridine-imidazole pharmacophore can be used as the basis of highly selective inhibitors of other therapeutically relevant protein kinases, e.g. p38α, as exemplified by the compound MU1299.

• Klíčová slova
• CK1, Chemical Probe, Inhibitor, Isoform Selectivity, Wnt Pathway,
• MeSH
• inhibitory proteinkinas chemie   MeSH
• kaseinkinasa I * metabolismus   MeSH
• lidé MeSH
• protein - isoformy metabolismus   MeSH
• signální transdukce * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibitory proteinkinas MeSH
• kaseinkinasa I * MeSH
• protein - isoformy MeSH

The pathogenic molecular mechanisms underlying the insurgence of nasal polyps has not been completely defined. In some patients, these lesions can have a recurrence after surgery removal, and the difference between recurrent and not recurrent patients is still unclear. To molecularly characterize and distinguish between these two classes, a cohort of patients affected by nasal polyposis was analysed. In all patients we analysed the p63 isoform expression using fresh tissues taken after surgery. Moreover, confocal immunofluorescence analysis of fixed sections was performed. The results show high ΔNp63 expression in samples from the nasal polyps of patients compared to the normal epithelia. Analysis of the expression level of the TAp63 isoform shows differential expression between the patients with recurrence compared to those not recurring. The data, considered as the ΔN/TAp63 ratio, really discriminate the two groups. In fact, even though ΔNp63 is expressed in non-recurrent patients, the resulting ratio ΔN/TAp63 is significantly lower in these patients. This clearly indicates that the status of TAp63 expression, represented by the ΔN/TAp63 ratio, could be considered a prognostic marker of low recurrence probability. In these samples we also investigated the expression of OTX2 transcription factor, known to be a selective activator of TAp63, detecting a significant correlation. Database analysis of HNSCC patients showed increased survival for the patients presenting OTX2 amplification and/or overexpression. These results, together with the fact that TAp63 can be selectively upregulated by HDAC inhibitors, open the possibility to consider local treatment of recurrent nasal polyps with these molecules.

• MeSH
• fluorescenční protilátková technika MeSH
• lidé MeSH
• nádorové supresorové proteiny genetika   metabolismus   MeSH
• nosní polypy genetika   metabolismus   MeSH
• protein - isoformy genetika   metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• transkripční faktory Otx genetika   metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• nádorové supresorové proteiny MeSH
• OTX2 protein, human MeSH Prohlížeč
• protein - isoformy MeSH
• TP63 protein, human MeSH Prohlížeč
• transkripční faktory Otx MeSH
• transkripční faktory MeSH

A small library of boron-cluster- and metallacarborane-cluster-based ligands was designed, prepared, and tested for isoform-selective activation or inhibition of the three nitric oxide synthase isoforms. On the basis of the concept of creating a hydrophobic analogue of a natural substrate, a stable and nontoxic basic boron cluster system, previously used for boron neutron capture therapy, was modified by the addition of positively charged moieties to its periphery, providing hydrophobic and nonclassical hydrogen bonding interactions with the protein. Several of these compounds show efficacy for inhibition of NO synthesis with differential effects on the various nitric oxide synthase isoforms.

• MeSH
• chemické modely MeSH
• kobalt chemie   MeSH
• lidé MeSH
• molekulární struktura MeSH
• organokovové sloučeniny chemická syntéza   farmakologie   MeSH
• protein - isoformy MeSH
• sloučeniny boru chemická syntéza   farmakologie   MeSH
• synthasa oxidu dusnatého antagonisté a inhibitory   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• kobalt MeSH
• organokovové sloučeniny MeSH
• protein - isoformy MeSH
• sloučeniny boru MeSH
• synthasa oxidu dusnatého MeSH

The seven-transmembrane-spanning receptors of the FZD1-10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD2, FZD4, or FZD5, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-β-catenin pathway through FZD2/4/5 as measured by phosphorylation of LRP6 and β-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs.

• Klíčová slova
• 32D Cells, Disheveled, Frizzled, Functional Selectivity, LDL Receptor-related Protein 6, Myeloid Cell, Receptor, WNT Pathway, WNT Signaling, β-Catenin (B-catenin),
• MeSH
• beta-katenin metabolismus   MeSH
• buněčné linie MeSH
• fosforylace MeSH
• frizzled receptory metabolismus   MeSH
• mapování interakce mezi proteiny MeSH
• mapy interakcí proteinů * MeSH
• myši MeSH
• protein - isoformy metabolismus   MeSH
• proteiny Wnt metabolismus   MeSH
• signální dráha Wnt * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• beta-katenin MeSH
• frizzled receptory MeSH
• protein - isoformy MeSH
• proteiny Wnt MeSH

