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1.
Cancer Res ; 81(12): 3347-3357, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33820800

RESUMO

In many human cancers, deregulation of the Notch pathway has been shown to play a role in the initiation and maintenance of the neoplastic phenotype. Aberrant Notch activity also plays a central role in the maintenance and survival of cancer stem cells (CSC), which underlie metastasis and resistance to therapy. For these reasons, inhibition of Notch signaling has become an exceedingly attractive target for cancer therapeutic development. However, attempts to develop Notch pathway-specific drugs have largely failed in the clinic, in part due to intestinal toxicity. Here, we report the discovery of NADI-351, the first specific small-molecule inhibitor of Notch1 transcriptional complexes. NADI-351 selectively disrupted Notch1 transcription complexes and reduced Notch1 recruitment to target genes. NADI-351 demonstrated robust antitumor activity without inducing intestinal toxicity in mouse models, and CSCs were ablated by NADI-351 treatment. Our study demonstrates that NADI-351 is an orally available and potent inhibitor of Notch1-mediated transcription that inhibits tumor growth with low toxicity, providing a potential therapeutic approach for improved cancer treatment. SIGNIFICANCE: This study showcases the first Notch1-selective inhibitor that suppresses tumor growth with limited toxicity by selectively ablating cancer stem cells.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Neoplasias Esofágicas/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptor Notch1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Apoptose , Proliferação de Células , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Humanos , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Clin Cancer Res ; 25(4): 1379-1388, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30487124

RESUMO

PURPOSE: Although most children with medulloblastoma are cured of their disease, Sonic Hedgehog (SHH) subgroup medulloblastoma driven by TRP53 mutations is essentially lethal. Casein kinase 1α (CK1α) phosphorylates and destabilizes GLI transcription factors, thereby inhibiting the key effectors of SHH signaling. We therefore tested a second-generation CK1α activator against TRP53-mutant, MYCN-amplified medulloblastoma. EXPERIMENTAL DESIGN: The ability of this CK1α activator to block SHH signaling was determined in vitro using GLI reporter cells, granular precursor primary cultures, and PATCHED1 (PTCH1)-mutant sphere cultures. While in vivo efficacy was tested using 2 different medulloblastoma mouse models: PTCH1 and ND2:SMOA1. Finally, the clinical relevance of CK1α activators was demonstrated using a TRP53-mutant, MYCN-amplified patient-derived xenograft. RESULTS: SSTC3 inhibited SHH activity in vitro, acting downstream of the vismodegib target SMOOTHENED (SMO), and reduced the viability of sphere cultures derived from SHH medulloblastoma. SSTC3 accumulated in the brain, inhibited growth of SHH medulloblastoma tumors, and blocked metastases in a genetically engineered vismodegib-resistant mouse model of SHH medulloblastoma. Importantly, SSTC3 attenuated growth and metastasis of orthotopic patient-derived TRP53-mutant, MYCN-amplified, SHH subgroup medulloblastoma xenografts, increasing overall survival. CONCLUSIONS: Using a newly described small-molecule, SSTC3, we show that CK1a activators could address a significant unmet clinical need for patients with SMO inhibitor-resistant medulloblastoma, including those harboring mutations in TRP53.


Assuntos
Benzoatos/farmacologia , Caseína Quinase Ialfa/genética , Meduloblastoma/tratamento farmacológico , Receptor Smoothened/genética , Anilidas/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Xenoenxertos , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Metástase Neoplásica , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína GLI1 em Dedos de Zinco/genética
3.
Sci Signal ; 10(485)2017 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-28655862

RESUMO

Constitutive WNT activity drives the growth of various human tumors, including nearly all colorectal cancers (CRCs). Despite this prominence in cancer, no WNT inhibitor is currently approved for use in the clinic largely due to the small number of druggable signaling components in the WNT pathway and the substantial toxicity to normal gastrointestinal tissue. We have shown that pyrvinium, which activates casein kinase 1α (CK1α), is a potent inhibitor of WNT signaling. However, its poor bioavailability limited the ability to test this first-in-class WNT inhibitor in vivo. We characterized a novel small-molecule CK1α activator called SSTC3, which has better pharmacokinetic properties than pyrvinium, and found that it inhibited the growth of CRC xenografts in mice. SSTC3 also attenuated the growth of a patient-derived metastatic CRC xenograft, for which few therapies exist. SSTC3 exhibited minimal gastrointestinal toxicity compared to other classes of WNT inhibitors. Consistent with this observation, we showed that the abundance of the SSTC3 target, CK1α, was decreased in WNT-driven tumors relative to normal gastrointestinal tissue, and knocking down CK1α increased cellular sensitivity to SSTC3. Thus, we propose that distinct CK1α abundance provides an enhanced therapeutic index for pharmacological CK1α activators to target WNT-driven tumors.


