RESUMEN
The conjugates of an adenosine mimetic and oligo-l-arginine or oligo-d-arginine (ARCs) were initially designed in our research group as inhibitors and photoluminescent probes targeting basophilic protein kinases. Here, we explored a panel of ARCs and their fluorescent derivatives in biochemical assays with members of the protein arginine methyltransferase (PRMT) family, focusing specifically on PRMT1. In the binding/displacement assay with detection of fluorescence anisotropy, we found that ARCs and arginine-rich peptides could serve as high-affinity ligands for PRMT1, whereas the equilibrium dissociation constant values depended dramatically on the number of arginine residues within the compounds. The fluorescently labeled probe ARC-1081 was displaced from its complex with PRMT1 by both S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH), indicating binding of the adenosine mimetic of ARCs to the SAM/SAH-binding site within PRMT1. The ARCs that had previously shown microsecond-lifetime photoluminescence in complex with protein kinases did not feature such property in complex with PRMT1, demonstrating the selectivity of the time-resolved readout format. When tested against a panel of PRMT family members in single-dose inhibition experiments, a micromolar concentration of ARC-902 was required for the inhibition of PRMT1 and PRMT7. Overall, our results suggest that the compounds containing multiple arginine residues (including the well-known cell-penetrating peptides) are likely to inhibit PRMT and thus interfere with the epigenetic modification status in complex biological systems, which should be taken into consideration during interpretation of the experimental data.
Asunto(s)
Adenosina , Proteína-Arginina N-Metiltransferasas , Adenosina/química , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Colorantes Fluorescentes , Arginina/química , Arginina/metabolismo , Péptidos/química , Proteínas QuinasasRESUMEN
Bivalent ligands, including bisubstrate inhibitors, are conjugates of pharmacophores, which simultaneously target two binding sites of the biomolecule. Such structures offer attainable means for the development of compounds whose ability to bind to the biological target could be modulated by an external trigger. In the present work, two deactivatable bisubstrate inhibitors of basophilic protein kinases (PKs) were constructed by conjugating the pharmacophores via linkers that could be cleaved in response to external stimuli. The inhibitor ARC-2121 incorporated a photocleavable nitrodibenzofuran-comprising ß-amino acid residue in the structure of the linker. The pharmacophores of the other deactivatable inhibitor ARC-2194 were conjugated via reduction-cleavable disulfide bond. The disassembly of the inhibitors was monitored by HPLC-MS. The affinity and inhibitory potency of the inhibitors toward cAMP-dependent PK (PKAcα) were established by an equilibrium competitive displacement assay and enzyme activity assay, respectively. The deactivatable inhibitors possessed remarkably high 1-2-picomolar affinity toward PKAcα. Irradiation of ARC-2121 with 365 nm UV radiation led to reaction products possessing a 30-fold reduced affinity. The chemical reduction of ARC-2194 resulted in the decrease of affinity of over four orders of magnitude. The deactivatable inhibitors of PKs are valuable tools for the temporal inhibition or capture of these pharmacologically important enzymes.
Asunto(s)
Inhibidores de Proteínas Quinasas , Proteínas Quinasas , Aminoácidos , Sitios de Unión , Disulfuros , Inhibidores Enzimáticos , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/metabolismoRESUMEN
Recent clinical success with targeted covalent inhibitors points to new possibilities for development of protein kinase (PK)-targeted drugs by exploiting reactive cysteine residues in and around the ATP-binding site. However, more than 300 human PKs lack cysteine residues in the ATP-binding site. Here, we report the first covalent bisubstrate PK inhibitor whose electrophilic warhead reaches outside the ATP-binding site and reacts with a distant cysteine residue. A series of covalent inhibitors and their reversible counterparts were synthesized and characterized. The most potent reversible inhibitor possessed picomolar affinity and its cysteine-reactive counterpart revealed high value of kinact/KI ratio (6.2 × 107 M-1 s-1) for the reaction with the catalytic subunit of cAMP-dependent PK (PKAc). Under optimized conditions, fluorescent dye-labeled covalent inhibitors demonstrated PKA-selectivity in the cell lysate and reacted with several proteins inside live cells, including PKAc. The disclosed compounds serve as leads for targeting PKs possessing an analogously positioned cysteine residue.
