RESUMO
A series of 1,4-benzoxazin-3-one analogs were investigated to discover mode-selective TRPV1 antagonists, since such antagonists are predicted to minimize target-based adverse effects. Using the high-affinity antagonist 2 as the lead structure, the structure activity relationship was studied by modifying the A-region through incorporation of a polar side chain on the benzoxazine and then by changing the C-region with a variety of substituted pyridine, pyrazole and thiazole moieties. The t-butyl pyrazole and thiazole C-region analogs provided high potency as well as mode-selectivity. Among them, antagonist 36 displayed potent and capsaicin-selective antagonism with IC50 = 2.31 nM for blocking capsaicin activation and only 47.5 % inhibition at 3 µM concentration toward proton activation, indicating that more than a 1000-fold higher concentration of 36 was required to inhibit proton activation than was required to inhibit capsaicin activation. The molecular modeling study of 36 with our homology model indicated that two π-π interactions with the Tyr511 and Phe591 residues by the A- and C-region and hydrogen bonding with the Thr550 residue by the B-region were critical for maintaining balanced and stable binding. Systemic optimization of antagonist 2, which has high-affinity but full antagonism for activators of all modes, led to the mode-selective antagonist 36 which represents a promising step in the development of clinical TRPV1 antagonists minimizing side effects such as hyperthermia and impaired heat sensation.
Assuntos
Benzoxazinas , Canais de Cátion TRPV , Ureia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo , Relação Estrutura-Atividade , Benzoxazinas/química , Benzoxazinas/farmacologia , Benzoxazinas/síntese química , Ureia/análogos & derivados , Ureia/química , Ureia/farmacologia , Ureia/síntese química , Humanos , Estrutura Molecular , Animais , Capsaicina/farmacologia , Capsaicina/química , Descoberta de Drogas , Relação Dose-Resposta a DrogaRESUMO
To discover mode-selective TRPV1 antagonists as thermoneutral drug candidates, the previous potent antagonist benzopyridone 2 was optimized based on the pharmacophore A- and C-regions. The structure activity relationship was investigated systematically by modifying the A-region by incorporating a polar side chain on the pyridone and then by changing the C-region with a variety of substituted pyridine and pyrazole moieties. The 3-t-butyl and 3-(1-methylcyclopropyl) pyrazole C-region analogs provided high potency as well as mode-selectivity. Among them, 51 and 54 displayed potent and capsaicin-selective antagonism with IC50 = 2.85 and 3.27 nM to capsaicin activation and 28.5 and 31.5 % inhibition at 3 µM concentration toward proton activation, respectively. The molecular modeling study of 51 with our homology model indicated that the hydroxyethyl side chain in the A-region interacted with Arg557 and Glu570, the urea B-region engaged in hydrogen bonding with Tyr511 and Thr550, respectively, and the pyrazole C-region made two hydrophobic interactions with the receptor. Optimization of antagonist 2, which has full antagonism for activators of all modes, lead to mode-selective antagonists 51 and 54. These observations will provide insight into the future development of clinical TRPV1 antagonists without target-based side effects.
Assuntos
Capsaicina , Ureia , Ureia/química , Capsaicina/farmacologia , Relação Estrutura-Atividade , Modelos Moleculares , Pirazóis/farmacologia , Canais de Cátion TRPVRESUMO
Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. In our previous work, we identified in racemate 1-2, based on the 2-benzyl-3-hydroxypropyl ester scaffold, two new potent and promising PKCα and PKCδ ligands, targeting the C1 domain of these two kinases. Herein, we report the resolution of the racemates by enantioselective semi-preparative HPLC. The attribution of the absolute configuration (AC) of homochirals 1 was performed by NMR, via methoxy-α-trifluoromethyl-α-phenylacetic acid derivatization (MTPA or Mosher's acid). Moreover, the match between the experimental and predicted electronic circular dichroism (ECD) spectra confirmed the assigned AC. These results proved that Mosher's esters can be properly exploited for the determination of the AC also for chiral primary alcohols. Lastly, homochiral 1 and 2 were assessed for binding affinity and functional activity against PKCα. No significative differences in the Ki of the enantiopure compounds was observed, thus suggesting that chirality does not seem to play a significant role in targeting PKC C1 domain. These results are in accordance with the molecular docking studies performed using a new homology model for the human PKCαC1B domain.