Assuntos
Antineoplásicos/farmacologia , Benzoatos/farmacologia , Caseína Quinase Ialfa/metabolismo , Ativadores de Enzimas/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Wnt/metabolismo , Animais , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Metástase Neoplásica , Técnicas de Cultura de Órgãos , Fosforilação , Compostos de Pirvínio/química , Transdução de Sinais , Ressonância de Plasmônio de Superfície , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto , Xenopus laevis
4.
Cancer Res ; 76(12): 3593-603, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27197169

RESUMO

In many cancers, aberrant Notch activity has been demonstrated to play a role in the initiation and maintenance of the neoplastic phenotype and in cancer stem cells, which may allude to its additional involvement in metastasis and resistance to therapy. Therefore, Notch is an exceedingly attractive therapeutic target in cancer, but the full range of potential targets within the pathway has been underexplored. To date, there are no small-molecule inhibitors that directly target the intracellular Notch pathway or the assembly of the transcriptional activation complex. Here, we describe an in vitro assay that quantitatively measures the assembly of the Notch transcriptional complex on DNA. Integrating this approach with computer-aided drug design, we explored potential ligand-binding sites and screened for compounds that could disrupt the assembly of the Notch transcriptional activation complex. We identified a small-molecule inhibitor, termed Inhibitor of Mastermind Recruitment-1 (IMR-1), that disrupted the recruitment of Mastermind-like 1 to the Notch transcriptional activation complex on chromatin, thereby attenuating Notch target gene transcription. Furthermore, IMR-1 inhibited the growth of Notch-dependent cell lines and significantly abrogated the growth of patient-derived tumor xenografts. Taken together, our findings suggest that a novel class of Notch inhibitors targeting the transcriptional activation complex may represent a new paradigm for Notch-based anticancer therapeutics, warranting further preclinical characterization. Cancer Res; 76(12); 3593-603. ©2016 AACR.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Neoplasias/tratamento farmacológico , Receptores Notch/antagonistas & inibidores , Tiazolidinas/farmacologia , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Somitos/embriologia , Peixe-Zebra
5.
Cancer Res ; 74(17): 4811-21, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24994715

RESUMO

The Hedgehog (HH) signaling pathway represents an important class of emerging developmental signaling pathways that play critical roles in the genesis of a large number of human cancers. The pharmaceutical industry is currently focused on developing small molecules targeting Smoothened (Smo), a key signaling effector of the HH pathway that regulates the levels and activity of the Gli family of transcription factors. Although one of these compounds, vismodegib, is now FDA-approved for patients with advanced basal cell carcinoma, acquired mutations in Smo can result in rapid relapse. Furthermore, many cancers also exhibit a Smo-independent activation of Gli proteins, an observation that may underlie the limited efficacy of Smo inhibitors in clinical trials against other types of cancer. Thus, there remains a critical need for HH inhibitors with different mechanisms of action, particularly those that act downstream of Smo. Recently, we identified the FDA-approved anti-pinworm compound pyrvinium as a novel, potent (IC50, 10 nmol/L) casein kinase-1α (CK1α) agonist. We show here that pyrvinium is a potent inhibitor of HH signaling, which acts by reducing the stability of the Gli family of transcription factors. Consistent with CK1α agonists acting on these most distal components of the HH signaling pathway, pyrvinium is able to inhibit the activity of a clinically relevant, vismodegib -resistant Smo mutant, as well as the Gli activity resulting from loss of the negative regulator suppressor of fused. We go on to demonstrate the utility of this small molecule in vivo, against the HH-dependent cancer medulloblastoma, attenuating its growth and reducing the expression of HH biomarkers.