Asunto(s)
Cisteína , Proteínas Quinasas , Adenosina Trifosfato , Sitios de Unión , Dominio Catalítico , Cisteína/química , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismoRESUMEN
STUDY QUESTION: Can a combination of the focussed protein kinase assays and a wide-scale proteomic screen pinpoint novel, clinically relevant players in decidualization in vitro and in vivo? SUMMARY ANSWER: Rho-dependent protein kinase (ROCK) activity is elevated in response to the combined treatment with progesterone and 8-Br-cAMP during in vitro decidualization, mirrored by increase of ROCK2 mRNA and protein levels and the phosphorylation levels of its downstream target Cofilin-1 (CFL1) in secretory versus proliferative endometrium. WHAT IS KNOWN ALREADY: Decidualization is associated with extensive changes in gene expression profile, proliferation, metabolism and morphology of endometrium, yet only a few underlying molecular pathways have been systematically explored. In vitro decidualization of endometrial stromal cells (ESCs) can be reportedly induced using multiple protocols with variable physiological relevance. In our previous studies, cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA)/prolactin axis that is classically upregulated during decidualization showed dampened activation in ESCs isolated from polycystic ovary syndrome (PCOS) patients as compared to controls. STUDY DESIGN, SIZE, DURATION: In vitro decidualization studies were carried out in passage 2 ESCs isolated from controls (N = 15) and PCOS patients (N = 9). In parallel, lysates of non-cultured ESCs isolated from proliferative (N = 4) or secretory (N = 4) endometrial tissue were explored. The observed trends were confirmed using cryo-cut samples of proliferative (N = 3) or secretory endometrium (N = 3), and in proliferative or secretory full tissue samples from controls (N = 8 and N = 9, respectively) or PCOS patients (N = 10 for both phases). PARTICIPANTS/MATERIALS, SETTING, METHODS: The activities of four target kinases were explored using kinase-responsive probes and selective inhibitors in lysates of in vitro decidualized ESCs and non-cultured ESCs isolated from tissue at different phases of the menstrual cycle. In the latter lysates, wide-scale proteomic and phosphoproteomic studies were further carried out. ROCK2 mRNA expression was explored in full tissue samples from controls or PCOS patients. The immunofluorescent staining of phosphorylated CFL1 was performed in full endometrial tissue samples, and in the in vitro decidualized fixed ESCs from controls or PCOS patients. Finally, the cellular migration properties were explored in live in vitro decidualized ESCs. MAIN RESULTS AND THE ROLE OF CHANCE: During in vitro decidualization, the activities of PKA, protein kinase B (Akt/PKB), and ROCK are increased while the activity of casein kinase 2 (CK2) is decreased; these initial trends are observable after 4-day treatment (P < 0.05) and are further augmented following the 9-day treatment (P < 0.001) with mixtures containing progesterone and 8-Br-cAMP or forskolin. The presence of progesterone is necessary for activation of ROCK, yet it is dispensable in the case of PKA and Akt/PKB; in comparison to controls, PCOS patient-derived ESCs feature dampened response to progesterone. In non-cultured ESCs isolated from secretory vs proliferative phase tissue, only activity of ROCK is increased (P < 0.01). ROCK2 protein levels are slightly elevated in secretory versus proliferative ESCs (relative mean standard deviation < 50%), and ROCK2 mRNA is elevated in mid-secretory versus proliferative full tissue samples (P < 0.05) obtained from controls but not PCOS patients. Activation of ROCK2 downstream signalling results in increase of phospho-S3 CFL1 in secretory endometrium (P < 0.001) as well as in vitro decidualized ESCs (P < 0.01) from controls but not PCOS patients. ROCK2-triggered alterations in the cytoskeleton are reflected by the significantly decreased motility of in vitro decidualized ESCs (P < 0.05). LARGE SCALE DATA: Proteomic and phosphoproteomic data are available via ProteomeXchange with identifier PXD026243. LIMITATIONS, REASONS FOR CAUTION: The number of biological samples was limited. The duration of protocol for isolation of non-cultured ESCs from tissue can potentially affect phosphorylation pathways in cells, yet the possible artefacts were minimized by the identical treatment of proliferative and secretory samples. WIDER IMPLICATIONS OF THE FINDINGS: The study demonstrated the benefits of combining the focussed kinase activity assay with wide-scale phosphoproteomics and showed the need for detailed elaboration of the in vitro decidualization protocols. ROCK was identified as the novel target of interest in decidualization, which requires closer attention in further studies-including the context of decidualization-related subfertility and infertility. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Estonian Ministry of Education and Research, and the Estonian Research Council (PRG1076, PRG454, PSG230 and PSG608), Enterprise Estonia (EU48695), Horizon 2020 innovation grant (ERIN, Grant no. EU952516) of the European Commission, the COMBIVET ERA Chair, H2020-WIDESPREAD-2018-04 (Grant agreement no. 857418), the Academy of Finland (Project grants 315921 and 321763), the Finnish Medical Foundation and The Sigrid Juselius Foundation. The authors confirm that they have no conflict of interest with respect to the content of this article.
Asunto(s)
Progesterona , Quinasas Asociadas a rho , Factores Despolimerizantes de la Actina , Endometrio , Femenino , Humanos , Proteómica , Células del Estroma , Quinasas Asociadas a rho/genéticaRESUMEN
We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.
Asunto(s)
Colorantes Fluorescentes , Imagen Molecular , Peptidomiméticos , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas c-pim-1 , Carbocianinas/química , Carbocianinas/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Humanos , Peptidomiméticos/química , Peptidomiméticos/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/metabolismoRESUMEN
We show that the antibody, clone mAb(D38C6), of the α isoform of the catalytic subunit of PKA (PKAcα) inhibits the kinase-catalyzed phosphorylation with low-nanomolar inhibitory potency (Ki = 2.4 nM). This property of the antibody was established by its capacity to displace a synthetic small-molecule active site-binding (orthosteric) photoluminescent ARC-Lum(Fluo) probe from the complex with PKAcα. Likely, the competitiveness of association of the two binders with the protein is coming from two excluding conformations of PKAcα to which the binders bind. mAb(D38C6) possesses a linear peptide epitope and it binds to the disordered C-tail of unliganded inactive conformer of PKAcα. ARC-Lum(Fluo) probes bind to the ordered and active conformation of PKAcα with Phe327 residue from the C-tail taking part in the formation of the active core. Consecutive application of these competitive PKAcα binders was used to develop an immunoassay allowing the determination of PKAcα concentration in complex biological solutions. At first, PKAcα was captured from the solution by the isoform-specific antibody and thereafter a high-affinity ARC-Lum(Fluo) probe was used to displace PKAcα from the binary complex. The developed immunoassay could be used for quantification of small amounts (starting from 93 pg, 2.3 fmol) of PKAcα in cell lysates.
Asunto(s)
Anticuerpos Monoclonales/química , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/análisis , Inmunoensayo , Sondas Moleculares/química , Péptidos/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Especificidad de Anticuerpos , Sitios de Unión , Unión Competitiva , Línea Celular Tumoral , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Células HeLa , Humanos , Cinética , Mediciones Luminiscentes , Modelos Moleculares , Péptidos/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de ProteínaRESUMEN
Protein kinase CK2, a heterotetrameric holoenzyme composed of two catalytic chains (CK2α) attached to a homodimer of regulatory subunits (CK2ß), is a target for drug development for cancer therapy. Here, we describe the tetraiodobenzimidazole derivative ARC-3140, a bisubstrate inhibitor addressing the ATP site and the substrate-binding site of CK2 with extraordinary affinity (Ki = 84 pM). In a crystal structure of ARC-3140 in complex with CK2α, three copies of the inhibitor are visible, one of them at the CK2ß interface of CK2α. Subsequent interaction studies based on microscale thermophoresis and fluorescence anisotropy changes revealed a significant impact of ARC-3140 and of its tetrabromo equivalent ARC-1502 on the CK2α/CK2ß interaction. A structural inspection revealed that ARC-3140, unlike CK2ß antagonists described so far, interferes with both sub-interfaces of the bipartite CK2α/CK2ß interaction. Thus, ARC-3140 is a lead for the further development of highly effective compounds perturbating the quaternary structure of the CK2α2ß2 holoenzyme.
