RESUMO
Circulating phosphate levels are tightly controlled within a narrow range in mammals. By using a novel small-molecule inhibitor, we show that the enzymatic activity of inositol hexakisphosphate kinases (IP6K) is essential for phosphate regulation in vivo. IP6K inhibition suppressed XPR1, a phosphate exporter, thereby decreasing cellular phosphate export, which resulted in increased intracellular ATP levels. The in vivo inhibition of IP6K decreased plasma phosphate levels without inhibiting gut intake or kidney reuptake of phosphate, demonstrating a pivotal role of IP6K-regulated cellular phosphate export on circulating phosphate levels. IP6K inhibition-induced decrease in intracellular inositol pyrophosphate, an enzymatic product of IP6K, was correlated with phosphate changes. Chronic IP6K inhibition alleviated hyperphosphataemia, increased kidney ATP, and improved kidney functions in chronic kidney disease rats. Our results demonstrate that the enzymatic activity of IP6K regulates circulating phosphate and intracellular ATP and suggest that IP6K inhibition is a potential novel treatment strategy against hyperphosphataemia.
Assuntos
Fosfatos/sangue , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Homeostase/efeitos dos fármacos , Humanos , Hiperfosfatemia/tratamento farmacológico , Fosfatos de Inositol/metabolismo , Mamíferos , Fosfatos/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/antagonistas & inibidores , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Receptor do Retrovírus Politrópico e XenotrópicoRESUMO
Full agonist-mediated activation of free fatty acid receptor 1 (FFAR1/GPR40) alleviates diabetes in rodents. Considering that diabetes is a chronic disease, assessment of treatment durability of chronic exposure to a GPR40 full agonist is pivotal for treating patients with diabetes. However, the physiologic significance of chronic in vitro and in vivo exposure to GPR40 full agonists is largely unclear. Here, we evaluated the in vitro and in vivo effects of chronic treatment with SCO-267, a GPR40 full agonist, on signal transduction and glucose control. In vitro experiments showed that SCO-267 is an allosteric full agonist for GPR40, which activates the Gα q, Gα s, and Gα 12/13 pathways and ß-arrestin recruitment. The calcium signal response was largely sustained in GPR40-overexpressing CHO cells even after prolonged incubation with SCO-267. To evaluate the in vivo relevance of chronic exposure to GPR40 full agonists, SCO-267 (1 and 10 mg/kg) was administered once daily to neonatally streptozotocin-induced diabetic rats for 15-33 days, and glucose control was evaluated. After 15 days of dosing followed by the drug washout period, SCO-267 improved glucose tolerance, most likely by increasing insulin sensitivity in rats. After 33 days, repeated exposure to SCO-267 was highly effective in improving glucose tolerance in rats. Furthermore, chronic exposure to SCO-267 increased pancreatic insulin content. These results demonstrated that even after chronic exposure, SCO-267 effectively activates GPR40 in cells and rats, suggesting the clinical application of SCO-267 in treating chronic diseases including diabetes. SIGNIFICANCE STATEMENT: GPR40 is a validated therapeutic target for diabetes. This study showed that even after chronic exposure, SCO-267, an allosteric GPR40 full agonist, effectively activates GPR40 in cells and rats; these results suggest a durable efficacy of SCO-267 in patients.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Controle Glicêmico/métodos , Piperidinas/administração & dosagem , Piridinas/administração & dosagem , Receptores Acoplados a Proteínas G/agonistas , Animais , Glicemia/metabolismo , Células CHO , Cricetinae , Cricetulus , Diabetes Mellitus Experimental/sangue , Relação Dose-Resposta a Droga , Humanos , Masculino , Ratos , Ratos Endogâmicos WKY , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Retinoic acid receptor-related orphan receptor γt (RORγt) agonists are expected to provide a novel class of immune-activating anticancer drugs via activation of Th17 cells and Tc17 cells. Herein, we describe a novel structure-based functionality switching approach from in house well-optimized RORγt inverse agonists to potent RORγt agonists. We succeeded in the identification of potent RORγt agonist 5 without major chemical structure change. The biochemical response was validated by molecular dynamics simulation studies that showed a helix 12 stabilization effect of RORγt agonists. These results indicate that targeting helix 12 is an attractive and novel medicinal chemistry strategy for switching existing RORγt inverse agonists to agonists.
