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1.
Nat Commun ; 12(1): 3856, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158470

RESUMO

The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles' heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well.


Assuntos
Motivos de Aminoácidos , DNA Helicases/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Sequência de Aminoácidos , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Instabilidade Genômica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
2.
ACS Chem Biol ; 16(4): 586-595, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33724769

RESUMO

Classic galactosemia is caused by loss-of-function mutations in galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic accumulation of its substrate, galactose-1-phosphate. One proposed therapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1) are primarily ATP-competitive with limited clinical utility to date. Here, we determined crystal structures of hGALK1 bound with reported ATP-competitive inhibitors of the spiro-benzoxazole series, to reveal their binding mode in the active site. Spurred by the need for additional chemotypes of hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also performed crystallography-based screening by soaking hundreds of hGALK1 crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Galactoquinase/antagonistas & inibidores , Galactosemias/tratamento farmacológico , Cristalografia por Raios X , Galactoquinase/química , Humanos , Conformação Proteica
3.
Biochimie ; 183: 100-107, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33476699

RESUMO

The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme's active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.


Assuntos
Inibidores Enzimáticos/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/antagonistas & inibidores , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , S-Adenosilmetionina/química , Regulação Alostérica , Humanos , Domínios Proteicos
4.
IUCrJ ; 7(Pt 4): 693-706, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32695416

RESUMO

DHTKD1 is a lesser-studied E1 enzyme among the family of 2-oxoacid de-hydrogenases. In complex with E2 (di-hydro-lipo-amide succinyltransferase, DLST) and E3 (dihydrolipo-amide de-hydrogenase, DLD) components, DHTKD1 is involved in lysine and tryptophan catabolism by catalysing the oxidative de-carboxyl-ation of 2-oxoadipate (2OA) in mitochondria. Here, the 1.9 Šresolution crystal structure of human DHTKD1 is solved in complex with the thi-amine diphosphate co-factor. The structure reveals how the DHTKD1 active site is modelled upon the well characterized homologue 2-oxoglutarate (2OG) de-hydrogenase but engineered specifically to accommodate its preference for the longer substrate of 2OA over 2OG. A 4.7 Šresolution reconstruction of the human DLST catalytic core is also generated by single-particle electron microscopy, revealing a 24-mer cubic scaffold for assembling DHTKD1 and DLD protomers into a megacomplex. It is further demonstrated that missense DHTKD1 variants causing the inborn error of 2-amino-adipic and 2-oxoadipic aciduria impact on the complex formation, either directly by disrupting the interaction with DLST, or indirectly through destabilizing the DHTKD1 protein. This study provides the starting framework for developing DHTKD1 modulators to probe the intricate mitochondrial energy metabolism.

5.
Nat Commun ; 11(1): 2813, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499479

RESUMO

5'-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates.


Assuntos
5-Aminolevulinato Sintetase/química , Regulação Enzimológica da Expressão Gênica , 5-Aminolevulinato Sintetase/deficiência , 5-Aminolevulinato Sintetase/genética , Acil Coenzima A/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Heme/química , Humanos , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Protoporfiria Eritropoética/genética , Especificidade por Substrato
6.
Clin Lymphoma Myeloma Leuk ; 16(6): 311-21, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27234438

