RESUMO
Histone deacetylases (HDACs) play diverse roles in many diseases including cancer, sarcopenia, and Alzheimer's. Different isoforms of HDACs appear to play disparate roles in the cell and are associated with specific diseases; as such, a substantial effort has been made to develop isoform-selective HDAC inhibitors. Our group focused on developing HDAC1/HDAC2-specific inhibitors as a cancer therapeutic. In the course of characterizing the mechanism of inhibition of a novel HDAC1/2-selective inhibitor, it was determined that it did not exhibit classical Michaelis-Menten kinetic behavior; this result is in contrast to the seminal HDAC inhibitor SAHA. Enzymatic assays, along with a newly developed binding assay, were used to determine the rates of binding and the affinities of both the HDAC1/2-selective inhibitor and SAHA. The mechanism of action studies identified a potential conformational change required for optimal binding by the selective inhibitor. A model of this putative conformational change is proposed.
Assuntos
Antineoplásicos/química , Benzoatos/química , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 2/antagonistas & inibidores , Inibidores de Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Xantenos/química , Animais , Antineoplásicos/farmacologia , Benzoatos/farmacologia , Linhagem Celular Tumoral , Feminino , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Xenoenxertos , Histona Desacetilase 1/química , Histona Desacetilase 2/química , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Cinética , Camundongos , Camundongos Nus , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Vorinostat , Xantenos/farmacologiaRESUMO
The successful application of both solid and solution phase library synthesis, combined with tight integration into the medicinal chemistry effort, resulted in the efficient optimization of a novel structural series of selective HDAC1/HDAC2 inhibitors by the MRL-Boston Parallel Medicinal Chemistry group. An initial lead from a small parallel library was found to be potent and selective in biochemical assays. Advanced compounds were the culmination of iterative library design and possess excellent biochemical and cellular potency, as well as acceptable PK and efficacy in animal models.
Assuntos
Inibidores de Histona Desacetilases , Animais , Técnicas de Química Combinatória , Cães , Desenho de Fármacos , Histona Desacetilase 1 , Histona Desacetilase 2 , Humanos , Estrutura Molecular , Ratos , Proteínas Repressoras/antagonistas & inibidores , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We report the preparation and structure-activity relationships of phosphorus-containing histone deacetylase inhibitors. A strong trend between decreasing phosphorus functional group size and superior mouse pharmacokinetic properties was identified. In addition, optimized candidates showed tumor growth inhibition in xenograft studies.
Assuntos
Antineoplásicos/farmacocinética , Inibidores Enzimáticos/farmacocinética , Inibidores de Histona Desacetilases , Organofosfonatos/farmacocinética , Proteínas Repressoras/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Histona Desacetilase 1 , Histona Desacetilase 2 , Histona Desacetilases/metabolismo , Camundongos , Camundongos Nus , Organofosfonatos/síntese química , Organofosfonatos/química , Proteínas Repressoras/metabolismo , Transplante HeterólogoRESUMO
A class of biaryl benzamides was identified and optimized as selective HDAC1&2 inhibitors (SHI-1:2). These agents exhibit selectivity over class II HDACs 4-7, as well as class I HDACs 3 and 8; providing examples of selective HDAC inhibitors for the HDAC isoforms most closely associated with cancer. The hypothesis for the increased selectivity is the binding of a pendant aromatic group in the internal cavity of the HDAC1&2 enzymes. SAR development based on an initial lead led to a series of potent and selective inhibitors with reduced off-target activity and tumor growth inhibition activity in a HCT-116 xenograft model.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Proteínas Repressoras/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Histona Desacetilase 1 , Histona Desacetilase 2 , Camundongos , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
An HTS screening campaign identified a series of low molecular weight phenols that showed excellent selectivity (>100-fold) for HDAC1/HDAC2 over other Class I and Class II HDACs. Evolution and optimization of this HTS hit series provided HDAC1-selective (SHI-1) compounds with excellent anti-proliferative activity and improved physical properties. Dose-dependent efficacy in a mouse HCT116 xenograft model was demonstrated with a phenylglycine SHI-1 analog.
Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Glicina/análogos & derivados , Inibidores de Histona Desacetilases , Fenilalanina/química , Acetilação , Amidas , Animais , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Cães , Canal de Potássio ERG1 , Inibidores Enzimáticos/farmacocinética , Canais de Potássio Éter-A-Go-Go/metabolismo , Glicina/química , Histona Desacetilase 1 , Humanos , Macaca mulatta , Camundongos , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A potent family of spirocyclic nicotinyl aminobenzamide selective HDAC1/HDAC2 inhibitors (SHI-1:2) is profiled. The incorporation of a biaryl zinc-binding motif into a nicotinyl scaffold resulted in enhanced potency and selectivity versus HDAC3, but also imparted hERG activity. It was discovered that increasing polar surface area about the spirocycle attenuates this liability. Compound 12 induced a 4-fold increase in acetylated histone H2B in an HCT-116 xenograft model study with acute exposure, and inhibited tumor growth in a 21-day efficacy study with qd dosing.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Inibidores de Histona Desacetilases , Niacinamida/síntese química , Niacinamida/farmacologia , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Animais , Antineoplásicos/química , Benzamidas/síntese química , Benzamidas/química , Benzamidas/farmacologia , Técnicas de Química Combinatória , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Células HCT116 , Histona Desacetilases , Histonas/análise , Humanos , Camundongos , Camundongos Nus , Estrutura Molecular , Niacinamida/química , Isoformas de Proteínas , Compostos de Espiro/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We report herein the initial exploration of novel selective HDAC1/HDAC2 inhibitors (SHI-1:2). Optimized SHI-1:2 structures exhibit enhanced intrinsic activity against HDAC1 and HDAC2, and are greater than 100-fold selective versus other HDACs, including HDAC3. Based on the SAR of these agents and our current understanding of the HDAC active site, we postulate that the SHI-1:2 extend the existing HDAC inhibitor pharmacophore to include an internal binding domain.
Assuntos
Derivados de Benzeno/síntese química , Derivados de Benzeno/farmacologia , Inibidores de Histona Desacetilases , Modelos Moleculares , Derivados de Benzeno/química , Sítios de Ligação/efeitos dos fármacos , Histona Desacetilase 1 , Histona Desacetilase 2 , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Estrutura Molecular , Isoformas de Proteínas , Proteínas Repressoras , Relação Estrutura-AtividadeRESUMO
Combination drug therapy is a widely used paradigm for managing numerous human malignancies. In cancer treatment, additive and/or synergistic drug combinations can convert weakly efficacious monotherapies into regimens that produce robust antitumor activity. This can be explained in part through pathway interdependencies that are critical for cancer cell proliferation and survival. However, identification of the various interdependencies is difficult due to the complex molecular circuitry that underlies tumor development and progression. Here, we present a high-throughput platform that allows for an unbiased identification of synergistic and efficacious drug combinations. In a screen of 22,737 experiments of 583 doublet combinations in 39 diverse cancer cell lines using a 4 by 4 dosing regimen, both well-known and novel synergistic and efficacious combinations were identified. Here, we present an example of one such novel combination, a Wee1 inhibitor (AZD1775) and an mTOR inhibitor (ridaforolimus), and demonstrate that the combination potently and synergistically inhibits cancer cell growth in vitro and in vivo This approach has identified novel combinations that would be difficult to reliably predict based purely on our current understanding of cancer cell biology. Mol Cancer Ther; 15(6); 1155-62. ©2016 AACR.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Neoplasias Experimentais/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Camundongos , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Pirimidinonas , Distribuição Aleatória , Sirolimo/administração & dosagem , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Induction of compensatory mechanisms and ERK reactivation has limited the effectiveness of Raf and MEK inhibitors in RAS-mutant cancers. We determined that direct pharmacologic inhibition of ERK suppressed the growth of a subset of KRAS-mutant pancreatic cancer cell lines and that concurrent phosphatidylinositol 3-kinase (PI3K) inhibition caused synergistic cell death. Additional combinations that enhanced ERK inhibitor action were also identified. Unexpectedly, long-term treatment of sensitive cell lines caused senescence, mediated in part by MYC degradation and p16 reactivation. Enhanced basal PI3K-AKT-mTOR signaling was associated with de novo resistance to ERK inhibitor, as were other protein kinases identified by kinome-wide siRNA screening and a genetic gain-of-function screen. Our findings reveal distinct consequences of inhibiting this kinase cascade at the level of ERK.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , TempoRESUMO
The identification and in vitro and in vivo characterization of a potent SHI-1:2 are described. Kinetic analysis indicated that biaryl inhibitors exhibit slow binding kinetics in isolated HDAC1 and HDAC2 preparations. Delayed histone hyperacetylation and gene expression changes were also observed in cell culture, and histone acetylation was observed in vivo beyond disappearance of drug from plasma. In vivo studies further demonstrated that continuous target inhibition was well tolerated and efficacious in tumor-bearing mice, leading to tumor growth inhibition with either once-daily or intermittent administration.