A significant drawback of the exogenous administration of insulin to diabetics is the non-physiological profile of insulin action resulting in the insufficient suppression of hepatic glucose production, which is the main contributing factor to diabetic hyperglycemia under fasting conditions and the basis of the challenge to restore a more physiological glucose profile in diabetes. The insulin receptor (IR) exists in two alternatively spliced variants, IR-A and IR-B, with different tissue distribution. While peripheral tissues contain different proportions of both isoforms, hepatic cells almost exclusively contain IR-B. In this respect, IR-B-selective insulin analogs would be of great interest for their potential to restore more natural metabolic homeostasis in diabetes. Recent advances in the structural biology of insulin and IR have provided new clues for understanding the interaction of both proteins. This article discusses and offers some structural perspectives for the design of specific insulin analogs with a preferential binding to IR-B.

• Klíčová slova
• CT-peptide, IR-A, IR-B, binding affinity, exon 11, insulin analog, insulin receptor isoform,
• Publikační typ
• časopisecké články MeSH

The p53 family member p63 exists as two major protein variants (TAp63 and ΔNp63) with distinct expression patterns and functional properties. Whilst downstream target genes of p63 have been studied intensively, how p63 variants are themselves controlled has been relatively neglected. Here, we review advances in understanding ΔNp63 and TAp63 regulation, highlighting their distinct pathways. TAp63 has roles in senescence and metabolism, and in germ cell genome maintenance, where it is activated post-transcriptionally by phosphorylation cascades after DNA damage. The function and regulation of TAp63 in mesenchymal and haematopoietic cells is less clear but may involve epigenetic control through DNA methylation. ΔNp63 functions to maintain stem/progenitor cells in various epithelia and is overexpressed in squamous and certain other cancers. ΔNp63 is transcriptionally regulated through multiple enhancers in concert with chromatin modifying proteins. Many signalling pathways including growth factors, morphogens, inflammation, and the extracellular matrix influence ΔNp63 levels, with inconsistent results reported. There is also evidence for reciprocal regulation, including ΔNp63 activating its own transcription. ΔNp63 is downregulated during cell differentiation through transcriptional regulation, while post-transcriptional events cause proteasomal degradation. Throughout the review, we identify knowledge gaps and highlight discordances, providing potential explanations including cell-context and cell-matrix interactions. Identifying individual p63 variants has roles in differential diagnosis and prognosis, and understanding their regulation suggests clinically approved agents for targeting p63 that may be useful combination therapies for selected cancer patients. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

• Klíčová slova
• HDAC inhibitors, chromatin modification, gene enhancers, p63, squamous cell cancer, stem cells,
• MeSH
• lidé MeSH
• nádorové supresorové proteiny * MeSH
• nádory * MeSH
• protein - isoformy MeSH
• transkripční faktory * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• nádorové supresorové proteiny * MeSH
• protein - isoformy MeSH
• TP63 protein, human MeSH Prohlížeč
• transkripční faktory * MeSH

Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling. ADSL deficiency is a selectively neuronopathic disorder with psychomotor retardation and epilepsy as leading traits. Both dephosphorylated enzyme substrates, succinylaminoimidazole-carboxamide riboside (SAICAr) and succinyladenosine (S-Ado), accumulate in the cerebrospinal fluid (CSF) of affected individuals with S-Ado/SAICAr concentration ratios proportional to the phenotype severity. We studied the disorder at various levels in a group of six patients with ADSL deficiency. We identified the complete ADSL cDNA and its alternatively spliced isoform resulting from exon 12 skipping. Both mRNA isoforms were expressed in all the tissues studied with the non-spliced form 10-fold more abundant. Both cDNAs were expressed in Escherichia coli and functionally characterized at the protein level. The results showed only the unspliced ADSL to be active. The gene consists of 13 exons spanning 23 kb. The promotor region shows typical features of the housekeeping gene. Eight mutations were identified in a group of six patients. The expression studies of the mutant proteins carried out in an attempt to study genotype-phenotype correlation showed that the level of residual enzyme activity correlates with the severity of the clinical phenotype. All the mutant enzymes studied in vitro displayed a proportional decrease in activity against both of their substrates. However, this was not concordant with strikingly different concentration ratios in the CSF of individual patients. This suggests either different in vivo enzyme activities against each of the substrates and/or their different turnover across the CSF-blood barrier, which may be decisive in determining disease severity.