RESUMO

BACKGROUND: Primary effusion lymphoma (PEL) is a rare non-Hodgkin lymphoma subtype primarily seen in human immunodeficiency virus (HIV)-infected individuals with low CD4(+) cell counts and elevated HIV viral loads. It has always been associated with human herpesvirus type 8 (HHV-8) and in 80% of cases has also been associated with Epstein-Barr virus (EBV). Less commonly, PEL has presented in patients with advanced age and other conditions associated with an altered immunity, including malignancy, liver cirrhosis, and immunosuppressive medications. It is a tumor of B-cell lineage; however, it shows a "null" phenotype, rarely expressing pan-B cell surface antigens. It will usually express CD45, CD30, CD38, CD138, and MUM1 and is characterized by lymphomatous effusions in body cavities but not lymphadenopathy. It is an aggressive lymphoma, with an average median survival of < 1 year. HHV-8-associated large B-cell lymphoma (HHV-8-LBL) is a second variant of PEL that is both solid and extracavitary. It has immunoblastic and/or anaplastic morphologic features and a distinct immunohistochemical staining pattern. It could also have a different clinical presentation than that of classic PEL. MATERIALS AND METHODS: We describe the case of a 57-year-old HIV-infected man who presented with a slow-growing and asymptomatic abdominal mass. Examination of an excisional biopsy specimen showed malignant large cells with prominent cytoplasm that were positive for pan-B cell antigen CD20, HHV-8, and EBV and negative for CD138, CD10, BCL-6, CD3, and CD30. The Ki-67 labeling index was 90%. The diagnosis was stage IIIA HHV-8-LBL, and he was treated with 6 cycles of R-EPOCH (rituximab, etoposide, vincristine, doxorubicin, cyclophosphamide, and prednisone) infusion chemotherapy. At 12 months after treatment, he was in complete remission. We also performed a Medline and Embase search to better understand the clinical findings of our patient and the unique attributes of HHV-8-LBL. Focusing our search on English language studies, we identified 83 cases of HHV-8-LBL without an effusion component. We compared these 83 cases with 118 reported cases of classic PEL. RESULTS: The median age of the patients with HHV-8-LBL was 41 years (range, 24-77), and 96% of the cases were associated with HIV. The median age of the patients with classic PEL was 41 years (range, 26-86), and 96% of the cases were associated with HIV. Of those with HHV-8-LBL, 31 of 61 (51%) had a pre-existing diagnosis of acquired immunodeficiency syndrome (AIDS) and 47 of 63 (75%) were coinfected with EBV. In contrast, 69 of 96 patients (72%) with classic PEL had a pre-existing AIDS diagnosis and 40 of 49 (82%) were coinfected with EBV. The mean CD4(+) count of the HHV-8-LBL patients was 256 cells/µL (range, 18-1126 cells/µL) compared with 139 cells/µL (range, 2-557 cells/µL) in the classic PEL patients. The median survival time for both groups was similar at 5.5 months (range, 25 days to ≥ 25 months) for patients with HHV-8-LBL and 4 months (range, 2 days to ≥ 113 months) for those with classic PEL. More patients with HHV-8-LBL were alive at the last follow-up point (59% vs. 18%). The percentage of patients achieving complete remission was 54% (30 of 56) and 36% (32 of 89) for HHV-8-LBL and classic PEL, respectively. CONCLUSION: Our patient's high CD4(+) cell count, the lack of a pre-existing AIDS diagnosis, and the excellent response to chemotherapy highlights that HHV-8-LBL might have distinct clinical features and possibly a better response to chemotherapy than classic PEL. HHV-8-LBL should be included in the differential diagnosis of HIV patients with solid lesions. It is essential that patients' Centers for Disease Control and Prevention HIV clinical status and HIV viral load at the diagnosis of PEL and HHV-8-LBL be reported and that the reported clinical results include longer term follow-up data. Only then will a more complete clinical picture of this little-appreciated and little-understood PEL variant be defined.


Assuntos
Infecções por Herpesviridae/complicações , Herpesvirus Humano 8 , Linfoma de Efusão Primária/diagnóstico , Linfoma de Efusão Primária/etiologia , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos B/metabolismo , Linfócitos B/patologia , Biópsia , Coinfecção , Infecções por HIV/complicações , Infecções por HIV/diagnóstico , Infecções por HIV/imunologia , Infecções por HIV/virologia , Homossexualidade Masculina , Humanos , Imuno-Histoquímica , Imunofenotipagem , Linfoma de Efusão Primária/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Adulto Jovem
7.
J Med Chem ; 57(18): 7550-64, 2014 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-25101488
8.
Bioorg Med Chem Lett ; 23(13): 3833-40, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23707259

RESUMO

A series of compounds which exhibited good human CCR1 binding and functional potency was modified resulting in the discovery of a novel series of high affinity, functionally potent antagonists of the CCR1 receptor. Issues of PXR activity, ion-channel potency, and poor metabolic stability were addressed by the addition of a hydroxyl group to an otherwise lipophilic area in the molecule resulting in the discovery of preclinical candidate BMS-457 for the treatment of rheumatoid arthritis.


Assuntos
Descoberta de Drogas , Piperidinas/farmacologia , Receptores CCR1/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Relação Estrutura-Atividade
9.
Prostate ; 71(5): 480-8, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20878947