RESUMO
This report documents the first example of a specific inhibitor of protein kinases with preferential binding to the activated kinase conformation: 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one 11r (MK-8033), a dual c-Met/Ron inhibitor under investigation as a treatment for cancer. The design of 11r was based on the desire to reduce time-dependent inhibition of CYP3A4 (TDI) by members of this structural class. A novel two-step protocol for the synthesis of benzylic sulfonamides was developed to access 11r and analogues. We provide a rationale for the observed selectivity based on X-ray crystallographic evidence and discuss selectivity trends with additional examples. Importantly, 11r provides full inhibition of tumor growth in a c-Met amplified (GTL-16) subcutaneous tumor xenograft model and may have an advantage over inactive form kinase inhibitors due to equal potency against a panel of oncogenic activating mutations of c-Met in contrast to c-Met inhibitors without preferential binding to the active kinase conformation.
Assuntos
Benzocicloeptenos/metabolismo , Benzocicloeptenos/farmacologia , Descoberta de Drogas , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Animais , Benzocicloeptenos/química , Linhagem Celular Tumoral , Cães , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/química , Ratos , Especificidade por Substrato , Sulfonamidas/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. METHODS: We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. RESULTS: The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. CONCLUSIONS: These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors.
Assuntos
Perfilação da Expressão Gênica , Neoplasias/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/metabolismo , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Cetuximab , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Bases de Dados Genéticas , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas ras/antagonistas & inibidores , Proteínas ras/genéticaRESUMO
The receptor tyrosine kinase c-Met is an attractive target for therapeutic blockade in cancer. Here, we describe MK-2461, a novel ATP-competitive multitargeted inhibitor of activated c-Met. MK-2461 inhibited in vitro phosphorylation of a peptide substrate recognized by wild-type or oncogenic c-Met kinases (N1100Y, Y1230C, Y1230H, Y1235D, and M1250T) with IC(50) values of 0.4 to 2.5 nmol/L. In contrast, MK-2461 was several hundredfold less potent as an inhibitor of c-Met autophosphorylation at the kinase activation loop. In tumor cells, MK-2461 effectively suppressed constitutive or ligand-induced phosphorylation of the juxtamembrane domain and COOH-terminal docking site of c-Met, and its downstream signaling to the phosphoinositide 3-kinase-AKT and Ras-extracellular signal-regulated kinase pathways, without inhibiting autophosphorylation of the c-Met activation loop. BIAcore studies indicated 6-fold tighter binding to c-Met when it was phosphorylated, suggesting that MK-2461 binds preferentially to activated c-Met. MK-2461 displayed significant inhibitory activities against fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor, and other receptor tyrosine kinases. In cell culture, MK-2461 inhibited hepatocyte growth factor/c-Met-dependent mitogenesis, migration, cell scatter, and tubulogenesis. Seven of 10 MK-2461-sensitive tumor cell lines identified from a large panel harbored genomic amplification of MET or FGFR2. In a murine xenograft model of c-Met-dependent gastric cancer, a well-tolerated oral regimen of MK-2461 administered at 100 mg/kg twice daily effectively suppressed c-Met signaling and tumor growth. Similarly, MK-2461 inhibited the growth of tumors formed by s.c. injection of mouse NIH-3T3 cells expressing oncogenic c-Met mutants. Taken together, our findings support further preclinical development of MK-2461 for cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Células Cultivadas , Cães , Sistemas de Liberação de Medicamentos/métodos , Ativação Enzimática/efeitos dos fármacos , Feminino , Haplorrinos , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Two genetically engineered, conditional mouse models of lung tumor formation, K-ras(LSL-G12D) and K-ras(LSL-G12D)/p53(LSL-R270H), are