Asunto(s)
Bencimidazoles/química , Bencimidazoles/farmacología , Quinasa de la Caseína II/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Quinasa de la Caseína II/química , Quinasa de la Caseína II/metabolismo , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Halogenación , Humanos , Simulación del Acoplamiento Molecular , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína/efectos de los fármacos , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
Photocaging of a tight-binding bisubstrate inhibitor of cAMP-dependent protein kinase (PKA) with a nitrodibenzofuran-based group fully abolished its inhibitory potency. The affinity difference between the photocaged and the active inhibitor was over 5 orders of magnitude. The photocaged inhibitor disrupted the PKA holoenzyme in cell lysates upon photolysis under a 398 nm LED.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dibenzofuranos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Animales , Células CHO , Cricetulus , Dibenzofuranos/síntesis química , Dibenzofuranos/química , Dibenzofuranos/efectos de la radiación , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/efectos de la radiación , Purinas/síntesis química , Purinas/química , Purinas/efectos de la radiación , Rayos UltravioletaRESUMEN
A previously disclosed protein kinase (PK) CK2-selective inhibitor 4-(2-amino-1,3-thiazol-5-yl)benzoic acid (ATB) and its selenium-containing counterpart (ASB) revealed remarkable room temperature phosphorescence when bound to the ATP pocket of the protein kinase CK2. Conjugation of these fragments with a mimic of CK2 substrate peptide resulted in bisubstrate inhibitors with increased affinity towards the kinase. Attachment of the fluorescent acceptor dye 5-TAMRA to the conjugates led to significant enhancement of intensity of long-lifetime (microsecond-scale) photoluminescence of both sulfur- and selenium-containing compounds. The developed photoluminescent probes make possible selective determination of the concentration of CK2 in cell lysates and characterization of CK2 inhibitors by means of time-gated measurement of photoluminescence.
Asunto(s)
Colorantes Fluorescentes/química , Compuestos de Organoselenio/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Tiazoles/farmacología , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Relación Dosis-Respuesta a Droga , Fluorescencia , Polarización de Fluorescencia , Humanos , Estructura Molecular , Compuestos de Organoselenio/química , Procesos Fotoquímicos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Tiazoles/química , Factores de TiempoRESUMEN
Cancer cells express high levels of CK2, and its inhibition leads to apoptosis. CK2 has therefore emerged as a new drug target for cancer therapy. A biligand inhibitor ARC-772 was constructed by conjugating 4-(2-amino-1,3-thiazol-5-yl)benzoic acid and a carboxylate-rich peptoid. ARC-772 was found to bind CK2 with a Kd value of 0.3â nm and showed remarkable CK2 inhibitory selectivity in a panel of 140 protein kinases (Gini coefficient: 0.75 at c=100â nm). ARC-775, the acetoxymethyl ester prodrug of ARC-772, was efficiently taken up by cells. Once internalized, the inhibitor is activated by cellular esterase activity. In HeLa cancer cells ARC-775 was found to activate caspase-3 (an apoptosis marker) at sub-micromolar concentrations (EC50 =0.3â µm), a 20-fold lower extracellular concentration than CX-4945, the only CK2 inhibitor under clinical trials. At micromolar concentrations, ARC-775 was also found to inhibit ADP-induced aggregation of human platelets. The overall results of this study demonstrate that oligo-anionic biligand inhibitors have good potential for drug development.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Quinasa de la Caseína II/antagonistas & inhibidores , Caspasa 3/metabolismo , Glicina/análogos & derivados , Agregación Plaquetaria/efectos de los fármacos , Tiazoles/síntesis química , Tiazoles/farmacología , Quinasa de la Caseína II/metabolismo , Caspasa 3/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glicina/síntesis química , Glicina/farmacología , Células HeLa , Humanos , Estructura Molecular , FosforilaciónRESUMEN