Assuntos
Ésteres , Proteína Quinase C-alfa , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Simulação de Acoplamento Molecular , EstereoisomerismoRESUMO
A series consisting of 117 2-(halogenated phenyl) acetamide and propanamide analogs were investigated as TRPV1 antagonists. The structure-activity analysis targeting their three pharmacophoric regions indicated that halogenated phenyl A-region analogs exhibited a broad functional profile ranging from agonism to antagonism. Among the compounds, antagonists 28 and 92 exhibited potent antagonism toward capsaicin for hTRPV1 with Ki[CAP] = 2.6 and 6.9 nM, respectively. Further, antagonist 92 displayed promising analgesic activity in vivo in both phases of the formalin mouse pain model. A molecular modeling study of 92 indicated that the two fluoro groups in the A-region made hydrophobic interactions with the receptor.
Assuntos
Acetamidas/farmacologia , Amidas/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Acetamidas/síntese química , Acetamidas/química , Amidas/síntese química , Amidas/química , Animais , Relação Dose-Resposta a Droga , Humanos , Camundongos , Estrutura Molecular , Relação Estrutura-Atividade , Canais de Cátion TRPV/metabolismoRESUMO
Among a series of benzopyridone-based scaffolds investigated as human transient receptor potential vanilloid 1 (TRPV1) ligands, two isomeric benzopyridone scaffolds demonstrated a consistent and distinctive functional profile in which 2-oxo-1,2-dihydroquinolin-5-yl analogues (e.g., 2) displayed high affinity and potent antagonism, whereas 1-oxo-1,2-dihydroisoquinolin-5-yl analogues (e.g., 3) showed full agonism with high potency. Our computational models provide insight into the agonist-antagonist boundary of the analogues suggesting that the Arg557 residue in the S4-S5 linker might be important for sensing the agonist binding and transmitting signals. These results provide structural insights into the TRPV1 and the protein-ligand interactions at a molecular level.
Assuntos
Descoberta de Drogas , Piridonas/química , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Animais , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Ureia/químicaRESUMO
A series of 1-indazol-3-(1-phenylpyrazol-5-yl)methyl ureas were investigated as hTRPV1 antagonists. The structure-activity relationship study was conducted systematically for both the indazole A-region and the 3-trifluoromethyl/t-butyl pyrazole C-region to optimize the antagonism toward the activation by capsaicin. Among them, the antagonists 26, 50 and 51 displayed highly potent antagonism with Ki(CAP) = 0.4-0.5 nM. Further, in vivo studies in mice indicated that these derivatives both antagonized capsaicin induced hypothermia, consistent with their in vitro activity, and themselves did not induce hyperthermia. In the formalin model, 51 showed anti-nociceptive activity in a dose-dependent manner.
Assuntos
Indazóis/farmacologia , Compostos de Metilureia/farmacologia , Pirazóis/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Analgésicos/síntese química , Analgésicos/farmacologia , Animais , Temperatura Corporal/efeitos dos fármacos , Células CHO , Capsaicina/farmacologia , Cricetulus , Humanos , Indazóis/síntese química , Compostos de Metilureia/síntese química , Camundongos , Estrutura Molecular , Pirazóis/síntese química , Relação Estrutura-Atividade , Canais de Cátion TRPV/agonistasRESUMO
A series of indane-type acetamide and propanamide analogues were investigated as TRPV1 antagonists. The analysis of structure-activity relationship indicated that indane A-region analogues exhibited better antagonism than did the corresponding 2,3-dihydrobenzofuran and 1,3-benzodioxole surrogates. Among them, antagonist 36 exhibited potent and selective antagonism toward capsaicin for hTRPV1 and mTRPV1. Further, in vivo studies indicated that antagonist 36 showed excellent analgesic activity in both phases of the formalin mouse pain model and inhibited the pain behavior completely at a dose of 1 mg/kg in the 2nd phase.
