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1.
Bioorg Med Chem Lett ; 28(2): 85-93, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29233651

RESUMO

We disclose the optimization of a high throughput screening hit to yield benzothiazine and tetrahydroquinoline sulfonamides as potent RORγt inverse agonists. However, a majority of these compounds showed potent activity against pregnane X receptor (PXR) and modest activity against liver X receptor α (LXRα). Structure-based drug design (SBDD) led to the identification of benzothiazine and tetrahydroquinoline sulfonamide analogs which completely dialed out LXRα activity and were less potent at PXR. Pharmacodynamic (PD) data for compound 35 in an IL-23 induced IL-17 mouse model is discussed along with the implications of a high Ymax in the PXR assay for long term preclinical pharmacokinetic (PK) studies.


Assuntos
Compostos Bicíclicos com Pontes/farmacologia , Desenho de Drogas , Propanóis/farmacologia , Receptores do Ácido Retinoico/agonistas , Receptores de Esteroides/agonistas , Sulfonamidas/farmacologia , Animais , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Receptores X do Fígado/agonistas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Estrutura Molecular , Receptor de Pregnano X , Propanóis/síntese química , Propanóis/química , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química
2.
Toxicol Sci ; 153(2): 396-408, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27466212

RESUMO

BMS-986094, the prodrug of a guanosine nucleotide analogue (2'-C-methylguanosine), was withdrawn from clinical trials due to serious safety issues. Nonclinical investigative studies were conducted as a follow up to evaluate the potential for BMS-986094-related mitochondrial-toxicity. In vitro, BMS-986094 was applied to human hepatoma cells (HepG2 and Huh-7) or cardiomyocytes (hiPSCM) up to 19 days to assess mitochondrial DNA content and specific gene expression. There were no mitochondrial DNA changes at concentrations ≤10 µM. Transcriptional effects, such as reductions in Huh-7 MT-ND1 and MT-ND5 mRNA content and hiPSCM MT-ND1, MT-COXII, and POLRMT protein expression levels, occurred only at cytotoxic concentrations (≥10 µM) suggesting these transcriptional effects were a consequence of the observed toxicity. Additionally, BMS-986094 has a selective weak affinity for inhibition of RNA polymerases as opposed to DNA polymerases. In vivo, BMS-986094 was given orally to cynomolgus monkeys for 3 weeks or 1 month at doses of 15 or 30 mg/kg/day. Samples of heart and kidney were collected for assessment of mitochondrial respiration, mitochondrial DNA content, and levels of high energy substrates. Although pronounced cardiac and renal toxicities were observed in some monkeys at 30 mg/kg/day treated for 3-4 weeks, there were no changes in mitochondrial DNA content or ATP/GTP levels. Collectively, these data suggest that BMS-986094 is not a direct mitochondrial toxicant.


Assuntos
DNA Mitocondrial/efeitos dos fármacos , Guanosina Monofosfato/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , DNA Mitocondrial/biossíntese , DNA Mitocondrial/fisiologia , Relação Dose-Resposta a Droga , Feminino , Guanosina Monofosfato/metabolismo , Guanosina Monofosfato/toxicidade , Guanosina Trifosfato/metabolismo , Coração/efeitos dos fármacos , Testes de Função Cardíaca , Humanos , Inosina Monofosfato/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Testes de Função Renal , Macaca fascicularis , Masculino
3.
Antimicrob Agents Chemother ; 57(12): 6205-12, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24080659

RESUMO

Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) remain the cornerstone of HIV treatment; however, they are associated with toxicities attributed in part to inhibition of mitochondrial DNA (mtDNA) polymerase γ. In this study, we compared the in vitro toxicity profiles of structurally similar NRTIs (BMS-986001 to stavudine and tenofovir to adefovir) that differ by the presence of an acetylene or methyl group, respectively. Primary cultures of human renal proximal tubule epithelium, skeletal muscle myotubes, and differentiated adipocytes were exposed to the NRTIs at the maximum concentration (Cmax) reported for the clinically approved dose (investigational dose for BMS-986001, 600 mg) and a high equimolar concentration (200 µM) for 19 days. After 19 days, BMS-986001 did not significantly decrease mtDNA or cell protein at either concentration in any cell line. In contrast, stavudine significantly decreased mtDNA in all cultures (1.5- to 2.5-fold) (except at Cmax in renal cells) and cell protein in renal cells (1.4- to 2.4-fold). By day 19, at 200 µM, tenofovir significantly reduced mtDNA in adipocytes (1.9-fold) and adefovir significantly decreased mtDNA in all cultures (3.7- to 10.2-fold); however, no significant reduction in mtDNA was observed at Cmax in any cell line. Adefovir also significantly reduced cell protein at both concentrations in renal cells (2.2- to 2.8-fold) and at 200 µM in muscle cells (2.0-fold). In conclusion, BMS-986001 and tenofovir were considerably less cytotoxic than their respective structural analogs, demonstrating that small structural differences can contribute to significant differences in toxicity.