Assuntos
Proteínas Hedgehog/metabolismo , Compostos de Pirvínio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Carcinoma Basocelular/tratamento farmacológico , Carcinoma Basocelular/metabolismo , Caseína Quinase Ialfa/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/metabolismo , Camundongos , Camundongos Nus , Células NIH 3T3 , Proteínas Oncogênicas , Receptores Acoplados a Proteínas G/metabolismo , Transativadores , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de Zinco
6.
PLoS One ; 9(7): e101969, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25003333

RESUMO

Mutations in the WNT-pathway regulator ADENOMATOUS POLYPOSIS COLI (APC) promote aberrant activation of the WNT pathway that is responsible for APC-associated diseases such as Familial Adenomatous Polyposis (FAP) and 85% of spontaneous colorectal cancers (CRC). FAP is characterized by multiple intestinal adenomas, which inexorably result in CRC. Surprisingly, given their common occurrence, there are few effective chemotherapeutic drugs for FAP. Here we show that the FDA-approved, anti-helminthic drug Pyrvinium attenuates the growth of WNT-dependent CRC cells and does so via activation of CK1α. Furthermore, we show that Pyrvinium can function as an in vivo inhibitor of WNT-signaling and polyposis in a mouse model of FAP: APCmin mice. Oral administration of Pyrvinium, a CK1α agonist, attenuated the levels of WNT-driven biomarkers and inhibited adenoma formation in APCmin mice. Considering its well-documented safe use for treating enterobiasis in humans, our findings suggest that Pyrvinium could be repurposed for the clinical treatment of APC-associated polyposes.


Assuntos
Polipose Adenomatosa do Colo/tratamento farmacológico , Antineoplásicos/farmacologia , Compostos de Pirvínio/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Aprovação de Drogas , Reposicionamento de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Via de Sinalização Wnt/efeitos dos fármacos
7.
PLoS One ; 8(8): e71508, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24013655

RESUMO

WNT signaling plays a key role in the self-renewal of tumor initiation cells (TICs). In this study, we used pyrvinium pamoate (PP), an FDA-approved antihelmintic drug that inhibits WNT signaling, to test whether pharmacologic inhibition of WNT signaling can specifically target TICs of aggressive breast cancer cells. SUM-149, an inflammatory breast cancer cell line, and SUM-159, a metaplastic basal-type breast cancer cell line, were used in these studies. We found that PP inhibited primary and secondary mammosphere formation of cancer cells at nanomolar concentrations, at least 10 times less than the dose needed to have a toxic effect on cancer cells. A comparable mammosphere formation IC50 dose to that observed in cancer cell lines was obtained using malignant pleural effusion samples from patients with IBC. A decrease in activity of the TIC surrogate aldehyde dehydrogenase was observed in PP-treated cells, and inhibition of WNT signaling by PP was associated with down-regulation of a panel of markers associated with epithelial-mesenchymal transition. In vivo, intratumoral injection was associated with tumor necrosis, and intraperitoneal injection into mice with tumor xenografts caused significant tumor growth delay and a trend toward decreased lung metastasis. In in vitro mammosphere-based and monolayer-based clonogenic assays, we found that PP radiosensitized cells in monolayer culture but not mammosphere culture. These findings suggest WNT signaling inhibition may be a feasible strategy for targeting aggressive breast cancer. Investigation and modification of the bioavailability and toxicity profile of systemic PP are warranted.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Compostos de Pirvínio/farmacologia , Radiossensibilizantes/farmacologia , Animais , Anti-Helmínticos/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Feminino , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Tolerância a Radiação/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/fisiologia , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo
8.
Cancer Discov ; 3(11): 1286-301, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23921231

RESUMO

UNLABELLED: Synovial sarcoma is an aggressive soft-tissue malignancy of children and young adults, with no effective systemic therapies. Its specific oncogene, SYT-SSX (SS18-SSX), drives sarcoma initiation and development. The exact mechanism of SYT-SSX oncogenic function remains unknown. In an SYT-SSX2 transgenic model, we show that a constitutive Wnt/ß-catenin signal is aberrantly activated by SYT-SSX2, and inhibition of Wnt signaling through the genetic loss of ß-catenin blocks synovial sarcoma tumor formation. In a combination of cell-based and synovial sarcoma tumor xenograft models, we show that inhibition of the Wnt cascade through coreceptor blockade and the use of small-molecule CK1α activators arrests synovial sarcoma tumor growth. We find that upregulation of the Wnt/ß-catenin cascade by SYT-SSX2 correlates with its nuclear reprogramming function. These studies reveal the central role of Wnt/ß-catenin signaling in SYT-SSX2-induced sarcoma genesis, and open new venues for the development of effective synovial sarcoma curative agents. SIGNIFICANCE: Synovial sarcoma is an aggressive soft-tissue cancer that afflicts children and young adults, and for which there is no effective treatment. The current studies provide critical insight into our understanding of the pathogenesis of SYT­SSX-dependent synovial sarcoma and pave the way for the development of effective therapeutic agents for the treatment of the disease in humans.