Assuntos
Desenho de Fármacos , Agonismo Inverso de Drogas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Animais , Ensaios de Triagem em Larga Escala , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade , Células Th17/efeitos dos fármacosRESUMO
Vasoactive intestinal peptide receptor 2 (VIPR2, also known as VPAC2) is a class B G-protein coupled receptor (GPCR) and plays important roles in the physiology of central nervous system (CNS) by interaction with natural ligands; vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Because it has been reported that high-expression and/or overactivation of VIPR2 link to schizophrenic symptoms, VIPR2 antagonists could be good drug candidates for schizophrenia therapeutics. In this study, we discovered several artificial peptides that antagonize both human and rodent VIPR2 with selectivities against receptor subtypes VIPR1 (also known as VPAC1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). Of them, the representative 16-mer cyclic peptide VIpep-3 (Ac-CPPYLPRRLCTLLLRS-OH) exhibited strong binding affinity with KD value of 41â¯nM to extracellular domain of human VIPR2 in SPR analysis and showed potent antagonist activity with IC50 values of 47â¯nM (human), 180â¯nM (mouse), and 44â¯nM (rat) against VIP-VIPR2 signal in cell-based Ca influx assay. This is not only the first report on artificial VIPR2-selective antagonist peptides but also good example of the effective approach to discover novel antagonist against class B GPCR. Our peptides will contribute to study and development of the novel CNS drugs targeting to VIPR2.
Assuntos
Descoberta de Drogas , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/antagonistas & inibidores , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Técnicas Biossensoriais , Células CHO , Cricetulus , Humanos , Ligantes , Camundongos , Biblioteca de Peptídeos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/síntese química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Ratos , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Peptídeo Intestinal Vasoativo/síntese química , Peptídeo Intestinal Vasoativo/químicaRESUMO
The retinoic acid-related orphan receptor gamma T (RORγt) plays an important role in Th17 cell proliferation and functionality. Thus, RORγt inverse agonists are thought to be potent therapeutic agents for Th17-mediated autoimmune diseases, such as rheumatoid arthritis, asthma, inflammatory bowel disease, and psoriasis. Although RORγt has constitutive activity, it is recognized that the receptor is physiologically regulated by various cholesterol derivatives. In this study, we sought to identify RORγt inverse agonists through a high-throughput screening campaign. To this end, we compared an apo-RORγt protein from Escherichia coli and a cholesterol-bound RORγt protein from insect cells. The IC50 of the known RORγt inverse agonist TO901317 was significantly lower for the apoprotein than for the cholesterol-bound RORγt. Through high-throughput screening using a fluorescence-based cholesterol binding assay with the apoprotein, we identified compound 1 as a novel cholesterol-competitive RORγt inverse agonist. Compound 1 inhibited the RORγt-TopFluor cholesterol interaction, coactivator recruitment, and transcriptional activity of RORγt. Cell-based reporter gene assay demonstrated that compound 1 showed higher potency by lipid depletion treatment. Collectively, our findings indicate that eliminating cholesterol from the RORγt protein is suitable for sensitive high-throughput screening to identify RORγt inverse agonists.
Assuntos
Colesterol/metabolismo , Avaliação Pré-Clínica de Medicamentos , Hidrocarbonetos Fluorados/farmacologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Sulfonamidas/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Hidrocarbonetos Fluorados/química , Estrutura Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Células Sf9 , Spodoptera , Sulfonamidas/química , Células Th17RESUMO
Aberrant expression of proteins often underlies many diseases, including cancer. A recently developed approach in drug development is small molecule-mediated, selective degradation of dysregulated proteins. We have devised a protein-knockdown system that utilizes chimeric molecules termed specific and nongenetic IAP-dependent protein erasers (SNIPERs) to induce ubiquitylation and proteasomal degradation of various target proteins. SNIPER(ER)-87 consists of an inhibitor of apoptosis protein (IAP) ligand LCL161 derivative that is conjugated to the estrogen receptor α (ERα) ligand 4-hydroxytamoxifen by a PEG linker, and we have previously reported that this SNIPER efficiently degrades the ERα protein. Here, we report that derivatization of the IAP ligand module yields SNIPER(ER)s with superior protein-knockdown activity. These improved SNIPER(ER)s exhibited higher binding affinities to IAPs and induced more potent degradation of ERα than does SNIPER(ER)-87. Further, they induced simultaneous degradation of cellular inhibitor of apoptosis protein 1 (cIAP1) and delayed degradation of X-linked IAP (XIAP). Notably, these reengineered SNIPER(ER)s efficiently induced apoptosis in MCF-7 human breast cancer cells that require IAPs for continued cellular survival. We found that one of these molecules, SNIPER(ER)-110, inhibits the growth of MCF-7 tumor xenografts in mice more potently than the previously characterized SNIPER(ER)-87. Mechanistic analysis revealed that our novel SNIPER(ER)s preferentially recruit XIAP, rather than cIAP1, to degrade ERα. Our results suggest that derivatized IAP ligands could facilitate further development of SNIPERs with potent protein-knockdown and cytocidal activities against cancer cells requiring IAPs for survival.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Antineoplásicos/farmacologia , Regulação para Baixo , Humanos , Ligantes , Células MCF-7 , Camundongos , Ligação Proteica , Proteólise , Tiazóis/farmacologia , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted protein degradation using small molecules is a novel strategy for drug development. We have developed hybrid molecules named specific and nongenetic inhibitor of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) that recruit IAP ubiquitin ligases to degrade target proteins. Here, we show novel SNIPERs capable of inducing proteasomal degradation of the androgen receptor (AR). Through derivatization of the SNIPER(AR) molecule at the AR ligand and IAP ligand and linker, we developed 42a (SNIPER(AR)-51), which shows effective protein knockdown activity against AR. Consistent with the degradation of the AR protein, 42a inhibits AR-mediated gene expression and proliferation of androgen-dependent prostate cancer cells. In addition, 42a efficiently induces caspase activation and apoptosis in prostate cancer cells, which was not observed in the cells treated with AR antagonists. These results suggest that SNIPER(AR)s could be leads for an anticancer drug against prostate cancers that exhibit AR-dependent proliferation.