Assuntos
Amidas/química , Indanos/química , Canais de Cátion TRPV/antagonistas & inibidores , Acetamidas/química , Acetamidas/metabolismo , Acetamidas/uso terapêutico , Amidas/metabolismo , Amidas/uso terapêutico , Analgésicos/química , Analgésicos/uso terapêutico , Animais , Capsaicina/química , Capsaicina/metabolismo , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Dor/induzido quimicamente , Dor/tratamento farmacológico , Piridinas/química , Relação Estrutura-Atividade , Canais de Cátion TRPV/metabolismoRESUMO
Paradoxically, some TRPV1 agonists are, at the organismal level, both nonpungent and clinically useful as topical analgesics. Here, we describe the scaled-up synthesis and characterization in mouse models of a novel, nonpungent vanilloid. Potent analgesic activity was observed in models of neuropathic pain, and the compound blocked capsaicin induced allodynia, showing dermal accumulation with little transdermal absorption. Finally, it displayed much weaker systemic toxicity compared to capsaicin and was negative in assays of genotoxicity.
Assuntos
Analgésicos/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Canais de Cátion TRPV/agonistas , Tiazóis/uso terapêutico , Analgésicos/síntese química , Analgésicos/farmacocinética , Analgésicos/toxicidade , Animais , Células CHO , Capsaicina , Cricetulus , Descoberta de Drogas , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Camundongos Endogâmicos ICR , Neuralgia/tratamento farmacológico , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/farmacocinética , Compostos de Fenilureia/toxicidade , Suínos , Tiazóis/síntese química , Tiazóis/farmacocinética , Tiazóis/toxicidadeRESUMO
In order to discover a novel type of analgesic, we investigated dual activity ligands with TRPV1 antagonism and mu-opioid receptor affinity with the goal of eliciting synergistic analgesia while avoiding the side effects associated with single targeting. Based on a combination approach, a series of 4-benzyl-4-(dimethylamino)piperidinyl analogues were designed, synthesized and evaluated for their receptor activities. Among them, compound 49 exhibited the most promising dual-acting activity toward TRPV1 and the mu-opioid receptor in vitro. In vivo,49 displayed potent, dose-dependent antinociceptive activity in both the 1st and 2nd phases in the formalin assay. Consistent with its postulated mechanism, we confirmed that in vivo, as in vitro, compound 49 both antagonized TRPV1 and functioned as a mu-opioid agonist. This result indicates that dual-acting TRPV1 antagonist/mu-opioid ligands can be made and represent a new and promising class of analgesic.
Assuntos
Analgésicos Opioides/farmacologia , Descoberta de Drogas , Dor/tratamento farmacológico , Receptores Opioides/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Animais , Células CHO , Células Cultivadas , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos ICR , Estrutura Molecular , Dor/metabolismo , Relação Estrutura-Atividade , Canais de Cátion TRPV/metabolismoRESUMO
The capsaicin receptor TRPV1 (transient receptor potential vanilloid 1) has been an object of intense interest for pharmacological development on account of its critical role in nociception. In the course of structure activity analysis, it has become apparent that TRPV1 ligands may vary dramatically in the rates at which they interact with TRPV1, presumably reflecting differences in their abilities to penetrate into the cell. Using a fast penetrating agonist together with an excess of a slower penetrating antagonist, we find that we can induce an agonist response of limited duration and, moreover, the duration of the agonist response remains largely independent of the absolute dose of agonist, as long as the ratio of antagonist to agonist is held constant. This general approach for limiting agonist duration under conditions in which absolute agonist dose is variable should have more general applicability.
RESUMO
Bryostatin 1 is a natural macrolide shown to improve neuronal connections and enhance memory in mice. Its mechanism of action is largely attributed to the modulation of novel and conventional protein kinase Cs (PKCs) by binding to their regulatory C1 domains. Munc13-1 is a C1 domain-containing protein that shares common endogenous and exogenous activators with novel and conventional PKC subtypes. Given the essential role of Munc13-1 in the priming of synaptic vesicles and neuronal transmission overall, we explored the potential interaction between bryostatin 1 and Munc13-1. Our results indicate that in vitro bryostatin 1 binds to both the isolated C1 domain of Munc13-1 ( Ki = 8.07 ± 0.90 nM) and the full-length Munc13-1 protein ( Ki = 0.45 ± 0.04 nM). Furthermore, confocal microscopy and immunoblot analysis demonstrated that in intact HT22 cells bryostatin 1 mimics the actions of phorbol esters, a previously established class of Munc13-1 activators, and induces plasma membrane translocation of Munc13-1, a hallmark of its activation. Consistently, bryostatin 1 had no effect on the Munc13-1H567K construct that is insensitive to phorbol esters. Effects of bryostatin 1 on the other Munc13 family members, ubMunc13-2 and bMunc13-2, resembled those of Munc13-1 for translocation. Lastly, we observed an increased level of expression of Munc13-1 following a 24 h incubation with bryostatin 1 in both HT22 and primary mouse hippocampal cells. This study characterizes Munc13-1 as a molecular target of bryostatin 1. Considering the crucial role of Munc13-1 in neuronal function, these findings provide strong support for the potential role of Munc13s in the actions of bryostatin 1.