Assuntos
Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Sarcoma Sinovial/genética , Sarcoma Sinovial/patologia , Via de Sinalização Wnt/efeitos dos fármacos , Adolescente , Adulto , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Nus , Camundongos Transgênicos , Compostos de Pirvínio/farmacologia , Sarcoma Experimental , Sarcoma Sinovial/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
9.
J Biomol Screen ; 16(9): 995-1006, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21859680

RESUMO

Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (ß-catenin and Axin) in opposing fashion. We have now fused ß-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.


Assuntos
Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Proteína Axina/metabolismo , Ensaios Enzimáticos , Flavonas/farmacologia , Células HEK293 , Células HeLa , Humanos , Luciferases/metabolismo , Reprodutibilidade dos Testes , Xenopus laevis/metabolismo , beta Catenina/metabolismo
10.
Nat Chem Biol ; 6(11): 829-36, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20890287

RESUMO

Wnt/ß-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote ß-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC(50) of ∼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and ß-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or ß-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.


Assuntos
Caseína Quinase Ialfa/metabolismo , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Compostos de Pirvínio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Animais , Proteína Axina , Caseína Quinase I/genética , Caseína Quinase I/metabolismo , Extratos Celulares , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Wnt/química , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteínas de Xenopus , Xenopus laevis , beta Catenina/genética , beta Catenina/metabolismo
11.
Mol Pharmacol ; 77(3): 459-68, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20026717

RESUMO

Phenotypic studies of mice lacking metabotropic glutamate receptor subtype 7 (mGluR7) suggest that antagonists of this receptor may be promising for the treatment of central nervous system disorders such as anxiety and depression. Suzuki et al. (J Pharmacol Exp Ther 323:147-156, 2007) recently reported the in vitro characterization of a novel mGluR7 antagonist called 6-(4-methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[ 4,5-c]pyridin-4(5H)-one (MMPIP), which noncompetitively inhibited the activity of orthosteric and allosteric agonists at mGluR7. We describe that MMPIP acts as a noncompetitive antagonist in calcium mobilization assays in cells coexpressing mGluR7 and the promiscuous G protein G alpha(15). Assessment of the activity of a small library of MMPIP-derived compounds using this assay reveals that, despite similar potencies, compounds exhibit differences in negative cooperativity for agonist-mediated calcium mobilization. Examination of the inhibitory activity of MMPIP and analogs using endogenous G(i/o)-coupled assay readouts indicates that the pharmacology of these ligands seems to be context-dependent, and MMPIP exhibits differences in negative cooperativity in certain cellular backgrounds. Electrophysiological studies reveal that, in contrast to the orthosteric antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxyclycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), MMPIP is unable to block agonist-mediated responses at the Schaffer collateral-CA1 synapse, a location at which neurotransmission has been shown to be modulated by mGluR7 activity. Thus, MMPIP and related compounds differentially inhibit coupling of mGluR7 in different cellular backgrounds and may not antagonize the coupling of this receptor to native G(i/o) signaling pathways in all cellular contexts. The pharmacology of this compound represents a striking example of the potential for context-dependent blockade of receptor responses by negative allosteric modulators.


Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/fisiologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Linhagem Celular , Cricetinae , Regulação para Baixo/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
12.
Mol Pharmacol ; 75(3): 577-88, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19047481

RESUMO

Activators of M(1) muscarinic acetylcholine receptors (mAChRs) may provide novel treatments for schizophrenia and Alzheimer's disease. Unfortunately, the development of M(1)-active compounds has resulted in nonselective activation of the highly related M(2) to M(5) mAChR subtypes, which results in dose-limiting side effects. Using a functional screening approach, we identified several novel ligands that potentiated agonist activation of M(1) with low micromolar potencies and induced 5-fold or greater leftward shifts of the acetylcholine (ACh) concentration-response curve. These ligands did not compete for binding at the ACh binding site, indicating that they modulate receptor activity by binding to allosteric sites. The two most selective compounds, cyclopentyl 1,6-dimethyl-4-(6-nitrobenzo[d][1,3]-dioxol-5-yl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (VU0090157) and (E)-2-(4-ethoxyphenylamino)-N'-((2-hydroxynaphthalen-1-yl)methylene)acetohydrazide (VU0029767), induced progressive shifts in ACh affinity at M(1) that were consistent with their effects in a functional assay, suggesting that the mechanism for enhancement of M(1) activity by these compounds is by increasing agonist affinity. These compounds were strikingly different, however, in their ability to potentiate responses at a mutant M(1) receptor with decreased affinity for ACh and in their ability to affect responses of the allosteric M(1) agonist, 1-[1'-(2-tolyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one. Furthermore, these two compounds were distinct in their abilities to potentiate M(1)-mediated activation of phosphoinositide hydrolysis and phospholipase D. The discovery of multiple structurally distinct positive allosteric modulators of M(1) is an exciting advance in establishing the potential of allosteric modulators for selective activation of this receptor. These data also suggest that structurally diverse M(1) potentiators may act by distinct mechanisms and differentially regulate receptor coupling to downstream signaling pathways.