Assuntos
Antineoplásicos/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Proteólise/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Células A549 , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Química Sintética , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Masculino , Neoplasias da Próstata/patologia , Relação Estrutura-AtividadeRESUMO
GPR39, a G-protein-coupled receptor activated by zinc, reportedly activates multiple intracellular signaling pathways via Gs, Gq, G12/13, and ß-arrestin, but little is known about downregulation of the receptor upon its activation. To our knowledge, this is the first report on the mechanism of feedback regulation of GPR39 function determined in GPR39-expressing HEK293 cells (HEK293-GPR39) as a model cell system. In HEK293-GPR39 cells, GPR39-C3, which is a positive allosteric modulator, activated cAMP production (downstream of Gs), IP1 accumulation (downstream of Gq), SRF-RE-dependent transcription (downstream of G12/13), and ß-arrestin recruitment. GPR39-C3 induced dose- and time-dependent loss of response in cAMP production by second challenge of the compound. This functional desensitization was blocked by the Rho kinase (ROCK) inhibitor, Y-27632, but not by Gq or Gs-pathway inhibitors or inhibition of ß-arrestin recruitment. In the receptor localization assay, GPR39-C3 induced internalization of GFP-tagged GPR39. This internalization was also inhibited by Y-27632, which suggested that ROCK activation is critical for internalization and desensitization of GPR39. A novel biased GPR39 positive allosteric modulator, 5-[2-[(2,4-dichlorophenyl)methoxy]phenyl]-2,2-dimethyl-1,3,5,6-tetrahydrobenzo[a]phenanthridin-4-one (GSB-118), which activated cAMP responses and ß-arrestin recruitment but showed no effect on SRF-RE-dependent transcription, did not induce desensitization. These results revealed a unique mechanism of desensitization of GPR39.
Assuntos
AMP Cíclico/metabolismo , Retroalimentação Fisiológica , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Sistemas do Segundo Mensageiro , Taquifilaxia , Zinco/metabolismo , Quinases Associadas a rho/metabolismo , Regulação Alostérica/efeitos dos fármacos , Amidas/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Cinética , Ligantes , Microscopia de Fluorescência , Fenantridinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sulfonamidas/farmacologia , beta-Arrestinas/metabolismo , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
BACKGROUND: Recent evidence suggests that androgen receptor (AR) splice variants, including AR-V7, play a pivotal role in resistance to androgen blockade in prostate cancer treatment. The development of new therapeutic agents that can suppress the transcriptional activities of AR splice variants has been anticipated as the next generation treatment of castration-resistant prostate cancer. METHODS: High-throughput screening of AR-V7 signaling inhibitors was performed using an AR-V7 reporter system. The effects of a glycogen synthase kinase-3 (GSK3) inhibitor, LY-2090314, on endogenous AR-V7 signaling were evaluated in an AR-V7-positive cell line, JDCaP-hr, by quantitative reverse transcription polymerase chain reaction. The relationship between AR-V7 signaling and ß-catenin signaling was assessed using RNA interference. The effect of LY-2090314 on cell growth in various prostate cancer cell lines was also evaluated. RESULTS: We identified GSK3 inhibitors as transcriptional suppressors of AR-V7 using a high-throughput screen with an AR-V7 reporter system. LY-2090314 suppressed the reporter activity and endogenous AR-V7 activity in JDCaP-hr cells. Because silencing of ß-catenin partly rescued the suppression, it was evident that the suppression was mediated, at least partially, via the activation of ß-catenin signaling. AR-V7 signaling and ß-catenin signaling reciprocally regulate each other in JDCaP-hr cells, and therefore, GSK3 inhibition can repress AR-V7 transcriptional activity by accumulating intracellular ß-catenin. Notably, LY-2090314 selectively inhibited the growth of AR-V7-positive prostate cancer cells in vitro. CONCLUSIONS: Our findings demonstrate the potential of GSK3 inhibitors in treating advanced prostate cancer driven by AR splice variants. In vivo evaluation of AR splice variant-positive prostate cancer models will help illustrate the overall significance of GSK3 inhibitors in treating prostate cancer.