commonly used to model human lung cancer. Developed by Tyler Jacks and colleagues, these models have been invaluable to study in vivo lung cancer initiation and progression in a genetically and physiologically relevant context. However, heterogeneity, multiplicity and complexity of tumor formation in these models make it challenging to monitor tumor growth in vivo and have limited the application of these models in oncology drug discovery. Here, we describe a novel analytical method to quantitatively measure total lung tumor burden in live animals using micro-computed tomography imaging. Applying this methodology, we studied the kinetics of tumor development and response to targeted therapy in vivo in K-ras and K-ras/p53 mice. Consistent with previous reports, lung tumors in both models developed in a time- and dose (Cre recombinase)-dependent manner. Furthermore, the compound K-ras(LSL-G12D)/p53(LSL-R270H) mice developed tumors faster and more robustly than mice harboring a single K-ras(LSL-G12D) oncogene, as expected. Erlotinib, a small molecule inhibitor of the epidermal growth factor receptor, significantly inhibited tumor growth in K-ras(LSL-G12D)/p53(LSL-R270H) mice. These results demonstrate that this novel imaging technique can be used to monitor both tumor progression and response to treatment and therefore supports a broader application of these genetically engineered mouse models in oncology drug discovery and development.
Assuntos
Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/patologia , Tomografia Computadorizada de Feixe Cônico/métodos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Carga Tumoral , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/tratamento farmacológico , Adenoviridae/genética , Animais , Antineoplásicos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Cloridrato de Erlotinib , Feminino , Genes p53 , Genes ras , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Camundongos Transgênicos , Neovascularização Patológica/diagnóstico por imagem , Quinazolinas/administração & dosagem , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genéticaRESUMO
This communication highlights the development of a nicotinamide series of histone deacetylase inhibitors within the benzamide structural class. Extensive exploration around the nicotinamide core led to the discovery of a class I selective HDAC inhibitor that possesses excellent intrinsic and cell-based potency, acceptable ancillary pharmacology, favorable pharmacokinetics, sustained pharmacodynamics in vitro, and achieves in vivo efficacy in an HCT116 xenograft model.
Assuntos
6-Aminonicotinamida/análogos & derivados , 6-Aminonicotinamida/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , 6-Aminonicotinamida/síntese química , Animais , Área Sob a Curva , Benzamidas/química , Disponibilidade Biológica , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cães , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacocinética , Meia-Vida , Humanos , Isoenzimas/antagonistas & inibidores , Modelos Moleculares , Transplante de Neoplasias , Ligação Proteica , Ratos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Interaction of the type 2 vasopressin receptor (V2R) with hormone causes desensitization and internalization. To study the role of the V2R NPxxY motif (which is involved in the clathrin-mediated endocytosis of several other receptors) in this process, we expressed FLAG-tagged wild-type V2R and a Y325F mutant V2R in LLC-PK1a epithelial cells that have low levels of endogenous V2R. Both proteins had a similar apical (35%) and basolateral (65%) membrane distribution. Substitution of Tyr325 with Phe325 prevented ligand-induced internalization of V2R determined by [3H]AVP binding and immunofluorescence but did not prevent ligand binding or signal transduction via adenylyl cyclase. Desensitization and resensitization of the V2R-Y325F mutation occurred independently of internalization. The involvement of clathrin in V2R downregulation was also shown by immunogold electron microscopy. We conclude that the NPxxY motif of the V2R is critically involved in receptor downregulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1a cells or for adenylyl cyclase-mediated signal transduction.