Assuntos
Agonistas Muscarínicos/química , Agonistas Muscarínicos/metabolismo , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Agonistas Muscarínicos/farmacologia , Ratos
13.
Int J Radiat Oncol Biol Phys ; 71(3): 873-9, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18514780

RESUMO

PURPOSE: SU11248 (sunitinib) is a small-molecule tyrosine kinase inhibitor which targets VEGFR and PDGFR isoforms. In the present study, the effects of SU11248 and ionizing radiation on pancreatic cancer were studied. METHODS AND MATERIALS: For in vitro studies human pancreatic adenocarcinoma cells lines were treated with 1 microM SU11248 1 h before irradiation. Western blot analysis was used to determine the effect of SU11248 on radiation-induced signal transduction. To determine if SU11248 sensitized pancreatic cancer to the cytotoxic effects of ionizing radiation, a clonogenic survival assay was performed using 0-6 Gy. For in vivo assays, CAPAN-1 cells were injected into the hind limb of nude mice for tumor volume and proliferation studies. RESULTS: SU11248 attenuated radiation-induced phosphorylation of Akt and ERK at 0, 5, 15, and 30 min. Furthermore, SU11248 significantly reduced clonogenic survival after treatment with radiation (p < 0.05). In vivo studies revealed that SU11248 and radiation delayed tumor growth by 6 and 10 days, respectively, whereas combined treatment delayed tumor growth by 30 days. Combined treatment with SU11248 and radiation further attenuated Brdu incorporation by 75% (p = 0.001) compared to control. CONCLUSIONS: SU11248 (sunitinib) sensitized pancreatic cancer to the cytotoxic effects of radiation. This compound is promising for future clinical trials with chemoradiation in pancreatic cancer.


Assuntos
Adenocarcinoma/fisiopatologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Indóis/administração & dosagem , Neoplasias Pancreáticas/fisiopatologia , Pirróis/administração & dosagem , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/administração & dosagem , Adenocarcinoma/radioterapia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Humanos , Neoplasias Pancreáticas/radioterapia , Sunitinibe
14.
Bioorg Med Chem Lett ; 18(9): 2845-9, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18424044
15.
Nat Chem Biol ; 4(1): 42-50, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18059262

RESUMO

Muscarinic acetylcholine receptors (mAChRs) provide viable targets for the treatment of multiple central nervous system disorders. We have used cheminformatics and medicinal chemistry to develop new, highly selective M4 allosteric potentiators. VU10010, the lead compound, potentiates the M4 response to acetylcholine 47-fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for acetylcholine and coupling to G proteins. Whole-cell patch clamp recordings revealed that selective potentiation of M4 with VU10010 increases carbachol-induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M4 knockout mice. Selective regulation of excitatory transmission by M4 suggests that targeting of individual mAChR subtypes could be used to differentially regulate specific aspects of mAChR modulation of function in this important forebrain structure.


Assuntos
Hipocampo/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Receptor Muscarínico M4/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Regulação Alostérica , Sítio Alostérico , Animais , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Eletrofisiologia , Hipocampo/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Knockout , Estrutura Molecular , Agonistas Muscarínicos/química , Antagonistas Muscarínicos/química , Ligação Proteica , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Ensaio Radioligante , Ratos , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inibidores , Receptor Muscarínico M4/genética , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
16.
Org Lett ; 5(3): 239-42, 2003 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-12556161

RESUMO

[reaction: see text] A short asymmetric synthesis of (2'S,5R,6R)-2'-[6-amino-5-hydroxy-1,3-cyclohexadiene-1-carbonyloxy]propionic acid, the enantiomer of the reported structure of (+)-oryzoxymycin is described. The reported spectral data does not match that obtained for synthetic "oryzoxymycin".


Assuntos
Ácidos Cicloexanocarboxílicos/química , Ácidos Cicloexanocarboxílicos/síntese química , Cicloexenos , Estrutura Molecular
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