RESUMO
High-affinity fluorescent derivatives of cyclic adenosine and guanosine monophosphate are powerful tools for investigating their natural targets. Cyclic nucleotide-regulated ion channels belong to these targets and are vital for many signal transduction processes, such as vision and olfaction. The relation of ligand binding to activation gating is still challenging, and there is a need for fluorescent probes that enable the process to be broken down to the single-molecule level. This inspired us to prepare fluorophore-labeled cyclic nucleotides, which are composed of a bright dye and a nucleotide derivative with a thiophenol motif at position 8 that has already been shown to enable superior binding affinity. These bioconjugates were prepared by a novel cross-linking strategy that involves substitution of the nucleobase with a modified thiophenolate in good yield. Both fluorescent nucleotides are potent activators of different cyclic nucleotide-regulated ion channels with respect to the natural ligand and previously reported substances. Molecular docking of the probes excluding the fluorophore reveals that the high potency can be attributed to additional hydrophobic and cation-π interactions between the ligand and the protein. Moreover, the introduced substances have the potential to investigate related target proteins, such as cAMP- and cGMP-dependent protein kinases, exchange proteins directly activated by cAMP or phosphodiesterases.
Assuntos
AMP Cíclico/química , AMP Cíclico/farmacologia , GMP Cíclico/química , GMP Cíclico/farmacologia , Corantes Fluorescentes/química , Canais Iônicos/agonistas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Conformação ProteicaRESUMO
Ionotropic purinergic receptors (P2X receptors) are non-specific cation channels that are activated by the binding of ATP at their extracellular side. P2X receptors contribute to multiple functions, including the generation of pain, inflammation, or synaptic transmission. The channels are trimers and structural information on several of their isoforms is available. In contrast, the cooperation of the subunits in the activation process is poorly understood. We synthesized a novel fluorescent ATP derivative, 2-[DY-547P1]-AET-ATP (fATP) to unravel the complex activation process in P2X2 and mutated P2X2 H319K channels with enhanced apparent affinity by characterizing the relation between ligand binding and activation gating. fATP is a full agonist with respect to ATP that reports the degree of binding by bright fluorescence. For quantifying the binding, a fast automated algorithm was employed on human embryonic kidney cell culture images. The concentrations of half maximum occupancy and activation as well as the respective Hill coefficients were determined. All Hill coefficients exceeded unity, even at an occupancy <10%, suggesting cooperativity of the binding even for the first and second binding step. fATP shows promise for continuative functional studies on other purinergic receptors and, beyond, any other ATP-binding proteins.
Assuntos
Trifosfato de Adenosina/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Agonistas do Receptor Purinérgico P2X/síntese química , Agonistas do Receptor Purinérgico P2X/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Animais , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Ligantes , Ligação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
A highly specific molecular interaction of diffusible ligands with their receptors belongs to the key processes in cellular signaling. Because an appropriate method to monitor the unitary binding events is still missing, most of our present knowledge is based on ensemble signals recorded from a big number of receptors, such as ion currents or fluorescence changes of suitably labeled receptors, and reasoning from these data to the ligand binding. To study the binding process itself, appropriately tagged ligands are required that fully activate the receptors and report the binding at the same time. Herein, we tailored a series of 18 novel fluorescent cyclic nucleotide derivatives by attaching 6 different dyes via different alkyl linkers to the 8-position of the purine ring of cGMP or cAMP. The biological activity was determined in inside-out macropatches containing either homotetrameric (CNGA2), heterotetrameric (CNGA2:CNGA4:CNGB1b), or hyperpolarization-activated cyclic nucleotide-modulated (HCN2) channels. All these novel fluorescent ligands are efficient to activate the channels, and the potency of most of them significantly exceeded that of the natural cyclic nucleotides cGMP or cAMP. Moreover, some of them showed an enhanced brightness when bound to the channels. The best of our derivatives bear great potential to systematically analyze the activation mechanism in CNG and HCN channels, at both the level of ensemble and single-molecule analyses.
Assuntos
AMP Cíclico/química , GMP Cíclico/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Corantes Fluorescentes/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica , Imagem Individual de MoléculaRESUMO
Synthetic derivatives of cyclic adenosine monophosphate, such as halogenated or other more hydrophobic analogs, are widely used compounds, to investigate diverse signal transduction pathways of eukaryotic cells. This inspired us to develop cyclic nucleotides, which exhibit chemical structures composed of brominated 7-deazaadenines and the phosphorylated ribosugar. The synthesized 8-bromo- and 7-bromo-7-deazaadenosine-3',5'-cyclic monophosphates rank among the most potent activators of cyclic nucleotide-regulated ion channels as well as cAMP-dependent protein kinase. Moreover, these substances bind tightly to exchange proteins directly activated by cAMP.
Assuntos
AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Adenina/análogos & derivados , Adenina/síntese química , Adenina/química , Adenina/farmacologia , Animais , AMP Cíclico/síntese química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/agonistas , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/agonistas , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Halogenação , Humanos , CamundongosRESUMO
Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are tetrameric non-specific cation channels in the plasma membrane that are activated by either cAMP or cGMP binding to specific binding domains incorporated in each subunit. Typical apparent affinities of these channels for these cyclic nucleotides range from several hundred nanomolar to tens of micromolar. Here we synthesized and characterized novel cAMP and cGMP derivatives by substituting either hydrophobic alkyl chains or similar-sized more hydrophilic heteroalkyl chains to the 8-position of the purine ring with the aim to obtain full agonists of higher potency. The compounds were tested in homotetrameric CNGA2, heterotetrameric CNGA2:CNGA4:CNGB1b and homotetrameric HCN2 channels. We show that nearly all compounds are full agonists and that longer alkyl chains systematically increase the apparent affinity, at the best more than 30 times. The effects are stronger in CNG than HCN2 channels which, however, are constitutively more sensitive to cAMP. Kinetic analyses reveal that the off-rate is significantly slowed by the hydrophobic alkyl chains. Molecular dynamics simulations and free energy calculations suggest that an intricate enthalpy - entropy compensation underlies the higher apparent affinity of the derivatives with the longer alkyl chains, which is shown to result from a reduced loss of configurational entropy upon binding.
Assuntos
AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/agonistas , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/agonistas , Animais , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Entropia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Ratos , Termodinâmica , XenopusRESUMO
C-X-C motif chemokine 12/C-X-C chemokine receptor type 4 (CXCL12/CXCR4) signaling is involved in ontogenesis, hematopoiesis, immune function and cancer. Recently, the orphan chemokine CXCL14 was reported to inhibit CXCL12-induced chemotaxis - probably by allosteric modulation of CXCR4. We thus examined the effects of CXCL14 on CXCR4 regulation and function using CXCR4-transfected human embryonic kidney (HEK293) cells and Jurkat T cells. CXCL14 did not affect dose-response profiles of CXCL12-induced CXCR4 phosphorylation, G protein-mediated calcium mobilization, dynamic mass redistribution, kinetics of extracellular signal-regulated kinase 1 (ERK1) and ERK2 phosphorylation or CXCR4 internalization. Hence, essential CXCL12-operated functions of CXCR4 are insensitive to CXCL14, suggesting that interactions of CXCL12 and CXCL14 pathways depend on a yet to be identified CXCL14 receptor.