Assuntos
Quinase 3 da Glicogênio Sintase , Neoplasias de Próstata Resistentes à Castração , Receptores Androgênicos/metabolismo , beta Catenina/metabolismo , Processamento Alternativo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Masculino , Próstata , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/terapia , Transdução de Sinais/efeitos dos fármacosRESUMO
Many diseases, especially cancers, result from aberrant or overexpression of pathogenic proteins. Specific inhibitors against these proteins have shown remarkable therapeutic effects, but these are limited mainly to enzymes. An alternative approach that may have utility in drug development relies on selective degradation of pathogenic proteins via small chimeric molecules linking an E3 ubiquitin ligase to the targeted protein for proteasomal degradation. To this end, we recently developed a protein knockdown system based on hybrid small molecule SNIPERs (Specific and Nongenetic IAP-dependent Protein Erasers) that recruit inhibitor of the apoptosis protein (IAP) ubiquitin ligases to specifically degrade targeted proteins. Here, we extend our previous study to show a proof of concept of the SNIPER technology in vivo By incorporating a high affinity IAP ligand, we developed a novel SNIPER against estrogen receptor α (ERα), SNIPER(ER)-87, that has a potent protein knockdown activity. The SNIPER(ER) reduced ERα levels in tumor xenografts and suppressed the growth of ERα-positive breast tumors in mice. Mechanistically, it preferentially recruits X-linked IAP (XIAP) rather than cellular IAP1, to degrade ERα via the ubiquitin-proteasome pathway. With this IAP ligand, potent SNIPERs against other pathogenic proteins, BCR-ABL, bromodomain-containing protein 4 (BRD4), and phosphodiesterase-4 (PDE4) could also be developed. These results indicate that forced ubiquitylation by SNIPERs is a useful method to achieve efficient protein knockdown with potential therapeutic activities and could also be applied to study the role of ubiquitylation in many cellular processes.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Mama/efeitos dos fármacos , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Descoberta de Drogas , Receptor alfa de Estrogênio/antagonistas & inibidores , Feminino , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Complexo de Endopeptidases do Proteassoma/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitinação/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Dedicator of cytokinesis 2 (DOCK2) is a key molecule for lymphocyte activation and migration. DOCK2 interacts with Ras-related C3 botulinus toxin substrate 1 (Rac1, GTPase) and mediates the GDP-GTP exchange reaction, indicating that inhibitors against protein-protein interaction (PPI) between DOCK2 and Rac1 would be good drug candidates for treating immune-related disorders. Here, we report DOCK2-selective PPI inhibitory peptides discovered using random peptide T7 phage display technology. These peptides inhibited DOCK2 activity at nanomolar concentrations and were delivered to intracellular compartments by combination with cell-penetrating peptide (CPP). Consequently, one peptide, R4-DCpep-2(V2W/K4R/ox)-NH2 (Ac-RRRRCWARYHGYPWCRRRR-NH2), inhibited migration in human B lymphocyte MINO cell line at IC50 = 120 nM. To our knowledge, this is the first report of a DOCK2-selective peptide inhibitor; this study will contribute to the development of novel DOCK2-targeting immunosuppressive drugs.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Linfoma de Células B/tratamento farmacológico , Peptídeos/química , Peptídeos/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sistema Livre de Células , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas Ativadoras de GTPase , Humanos , Linfoma de Células B/patologia , Biblioteca de Peptídeos , Peptídeos/metabolismo , Mapas de Interação de Proteínas , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The unique function of 4-hydroxyisoleucine (4-HIL) is to stimulate glucose-induced insulin secretion in a glucose-dependent manner. 4-HIL is distributed only in certain kinds of plants and mushrooms, but the biosynthetic mechanism of 4-HIL has not been elucidated. Moreover, 4-HIL-producing microorganisms have not been reported. l-isoleucine (l-Ile) hydroxylating activity producing 4-HIL was detected in a cell lysate of Bacillus thuringiensis strain 2e2 AKU 0251 obtained from the mid-late exponential phase of growth. Properties of the purified hydroxylase demonstrated that it is a alpha-ketoglutaric acid (alpha-KG) dependent l-Ile dioxygenase (IDO) and requires alpha-KG, ferric ion, and ascorbic acid for its maximum activity. IDO showed high stereoselectivity in l-Ile hydroxylation producing only (2S,3R,4S)-4-HIL. The N-terminal 22 amino acids sequence revealed high homology to a hypothetical protein (GenBank ID: RBTH_06809) in B. thuringiensis serovar israelensis ATCC 35646. The histidine motif, which is conserved in alpha-KG dependent dioxygenases, is found in RBTH_06809.