Assuntos
Adenina/análogos & derivados , DNA Mitocondrial/efeitos dos fármacos , Organofosfonatos/farmacologia , Organofosfonatos/toxicidade , Inibidores da Transcriptase Reversa/farmacologia , Estavudina/toxicidade , Timidina/análogos & derivados , Adenina/farmacologia , Adenina/toxicidade , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , DNA Mitocondrial/ultraestrutura , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Cultura Primária de Células , Relação Estrutura-Atividade , Tenofovir , Timidina/farmacologia
4.
Stem Cells Transl Med ; 2(7): 505-9, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23757504

RESUMO

Faithfully recapitulating human physiology "in a dish" from a renewable source remains a holy grail for medicine and pharma. Many procedures have been described that, to a limited extent, exhibit human tissue-specific function in vitro. In particular, incomplete cellular differentiation and/or the loss of cell phenotype postdifferentiation play a major part in this void. We have developed an interdisciplinary approach to address this problem, using skill sets in cell biology, materials chemistry, and pharmacology. Pluripotent stem cells were differentiated to hepatocytes before being replated onto a synthetic surface. Our approach yielded metabolically active hepatocyte populations that displayed stable function for more than 2 weeks in vitro. Although metabolic activity was an important indication of cell utility, the accurate prediction of cellular toxicity in response to specific pharmacological compounds represented our goal. Therefore, detailed analysis of hepatocellular toxicity was performed in response to a custom-built and well-defined compound set and compared with primary human hepatocytes. Importantly, stem cell-derived hepatocytes displayed equivalence to primary human material. Moreover, we demonstrated that our approach was capable of modeling metabolic differences observed in the population. In conclusion, we report that pluripotent stem cell-derived hepatocytes will model toxicity predictably and in a manner comparable to current gold standard assays, representing a major advance in the field.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Hepatócitos/citologia , Células-Tronco Pluripotentes/citologia , Testes de Toxicidade/métodos , Testes de Toxicidade/normas , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Humanos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/fisiologia , Cultura Primária de Células , Padrões de Referência , Reprodutibilidade dos Testes
5.
Clin Cancer Res ; 11(5): 2063-73, 2005 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15756033

RESUMO

PURPOSE: The insulin-like growth factor (IGF) signaling pathway is implicated in cellular mitogenesis, angiogenesis, tumor cell survival, and tumorigenesis. Inhibition of this pathway results in decreased cell growth, inhibition of tumor formation in animal models, and increased apoptosis in cells treated with cytotoxic chemotherapy. We generated and characterized a human monoclonal antibody that targeted the IGF receptor. EXPERIMENTAL DESIGN: By use of XenoMouse technology, we generated CP-751,871, a fully human IgG2 antibody with high affinity (K(d) = 1.5 nmol/L) for human IGF-1R and evaluated its biological, pharmacologic, and antitumor properties. RESULTS: This antibody blocks binding of IGF-1 to its receptor (IC(50) 1.8 nmol/L), IGF-1-induced receptor autophosphorylation (IC(50) 0.42 nmol/L) and induced the down-regulation of IGF-1R in vitro and in tumor xenografts. The extent of IGF-1R down-regulation in vivo was proportional to CP-751,871 concentrations in the serum of tumor-bearing mice. Pharmacokinetic profiles in cynomolgus monkeys indicated a close to linear increase of exposure following i.v. dosing of antibody in the range of 3 to 100 mg/kg. CP-751,871 showed significant antitumor activity both as a single agent and in combination with Adriamycin, 5-fluorouracil, or tamoxifen in multiple tumor models. A biomarker assay was developed to establish the relationship between circulating antibody concentrations and down-regulation of IGF-1R in peripheral blood cells. The concentration of CP-751,871 required to down-regulate 50% of IGF-1R on peripheral blood cells was 0.3 nmol/L. CONCLUSION: These data suggest that inhibition of the IGF cascade by use of this monoclonal antibody may be of clinical benefit in the treatment of human cancers.