Assuntos
Briostatinas/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Animais , Sítios de Ligação , Linhagem Celular , Células Cultivadas , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Ésteres de Forbol/farmacologia , Ligação ProteicaRESUMO
Despite our extensive knowledge on the biology of protein kinase C (PKC) and its involvement in disease, limited success has been attained in the generation of PKC isozyme-specific modulators acting via the C1 domain, the binding site for the lipid second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. Synthetic efforts had recently led to the identification of AJH-836, a DAG-lactone with preferential affinity for novel isozymes (nPKCs) relative to classical PKCs (cPKCs). Here, we compared the ability of AJH-836 and a prototypical phorbol ester (phorbol 12-myristate 13-acetate, PMA) to induce changes in gene expression in a lung cancer model. Gene profiling analysis using RNA-Seq revealed that PMA caused major changes in gene expression, whereas AJH-836 only induced a small subset of genes, thus providing a strong indication for a major involvement of cPKCs in their control of gene expression. MMP1, MMP9, and MMP10 were among the genes most prominently induced by PMA, an effect impaired by RNAi silencing of PKCα, but not PKCδ or PKCε. Comprehensive gene signature analysis and bioinformatics efforts, including functional enrichment and transcription factor binding site analyses of dysregulated genes, identified major differences in pathway activation and transcriptional networks between PMA and DAG-lactones. In addition to providing solid evidence for the differential involvement of individual PKC isozymes in the control of gene expression, our studies emphasize the importance of generating targeted C1 domain ligands capable of differentially regulating PKC isozyme-specific function in cellular models.
Assuntos
Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lactonas/farmacologia , Neoplasias Pulmonares/genética , Ésteres de Forbol/farmacologia , Proteína Quinase C/metabolismo , Células A549 , Diglicerídeos/farmacologia , Descoberta de Drogas , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ligantes , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Proteína Quinase C/genética , Transcriptoma/efeitos dos fármacosRESUMO
A series of 2-(3,5-substituted 4-aminophenyl)acetamide and propanamide derivatives were investigated as human TRPV1 antagonists. The analysis of the structure-activity relationship indicated that 2-(3,5-dihalo 4-aminophenyl)acetamide analogues displayed excellent antagonism of hTRPV1 activation by capsaicin and showed improved potency compared to the corresponding propanamides. The most potent antagonist (36) exhibited potent and selective antagonism for hTRPV1 not only to capsaicin but also to NADA and elevated temperature; however, it only displayed weak antagonism to low pH. Further studies indicated that oral administration of antagonist 36 blocked the hypothermic effect of capsaicin in vivo but demonstrated hyperthermia at that dose. A docking study of 36 was performed in our established hTRPV1 homology model to understand its binding interactions with the receptor and to compare with that of previous antagonist 1.
Assuntos
Amidas/química , Canais de Cátion TRPV/antagonistas & inibidores , Acetamidas/química , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Amidas/farmacologia , Amidas/uso terapêutico , Sítios de Ligação , Capsaicina/química , Capsaicina/toxicidade , Humanos , Concentração de Íons de Hidrogênio , Hipotermia/patologia , Hipotermia/prevenção & controle , Ligantes , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Canais de Cátion TRPV/metabolismoRESUMO
Diacylglycerol-lactones have proven to be a powerful template for the design of potent ligands targeting C1 domains, the recognition motif for the cellular second messenger diacylglycerol. A major objective has been to better understand the structure activity relations distinguishing the seven families of signaling proteins that contain such domains, of which the protein kinase C (PKC) and RasGRP families are of particular interest. Here, we synthesize a series of aryl- and alkyl-substituted diacylglycerol-lactones and probe their relative selectivities for RasGRP3 versus PKC. Compound 96 showed 73-fold selectivity relative to PKCα and 45-fold selectivity relative to PKCε for in vitro binding activity. Likewise, in intact cells, compound 96 induced Ras activation, a downstream response to RasGRP stimulation, with 8-29 fold selectivity relative to PKCδ S299 phosphorylation, a measure of PKCδ stimulation.