High demand for inhibitors regulating the activity of protein kinases has stimulated the quest for high throughput and reliable compound screening assays. Here we introduce a method applying a non-metal photoluminescent probe ARC-Lum(Fluo) for determination of dissociation constants of competitive inhibitors of protein kinases. Employing a single probe instead of a combination of antibody and fluorescent tracer makes the assay simpler, cheaper, and more accurate than several other inhibitor-screening technologies. High affinity (20 pM) and low background signal of the free probe supports the determination of dissociation constants of tight-binding as well as low affinity inhibitors. The calculated lowest Kd value that can be accurately determined with the method is 60 fM. We also introduce graphical presentation of the linearized Cheng-Prusoff equation and demonstrate multiple possibilities for its application (deciding upon the assay formats, calculation of the limits of Kd determination, etc.). The open toolbox (http://www.ut.ee/medchem/toolbox-fluorescence-probes) is available for creating the map of resolvable affinities if applying the competitive probes at defined assay conditions.
Asunto(s)
Técnicas Biosensibles/métodos , Proteínas Quinasas Dependientes de AMP Cíclico/química , Fluorescencia , Inhibidores de Proteínas Quinasas/análisis , Humanos , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Structurally diverse inhibitors of the protein kinase CK2 are required for regulation of this ubiquitous protein to establish biological roles of the enzyme which catalyzes the phosphorylation of a vast number of substrate proteins. In this article we disclose a series of new bisubstrate inhibitors of CK2 that are structurally represented by the oligo(l-Asp) peptide conjugates of benzo[c][2,6]naphthyridine-8-carboxylic acid. This fragment originated from CX-4945, the first in class inhibitor taken to clinical trials. The most potent conjugates possessed two-digit picomolar affinity and clear selectivity for CK2α in a panel of 140 protein kinases. Labeling of the inhibitors with a fluorescent dye yielded probes for a fluorescence anisotropy-based binding/displacement assay which can be used for analysis of CK2 and precise determination of affinity of the highly potent (tight-binding) CK2-targeting inhibitors.
Asunto(s)
Ácido Aspártico/química , Quinasa de la Caseína II/antagonistas & inhibidores , Naftiridinas/química , Naftiridinas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
The atypical protein kinase haspin is a key player in mitosis by catalysing the phosphorylation of Thr3 in histoneâ H3, and thus ensuring the normal function of the chromosomal passenger complex. Here, we report the development of bisubstrate-analogue inhibitors targeting haspin. The compounds were constructed by linking 5-iodotubercidin to the Nâ terminus of histoneâ H3 peptide. The new conjugates show high affinity (sub-nanomolar KD ) towards haspin as well as slow kinetics of association and dissociation (residence time of several hours). This reflects a unique binding mode and translated into improved selectivity. The latter was confirmed in a biochemical binding/displacement assay with a panel of ten protein kinases, in a thermal shift assay with off-targets of 5-iodotubercidin (adenosine kinase and the Cdc2-like kinase family) and in assay with spiked HeLa cell lysate.