RESUMO

BACKGROUND: Androgen receptor (AR) antagonists are part of the standard of care for prostate cancer. Despite the almost inevitable development of resistance in prostate tumors to AR antagonists, no new AR antagonists have been approved for over a decade. Treatment failure is due in part to mutations that increase activity of AR in response to lower ligand concentrations as well as to mutations that result in AR response to a broader range of ligands. The failure to discover new AR antagonists has occurred in the face of continued research; to enable progress, a clear understanding of the reasons for failure is required. METHODS: Non-clinical drug safety studies and safety pharmacology assays were performed on previously approved AR antagonists (bicalutamide, flutamide, nilutamide), next generation antagonists in clinical testing (MDV3100, BMS-641988), and a pre-clinical drug candidate (BMS-501949). In addition, non-clinical studies with AR mutant mice, and EEG recordings in rats were performed. Non-clinical findings are compared to disclosures of clinical trial results. RESULTS: As a drug class, AR antagonists cause seizure in animals by an off-target mechanism and are found in vitro to inhibit GABA-A currents. Clinical trials of candidate next generation AR antagonists identify seizure as a clinical safety risk. CONCLUSIONS: Non-clinical drug safety profiles of the AR antagonist drug class create a significant barrier to the identification of next generation AR antagonists. GABA-A inhibition is a common off-target activity of approved and next generation AR antagonists potentially explaining some side effects and safety hazards of this class of drugs.


Assuntos
Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/toxicidade , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Antagonistas de Androgênios/farmacocinética , Animais , Cães , Descoberta de Drogas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neoplasias da Próstata/metabolismo , Ratos , Ratos Sprague-Dawley
10.
Cancer Res ; 69(16): 6522-30, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19654297

RESUMO

Despite an excellent initial response to first-line hormonal treatment, most patients with metastatic prostate cancer will succumb to a hormone-refractory form of the disease. Because these tumors are still dependent on a functional androgen receptor (AR), there is a need to find novel and more potent antiandrogens. While searching for small molecules that bind to the AR and inhibit its transcriptional activity, BMS-641988 was discovered. This novel antiandrogen showed an increased (>1 log) potency compared with the standard antiandrogen, bicalutamide, in both binding affinity to the AR and inhibition of AR-mediated transactivation in cell-based reporter assays. In mature rats, BMS-641988 strongly inhibited androgen-dependent growth of the ventral prostate and seminal vesicles. In the CWR-22-BMSLD1 human prostate cancer xenograft model, BMS-641988 showed increased efficacy over bicalutamide (average percent tumor growth inhibition >90% versus <50%), even at exposure levels of bicalutamide 3-fold greater than what can be attained in humans. Furthermore, BMS-641988 was efficacious in CWR-22-BMSLD1 tumors initially refractory to treatment with bicalutamide. BMS-641988 was highly efficacious in the LuCaP 23.1 human prostate xenograft model, inducing stasis throughout the approximately 30-day dosing. To explore the functional mechanisms of BMS-641988, gene expression profiling analysis was done on CWR-22-BMSLD1 xenograft models in mice. Treatment with BMS-641988 resulted in a global gene expression profile more similar to castration compared with that of bicalutamide. Overall, these data highlight that the unique preclinical profile of BMS-641988 may provide additional understanding for the hormonal treatment of prostate cancer.


Assuntos
Antagonistas de Receptores de Andrógenos , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Imidas/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/uso terapêutico , Animais , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Imidas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Vet Immunol Immunopathol ; 126(3-4): 377-81, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-18771806

RESUMO

CD137 plays an important role as a co-stimulatory molecule in activated T cells. Agonistic CD137 specific antibodies have been investigated as therapeutic agents to promote tumor-specific immune responses by direct activation of T cells. As part of the pre-clinical pharmacological evaluation of cynomolgus monkeys, monkey CD137 was cloned and characterized. The deduced amino acid sequence encoded a full-length gene of 254 amino acids 95% identical to human CD137. Sequence variants identified in monkey CD137 include four splice variants lacking the transmembrane domain. These variants were detectable in human including two previously unreported variants. Two missense single nucleotide polymorphisms were detected present in 42 and 50% of 36 monkeys tested. In both monkey and human, mRNA expression of full-length CD137 and splice variants were significantly increased in peripheral blood mononuclear cells (PBMCs) upon stimulation by anti-CD3 antibodies. Recombinant monkey CD137 protein was bound with high affinity by an agonistic anti-human CD137 antibody but not by an anti-mouse CD137 antibody. In summary, compared to human, monkey CD137 showed distinct extracellular domain amino acid sequence and sequence polymorphisms. Thus, antibodies directed against epitopes in this extracellular domain could have differences in pharmacologic activity between cynomolgus monkeys and human or across individual cynomolgus monkeys.