Assuntos
Diglicerídeos/química , Desenho de Fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Lactonas/química , Lactonas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Domínios Proteicos , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-épsilon/metabolismo , Especificidade por Substrato , Fatores ras de Troca de Nucleotídeo GuaninaRESUMO
A series of A-region analogues of 2-(3-fluoro-4-methylsufonamidophenyl) propanamide 1 were investigated as TRPV1 antagonists. The analysis of structure-activity relationship indicated that a fluoro group at the 3- (or/and) 5-position and a methylsulfonamido group at the 4-position were optimal for antagonism of TRPV1 activation by capsaicin. The most potent antagonist 6 not only exhibited potent antagonism of activation of hTRPV1 by capsaicin, low pH and elevated temperature but also displayed highly potent antagonism of activation of rTRPV1 by capsaicin. Further studies demonstrated that antagonist 6 blocked the hypothermic effect of capsaicin in vivo, consistent with its in vitro mechanism, and it showed promising analgesic activity in the formalin animal model.
Assuntos
Amidas/farmacologia , Descoberta de Drogas , Canais de Cátion TRPV/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Animais , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Canais de Cátion TRPV/metabolismoRESUMO
Diacylglycerol (DAG) is a key lipid second messenger downstream of cellular receptors that binds to the C1 domain in many regulatory proteins. Protein kinase C (PKC) isoforms constitute the most prominent family of signaling proteins with DAG-responsive C1 domains, but six other families of proteins, including the chimaerins, Ras-guanyl nucleotide-releasing proteins (RasGRPs), and Munc13 isoforms, also play important roles. Their significant involvement in cancer, immunology, and neurobiology has driven intense interest in the C1 domain as a therapeutic target. As with other classes of targets, however, a key issue is the establishment of selectivity. Here, using [3H]phorbol 12,13-dibutyrate ([3H]PDBu) competition binding assays, we found that a synthetic DAG-lactone, AJH-836, preferentially binds to the novel PKC isoforms PKCδ and PKCϵ relative to classical PKCα and PKCßII. Assessment of intracellular translocation, a hallmark for PKC activation, revealed that AJH-836 treatment stimulated a striking preferential redistribution of PKCϵ to the plasma membrane relative to PKCα. Moreover, unlike with the prototypical phorbol ester phorbol 12-myristate 13-acetate (PMA), prolonged exposure of cells to AJH-836 selectively down-regulated PKCδ and PKCϵ without affecting PKCα expression levels. Biologically, AJH-836 induced major changes in cytoskeletal reorganization in lung cancer cells, as determined by the formation of membrane ruffles, via activation of novel PKCs. We conclude that AJH-836 represents a C1 domain ligand with PKC-activating properties distinct from those of natural DAGs and phorbol esters. Our study supports the feasibility of generating selective C1 domain ligands that promote novel biological response patterns.
Assuntos
Diglicerídeos/química , Lactonas/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/metabolismo , Proteína Quinase C-épsilon/metabolismo , Células A549 , Ligação Competitiva , Células HeLa , Humanos , Ligantes , Ligação Proteica , Transporte Proteico , Especificidade por SubstratoRESUMO
Important strides are being made in understanding the effects of structural features of bryostatinâ 1, a candidate therapeutic agent for cancer and dementia, in conferring its potency toward protein kinaseâ C and the unique spectrum of biological responses that it induces. A critical pharmacophoric element in bryostatinâ 1 is the secondary hydroxy group at the C26 position, with a corresponding primary hydroxy group playing an analogous role in binding of phorbol esters to protein kinase C. Herein, we describe the synthesis of a bryostatin homologue in which the C26 hydroxy group is primary, as it is in the phorbol esters, and show that its biological activity is almost indistinguishable from that of the corresponding compound with a secondary hydroxy group.