Asunto(s)
Histonas/química , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Fragmentos de Péptidos/química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Tubercidina/análogos & derivados , Colorantes Fluorescentes/química , Células HeLa , Histonas/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Cinética , Fragmentos de Péptidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/química , Rodaminas/química , Temperatura , Tubercidina/química , Tubercidina/farmacologíaRESUMEN
CK2 is a ubiquitous serine/threonine protein kinase, which has the potential to catalyze the generation of a large proportion of the human phosphoproteome. Due to its role in numerous cellular functions and general anti-apoptotic activity, CK2 is an important target of research with therapeutic potential. This emphasizes the need for cell-permeable highly potent and selective inhibitors and photoluminescence probes of CK2 for investigating the protein phosphorylation networks in living cells. Previously, we had developed bisubstrate inhibitors for CK2 (CK2-targeted ARCs) that showed remarkable affinity (KD < 1 nM) and selectivity, but lacked proteolytic stability and plasma membrane permeability. In this report, the structures of CK2-targeted ARCs were modified for the application in live cells. Based on structure-activity studies, proteolytically stable achiral oligoanionic peptoid conjugates of 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz) were constructed. Affinity of the conjugates toward CK2 reached subnanomolar range. Acetoxymethyl (AM) prodrug strategy was applied for loading TBBz-peptoid conjugates into living cells. The uptake of inhibitors was visualized by live cell imaging and the reduction of the phosphorylation levels of two CK2-related phosphosites, Cdc37 pSer13 and NFκB pSer529, was demonstrated by Western blot analysis.
Asunto(s)
Bencimidazoles/química , Bencimidazoles/farmacología , Peptoides/química , Peptoides/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Bencimidazoles/farmacocinética , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Supervivencia Celular/efectos de los fármacos , Esterificación , Células HeLa , Humanos , Modelos Moleculares , Peptoides/farmacocinética , Inhibidores de Proteínas Quinasas/farmacocinéticaRESUMEN
An assay was developed for the characterization of protein kinase inhibitors in lysates of mammalian cells based on the measurement of FRET between overexpressed red fluorescent protein (TagRFP)-fused protein kinases (PKs) and luminophore-labeled small-molecule inhibitors (ARC-Photo probes). Two types of the assay, one using TagRFP as the photoluminescence donor together with ARC-Photo probes containing a red fluorophore dye as acceptor, and the other using TagRFP as the acceptor fluorophore in combination with a terbium cryptate-based long-lifetime photoluminescence donor, were used for FRET-based measurements in lysates of the cells overexpressing TagRFP-fused PKs. The second variant of the assay enabled the performance of the measurements under time-resolved conditions that led to substantially higher values of the signal/background ratio and further improved the reliability of the assay.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Animales , Clonación Molecular , Células HeLa , Humanos , Sustancias Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Células 3T3 NIH , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Regulación hacia Arriba , Proteína Fluorescente RojaRESUMEN
Benzoselenadiazole-containing inhibitors of protein kinases were constructed and their capability to emit phosphorescence in the kinase-bound state was established. Labelling of the inhibitors with a red fluorescent dye led to sensitive responsive photoluminescent probes for protein kinase CK2 that emitted red light with a long (microsecond-scale) decay time upon excitation of the probes with a pulse of near-UV light.
Asunto(s)
Azoles/química , Quinasa de la Caseína II/química , Colorantes/química , Compuestos de Organoselenio/química , Péptidos/química , Azoles/efectos de la radiación , Colorantes/efectos de la radiación , Proteínas Quinasas Dependientes de AMP Cíclico/química , Luz , Compuestos de Organoselenio/efectos de la radiación , Péptidos/efectos de la radiación , Proteínas Proto-Oncogénicas c-pim-1/químicaRESUMEN