Assuntos
Macaca fascicularis/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência , Análise de Sequência de DNA/veterinária
12.
Toxicol Sci ; 103(1): 28-34, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18281259

RESUMO

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints. The Carcinogenicity Working Group of the Predictive Safety Testing Consortium has concentrated on sharing data to test the predictivity of two published hepatic gene expression signatures, including the signature by Fielden et al. (2007, Toxicol. Sci. 99, 90-100) for predicting nongenotoxic hepatocarcinogens, and the signature by Nie et al. (2006, Mol. Carcinog. 45, 914-933) for predicting nongenotoxic carcinogens. Although not a rigorous prospective validation exercise, the consortium approach created an opportunity to perform a meta-analysis to evaluate microarray data from short-term rat studies on over 150 compounds. Despite significant differences in study designs and microarray platforms between laboratories, the signatures proved to be relatively robust and more accurate than expected by chance. The accuracy of the Fielden et al. signature was between 63 and 69%, whereas the accuracy of the Nie et al. signature was between 55 and 64%. As expected, the predictivity was reduced relative to internal validation estimates reported under identical test conditions. Although the signatures were not deemed suitable for use in regulatory decision making, they were deemed worthwhile in the early assessment of drugs to aid decision making in drug development. These results have prompted additional efforts to rederive and evaluate a QPCR-based signature using these samples. When combined with a standardized test procedure and prospective interlaboratory validation, the accuracy and potential utility in preclinical applications can be ascertained.


Assuntos
Testes de Carcinogenicidade/métodos , Genômica , Animais , Perfilação da Expressão Gênica , Masculino , Ratos , Ratos Sprague-Dawley
13.
Toxicol Pathol ; 35(5): 621-35, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17654404

RESUMO

Toxicogenomics is considered a valuable tool for reducing pharmaceutical candidate attrition by facilitating earlier identification, prediction and understanding of toxicities. A retrospective evaluation of 3 years of routine transcriptional profiling in non-clinical safety studies was undertaken to assess the utility of toxicogenomics in drug safety assessment. Based on the analysis of studies with 33 compounds, marked global transcriptional changes (> 4% transcripts at p < 0.01) were shown to be a robust biomarker for dosages considered to be toxic . In general, there was an inconsistent correlation between transcription and histopathology, most likely due to differences in sensitivity to focal microscopic lesions, to secondary effects, and to events that precede structural tissue changes. For 60% of toxicities investigated with multiple time-point data, transcriptional changes were observed prior to changes in traditional study endpoints. Candidate transcriptional markers of pharmacologic effects were detected in 40% of targets profiled. Mechanistic classification of toxicity was obtained for 30% of targets. Furthermore, data comparison to compendia of transcriptional changes provided assessments of the specificity of transcriptional responses. Overall, our experience suggests that toxicogenomics has contributed to a greater understanding of mechanisms of toxicity and to reducing drug attrition by empiric analysis where safety assessment combines toxicogenomic and traditional evaluations.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Toxicogenética , Animais , Biomarcadores , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Humanos , Estudos Retrospectivos , Medição de Risco , Segurança
14.
Toxicol Appl Pharmacol ; 223(3): 246-56, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17663016

RESUMO

We previously reported prevention of urolithiasis and associated rat urinary bladder tumors by urine acidification (via diet acidification) in male rats treated with the dual peroxisome proliferator-activated receptor (PPAR)alpha/gamma agonist muraglitazar. Because urine acidification could potentially alter PPAR signaling and/or cellular proliferation in urothelium, we evaluated urothelial cell PPARalpha, PPARdelta, PPARgamma, and epidermal growth factor receptor (EGFR) expression, PPAR signaling, and urothelial cell proliferation in rats fed either a normal or an acidified diet for 5, 18, or 33 days. A subset of rats in the 18-day study also received 63 mg/kg of the PPARgamma agonist pioglitazone daily for the final 3 days to directly assess the effects of diet acidification on responsiveness to PPARgamma agonism. Urothelial cell PPARalpha and gamma expression and signaling were evaluated in the 18- and 33-day studies by immunohistochemical assessment of PPAR protein (33-day study only) and quantitative real-time polymerase chain reaction (qRT-PCR) measurement of PPAR-regulated gene expression. In the 5-day study, EGFR expression and phosphorylation status were evaluated by immunohistochemical staining and egfr and akt2 mRNA levels were assessed by qRT-PCR. Diet acidification did not alter PPARalpha, delta, or gamma mRNA or protein expression, PPARalpha- or gamma-regulated gene expression, total or phosphorylated EGFR protein, egfr or akt2 gene expression, or proliferation in urothelium. Moreover, diet acidification had no effect on pioglitazone-induced changes in urothelial PPARgamma-regulated gene expression. These results support the contention that urine acidification does not prevent PPARgamma agonist-induced bladder tumors by altering PPARalpha, gamma, or EGFR expression or PPAR signaling in rat bladder urothelium.