Assuntos
Briostatinas/química , Briostatinas/farmacologia , Desenho de Fármacos , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Briostatinas/síntese química , Briostatinas/farmacocinética , Linhagem Celular Tumoral , Humanos , Metilação , Camundongos , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Relação Estrutura-AtividadeRESUMO
To investigate the cellular distribution of tumor-promoting vs. non-tumor-promoting bryostatin analogues, we synthesized fluorescently labeled variants of two bryostatin derivatives that have previously shown either phorbol ester-like or bryostatin-like biological activity in U937 leukemia cells. These new fluorescent analogues both displayed high affinity for protein kinaseâ C (PKC) binding and retained the basic properties of the parent unlabeled compounds in U937 assays. The fluorescent compounds showed similar patterns of intracellular distribution in cells, however; this argues against an existing hypothesis that various patterns of intracellular distribution are responsible for differences in biological activity. Upon further characterization, the fluorescent compounds revealed a slow rate of cellular uptake; correspondingly, they showed reduced activity for cellular responses that were only transient upon treatment with phorbol ester or bryostatinâ 1.
Assuntos
Briostatinas/química , Corantes Fluorescentes/química , Humanos , Ésteres de Forbol/química , Ligação Proteica , Proteína Quinase C/metabolismo , Células U937RESUMO
The PKC isozymes represent the most prominent family of signaling proteins mediating response to the ubiquitous second messenger diacylglycerol. Among them, PKCθ is critically involved in T-cell activation. Whereas all the other conventional and novel PKC isoforms have twin C1 domains with potent binding activity for phorbol esters, in PKCθ only the C1b domain possesses potent binding activity, with little or no activity reported for the C1a domain. In order to better understand the structural basis accounting for the very weak ligand binding of the PKCθ C1a domain, we assessed the effect on ligand binding of twelve amino acid residues which differed between the C1a and C1b domains of PKCθ. Mutation of Pro9 of the C1a domain of PKCθ to the corresponding Lys9 found in C1b restored in vitro binding activity for [3H]phorbol 12,13-dibutyrate to 3.6â¯nM, whereas none of the other residues had substantial effect. Interestingly, the converse mutation in the C1b domain of Lys9 to Pro9 only diminished binding affinity to 11.7â¯nM, compared to 254â¯nM in the unmutated C1a. In confocal experiments, deletion of the C1b domain from full length PKCθ diminished, whereas deletion of the C1a domain enhanced 5-fold (at 100â¯nM PMA) the translocation to the plasma membrane. We conclude that the Pro168 residue in the C1a domain of full length PKCθ plays a critical role in the ligand and membrane binding, while exchanging the residue (Lys240) at the same position in C1b domain of full length PKCθ only modestly reduced the membrane interaction.
Assuntos
Ésteres de Forbol/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C-theta/química , Proteína Quinase C-theta/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Proteína Quinase C-theta/genética , Células Tumorais CultivadasRESUMO
Munc13-1 is a presynaptic active-zone protein essential for neurotransmitter release and presynaptic plasticity in the brain. This multidomain scaffold protein contains a C1 domain that binds to the activator diacylglycerol/phorbol ester. Although the C1 domain bears close structural homology with the C1 domains of protein kinase C (PKC), the tryptophan residue at position 22 (588 in the full-length Munc13-1) occludes the activator binding pocket, which is not the case for PKC. To elucidate the role of this tryptophan, we generated W22A, W22K, W22D, W22Y, and W22F substitutions in the full-length Munc13-1, expressed the GFP-tagged constructs in Neuro-2a cells, and measured their membrane translocation in response to phorbol ester treatment by imaging of the live cells using confocal microscopy. The extent of membrane translocation followed the order, wild-type > W22K > W22F > W22Y > W22A > W22D. The phorbol ester binding affinity of the wild-type Munc13-1C1 domain and its mutants was phosphatidylserine (PS)-dependent following the order, wild-type > W22K > W22A ⫠W22D in both 20% and 100% PS. Phorbol ester affinity was higher for Munc13-1 than the C1 domain. While Munc13-1 translocated to the plasma membrane, the C1 domain translocated to internal membranes in response to phorbol ester. Molecular dynamics (80 ns) studies reveal that Trp-22 is relatively less flexible than the homologous Trp-22 of PKCδ and PKCθ. Results are discussed in terms of the overall negative charge state of the Munc13-1C1 domain and its possible interaction with the PS-rich plasma membrane. This study shows that Trp-588 is an important structural element for ligand binding and membrane translocation in Munc13-1.