• MeSH
• adenylsukcinátlyasa biosyntéza   chemie   nedostatek   genetika   MeSH
• alternativní sestřih MeSH
• dítě MeSH
• Escherichia coli metabolismus   MeSH
• exony MeSH
• fenotyp MeSH
• fibroblasty metabolismus   MeSH
• genotyp MeSH
• kinetika MeSH
• klonování DNA MeSH
• kojenec MeSH
• komplementární DNA metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• předškolní dítě MeSH
• promotorové oblasti (genetika) MeSH
• protein - isoformy MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• teplota MeSH
• tkáňová distribuce MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylsukcinátlyasa MeSH
• komplementární DNA MeSH
• protein - isoformy MeSH

Topoisomerase II alpha and beta (TOP2A and TOP2B) isoenzymes perform essential and non-redundant cellular functions. Anthracyclines induce their potent anti-cancer effects primarily via TOP2A, but at the same time they induce a dose limiting cardiotoxicity through TOP2B. Here we describe the development of the obex class of TOP2 inhibitors that bind to a previously unidentified druggable pocket in the TOP2 ATPase domain to act as allosteric catalytic inhibitors by locking the ATPase domain conformation with the capability of isoform-selective inhibition. Through rational drug design we have developed topobexin, which interacts with residues that differ between TOP2A and TOP2B to provide inhibition that is both selective for TOP2B and superior to dexrazoxane. Topobexin is a potent protectant against chronic anthracycline cardiotoxicity in an animal model. This demonstration of TOP2 isoform-specific inhibition underscores the broader potential to improve drug specificity and minimize adverse effects in various medical treatments.

• MeSH
• antracykliny * škodlivé účinky   farmakologie   MeSH
• DNA-topoisomerasy typu II * metabolismus   chemie   MeSH
• inhibitory topoisomerasy II * farmakologie   chemie   MeSH
• kardiotonika * farmakologie   chemie   MeSH
• kardiotoxicita * prevence a kontrola   MeSH
• lidé MeSH
• myši MeSH
• proteiny vázající poly-ADP-ribosu antagonisté a inhibitory   metabolismus   chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antracykliny * MeSH
• DNA-topoisomerasy typu II * MeSH
• inhibitory topoisomerasy II * MeSH
• kardiotonika * MeSH
• proteiny vázající poly-ADP-ribosu MeSH
• TOP2A protein, human MeSH Prohlížeč
• TOP2B protein, human MeSH Prohlížeč

The study addressed the effect of colchicine and its derivatives on the protein levels of cytochrome P450 (CYP) 1A2, 2A6, 3A4, 2C9/19, and 2E1 isoforms. Primary human hepatocyte culture was the model of choice. Levels of individual CYP isoforms were detected using immunoblotting. Colchicine caused an increase of CYP2E1 protein content, colchiceine and N-deacetylcolchiceine induced isoforms CYP2C9, 2E1 and 3A4 whereas colchicoside induced CYP2C9 and 2E1. The levels of CYP1A2 and 2A6 were unaffected by any of tested compounds. Demecolcine and 3-demethylcolchicine had no effect on any studied P450 isoform. Since colchicine is an exclusive substrate of CYP3A4 whereas it induces CYP2E1, there is a suspicion rather at protein stabilization than at gene induction concerning induction origin.

• MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• kolchicin farmakologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• protein - isoformy metabolismus   MeSH
• systém (enzymů) cytochromů P-450 metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kolchicin MeSH
• protein - isoformy MeSH
• systém (enzymů) cytochromů P-450 MeSH

Cardiac fibrotization is a well-known process characteristic of many cardiac pathological conditions. The key element is excessive activation of cardiac fibroblasts, their transdifferentiation into myofibroblasts, increased production, and accumulation of extracellular matrix proteins, resulting in cardiac stiffness. The exact cellular mechanisms and molecular components involved in the process are not fully elucidated, but the SOCE mechanism could play an important role. Its key molecules are the molecular sensor of calcium in ER/SR - STIM and the highly selective calcium channels Orai located in the plasma membrane. This study aims to evaluate selected SOCE-associated genes in the activation of HCF cell culture by several known substances (phenylephrine, isoprenaline) that represent cardiovascular overload. After cell cultivation, cell medium was collected to measure the soluble collagen content. From the harvested cells, qRT-PCR was performed to determine the mRNA levels of the corresponding genes. The activation of cells was based on changes in the relative expression of collagen genes as well as the collagen content in the medium of the cell culture. We detected an increase in the expression of the Orai2 isoform, a change in the Orai1/Orai3 ratio and also an increase in the expression of the STIM2 isoform. These results suggest an increased activation of the SOCE mechanism under stress conditions of fibroblasts, which supports the hypothesis of fibroblast activation in pathological processes by altering calcium homeostasis through the SOCE mechanism.

• MeSH
• fibroblasty metabolismus   MeSH
• kanály aktivované uvolněním vápníku metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• myokard cytologie   metabolismus   MeSH
• protein - isoformy metabolismus   MeSH
• proteiny STIM metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kanály aktivované uvolněním vápníku MeSH
• protein - isoformy MeSH
• proteiny STIM MeSH