We report the development of three fluorescent probes for protein kinase Aurora A that are derived from the well-known inhibitors MLN8237 and VX-689 (MK-5108). Two of these probes target the ATP site of Aurora A, and one targets simultaneously the ATP and substrate sites of the kinase. The probes were tested in an assay with fluorescence polarisation/anisotropy readout, and we demonstrated slow association kinetics and long residence time of the probes (kon 10(5)-10(7) M(-1) s(-1), koff 10(-3)-10(-4) s(-1); residence time 500-3000 s). The presence of the Aurora A activator TPX2 caused a significant reduction in the on-rate and increase in the off-rate of fluorescent probes targeting ATP site. These observations were supported by Aurora A inhibition assays with MLN8237 and VX-689. Overall, our results emphasise the importance of rational design of experiments with these compounds and correct interpretation of the obtained data.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Azepinas/química , Ácidos Ciclohexanocarboxílicos/química , Colorantes Fluorescentes/química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química , Tiazoles/química , Aurora Quinasa A/metabolismo , Azepinas/metabolismo , Ácidos Ciclohexanocarboxílicos/metabolismo , Polarización de Fluorescencia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Semivida , Cinética , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Pirimidinas/metabolismo , Termodinámica , Tiazoles/metabolismoRESUMEN
Responsive ARC-Lum probes were used for measurement of the concentration of active protein kinases (PKs) and determination of affinity of inhibitors of PKs. ARC-Lum probes incorporate thiophene or a selenophene heterocycle and a fluorophore conjugated to the lysine residue in the peptide fragment. In the complex with a PK, ARC-Lum probes emit long-lifetime (microsecond-scale) luminescence at the emission wavelengths of the fluorescent label if the complex is illuminated at the excitation wavelength of the thiophene- or selenophene-containing phosphorescence donors. Bisubstrate ARC-Lum probes bind with sub-nanomolar affinity with several PKs of the AGC group. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
Asunto(s)
Colorantes Fluorescentes/metabolismo , Lisina/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Algoritmos , Anisotropía , Sitios de Unión , Unión Competitiva , Biocatálisis/efectos de los fármacos , Colorantes Fluorescentes/química , Cinética , Lisina/química , Modelos Químicos , Estructura Molecular , Unión Proteica , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Espectrofotometría , Estaurosporina/metabolismo , Estaurosporina/farmacología , Tiofenos/química , Tiofenos/metabolismo , Factores de TiempoRESUMEN
Up-regulation of an acidophilic protein kinase, CK2, has been established in several types of cancer. This cognition has made CK2 an important target for drug development for cancer chemotherapy. The characterization of potential drug candidates, determination of the structure and clarification of the functions of CK2 could be facilitated by the application of small-molecule fluorescent probes that bind to the active site of the enzyme with high affinity and selectivity. We have used a bisubstrate approach for the development of a highly potent inhibitor of CK2. 4,5,6,7-Tetrabromo-1H-benzimidazole was conjugated with peptides containing multiple aspartate residues via different linkers. The design of the inhibitors was by crystallographic analysis of the complex of an inhibitor with the catalytic subunit of the enzyme (CK2α). The inhibitory potency of the synthesized compounds was established in a kinetic assay that used thin layer chromatography for the measurement of the rate of phosphorylation of fluorescently labelled peptide 5-TAMRA-RADDSDDDDD. The most potent inhibitor, ARC-1502 (K(i) = 0.5 nM), revealed high selectivity for CK2α in a panel of 140 protein kinases. Labelling of ARC-1502 with PromoFluor-647 gave the fluorescent probe ARC-1504 that possessed subnanomolar affinity towards both CK2α and the holoenzyme. The probe was used in a fluorescence anisotropy-based binding assay to measure the concentration of CK2α and characterize non-labelled ligands binding to the active site of CK2α.
Asunto(s)
Bencimidazoles/farmacología , Quinasa de la Caseína II/antagonistas & inhibidores , Colorantes Fluorescentes/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Bencimidazoles/síntesis química , Bencimidazoles/química , Quinasa de la Caseína II/química , Quinasa de la Caseína II/metabolismo , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Cinética , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
Conjugates of adenosine mimics and d-arginine-rich peptides (ARCs) are potent inhibitors of protein kinases (PKs) from the AGC group. Labeling ARCs with fluorescent dyes or immobilizing on chip surfaces gives fluorescent probes (ARC-Photo) and biosensors that can be used for high-throughput screening (HTS) of inhibitors of protein kinases. The bisubstrate character (simultaneous association with both binding sites of the kinase) and high affinity of ARCs allow ARC-based probes and sensors to be used for characterization of inhibitors targeted to either binding site of the kinase with affinities in whole nanomolar to micromolar range. The ability to penetrate cell plasma membrane and bind to the target kinase fused with a fluorescent protein leads to the possibility to use ARC-Photo probes for high content screening (HCS) of inhibitors in cellular milieu with detection of intensity of Förster resonance energy transfer (FRET) between two fluorophores.