Assuntos
Ácidos/urina , Dieta , Fator de Crescimento Epidérmico/biossíntese , Receptores Ativados por Proliferador de Peroxissomo/biossíntese , Bexiga Urinária/metabolismo , Urotélio/metabolismo , Animais , Proliferação de Células , Imuno-Histoquímica , Masculino , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Fosforilação , Pioglitazona , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Tiazolidinedionas/farmacologia , Bexiga Urinária/citologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/prevenção & controle , Urotélio/citologia
15.
Proc Natl Acad Sci U S A ; 104(16): 6846-51, 2007 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-17428923

RESUMO

Once-daily s.c. administration of either human parathyroid hormone (PTH)-(1-84) or recombinant human PTH-(1-34) provides for dramatic increases in bone mass in women with postmenopausal osteoporosis. We initiated a program to discover orally bioavailable small molecule equivalents of these peptides. A traditional high-throughput screening approach using cAMP activation of the PTH/PTH-related peptide receptor (PPR) as a readout failed to provide any lead compounds. Accordingly, we designed a new screen for this receptor that used a modified N-terminal fragment of PTH as a probe for small molecule binding to the transmembrane region of the PPR, driven by the assumption that the pharmacological properties (agonist/antagonist) of compounds that bound to this putative signaling domain of the PPR could be altered by chemical modification. We developed DPC-AJ1951, a 14 amino acid peptide that acts as a potent agonist of the PPR, and characterized its activity in ex vivo and in vivo assays of bone resorption. In addition, we studied its ability to initiate gene transcription by using microarray technology. Together, these experiments indicated that the highly modified 14 amino acid peptide induces qualitatively similar biological responses to those produced by PTH-(1-34), albeit with lower potency relative to the parent peptide. Encouraged by these data, we performed a screen of a small compound collection by using DPC-AJ1951 as the ligand. These studies led to the identification of the benzoxazepinone SW106, a previously unrecognized small molecule antagonist for the PPR. The binding of SW106 to the PPR was rationalized by using a homology receptor model.


Assuntos
Sondas Moleculares/fisiologia , Oxazepinas/farmacologia , Hormônio Paratireóideo/fisiologia , Fragmentos de Peptídeos/fisiologia , Receptor Tipo 1 de Hormônio Paratireóideo/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Ligação Competitiva , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Técnicas de Sonda Molecular , Dados de Sequência Molecular , Oxazepinas/agonistas , Hormônio Paratireóideo/agonistas , Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo
16.
Toxicol Sci ; 94(2): 272-80, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17000718

RESUMO

Murine double minute 2 (Mdm2) negatively regulates p53 by mediating its ubiquitination and proteosomal degradation, and Mdm2 is recognized as a proto-oncogene. In the present study, hepatic gene expression patterns induced by phenobarbital (PB; 100 mg/kg) and pregnenolone 16alpha-carbonitrile (PCN, 100 mg/kg) were evaluated in male and female Sprague-Dawley rats using Affymetrix Rat Genome U34A gene arrays. In addition to changes in the hepatic expression of well-characterized drug-metabolizing enzymes, an increase in Mdm2 mRNA was observed with both compounds after single or repeat dosing (5 days). However, gene array analyses did not reveal changes in other p53-dependent genes, suggesting that induction of Mdm2 occurred in a p53-independent manner. Real-time polymerase chain reaction confirmed the microarray results, as PB increased Mdm2 mRNA approximately twofold after single or repeat doses in male and female rats. PCN treatment increased Mdm2 mRNA levels up to 5- and 12-fold in male and female rats, respectively, after 5 days of dosing. Hepatic Mdm2 protein levels were increased, and immunohistochemical evaluation of rat liver demonstrated nuclear localization of Mdm2, suggesting an interaction with p53. Consequently, p53 protein levels were also decreased by approximately 35 and 50% after 5 days of PB and PCN treatment, respectively. In direct contrast to rats, PB and PCN (100 mg/kg) did not induce Mdm2 mRNA in mouse liver after 5 days of dosing. Finally, although Mdm2 in mice and humans is reported to migrate electrophoretically as two proteins with molecular weights of 76 and 90 kDa, rat Mdm2 protein was detected primarily as a 120-kDa species. Follow-up experiments indicated that rat hepatic Mdm2 was subject to posttranslational modification with small ubiquitin-modifying (SUMO) proteins. Although the molecular mechanisms controlling Mdm2 induction by PB and PCN in rats have not yet been determined, these results suggest that early effects on cell cycle regulation, response to DNA damage or cell transformation may contribute to liver tumor development.


Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fenobarbital/farmacologia , Carbonitrila de Pregnenolona/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Administração Oral , Animais , Indução Enzimática/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Imuno-Histoquímica , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína Supressora de Tumor p53/genética
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