Assuntos
Anticorpos Monoclonais/imunologia , Neoplasias Colorretais/patologia , Mieloma Múltiplo/patologia , Receptor IGF Tipo 1/imunologia , Receptor IGF Tipo 1/fisiologia , Animais , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Modelos Animais de Doenças , Regulação para Baixo , Imunoglobulina G/imunologia , Fator de Crescimento Insulin-Like I/metabolismo , Macaca fascicularis , Camundongos , Fosforilação , Receptor IGF Tipo 1/biossíntese , Transdução de Sinais , Transplante Heterólogo , Células Tumorais Cultivadas
6.
Mol Pharmacol ; 67(6): 1909-19, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15755908

RESUMO

The lipid and metabolic disturbances associated with human immunodeficiency virus (HIV) protease inhibitor therapy in AIDS have stimulated interest in developing new agents that minimize these side effects in the clinic. The underlying explanation of mechanism remains enigmatic, but a recently described link between endoplasmic reticulum (ER) stress and dysregulation of lipid metabolism suggests a provocative integration of existing and emerging data. We provide new evidence from in vitro models indicating that proteasome inhibition and differential glucose transport blockade by protease inhibitors are proximal events eliciting an ER stress transcriptional response that can regulate lipogenic pathways in hepatocytes or adipocytes. Proteasome activity was inhibited in vitro by several protease inhibitors at clinically relevant (micromolar) levels. In the intact cells, protease inhibitors rapidly elicited a pattern of gene expression diagnostic of intracellular proteasome inhibition and activation of an ER stress response. This included induction of transcription factors GADD153, ATF4, and ATF3; amino acid metabolic enzymes; proteasome components; and certain ER chaperones. In hepatocyte lines, the ER stress response was closely linked to moderate increases in lipogenic and cholesterogenic gene expression. However, in adipocytes where GLUT4 was directly inhibited by some protease inhibitors, time-dependent suppression of lipogenic genes and triglyceride synthesis was observed in coordination with the ER stress response. These results further link ER stress to dyslipidemia and contribute to a unifying mechanism for the pathophysiology of protease inhibitor-associated lipodystrophy, helping explain differences in clinical metabolic profiles among protease inhibitors.


Assuntos
Retículo Endoplasmático/metabolismo , Inibidores da Protease de HIV/farmacologia , Hiperlipidemias/metabolismo , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Inibidores de Proteassoma , Estresse Fisiológico/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Humanos , Hiperlipidemias/enzimologia , Hiperlipidemias/genética , Camundongos , Proteínas de Transporte de Monossacarídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Ratos Wistar , Estresse Fisiológico/genética
7.
Biochem Pharmacol ; 66(9): 1755-68, 2003 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-14563486

RESUMO

Inhibition of HbS polymerization is a major target for therapeutic approaches in sickle cell anemia. Toward this goal, initial efforts at pharmacological elevation of fetal hemoglobin (HbF) has shown therapeutic efficacy. In order to identify well-tolerated, novel agents that induce HbF in patients, we developed a high-throughput screening approach based on induction of gamma-globin gene expression in erythroid cells. We measured gamma-globin transcription in K562 cells transfected with either gamma promoter elements fused with the locus control region hypersensitivity site 2 and luciferase reporter gene (HS2 gamma) or a beta-yeast artificial chromosome in which the luciferase reporter gene was recombined into the gamma-globin coding sequences (gamma YAC). Corresponding pharmacological increases in HbF protein were confirmed in both K562 cells and in human primary erythroid progenitor cells. Approximately 186,000 defined chemicals and fungal extracts were evaluated for their ability to increase gamma gene transcription in either HS2 gamma or gamma YAC models. Eleven distinct classes of compounds were identified, the majority of which were active within 24-48 hr. The short chain hydroxamate-containing class generally exhibited delayed maximal activity, which continued to increase transcription up to 120 hr. The cyclic tetrapeptide OSI-2040 and the hydroxamates were shown to have histone deacetylase inhibitory activity. In primary hematopoietic progenitor cell cultures, OSI-2040 increased HbF by 4.5-fold at a concentration of only 40 nM, comparable to the effects of hydroxyurea at 100 microM. This screening methodology successfully identifies active compounds for further mechanistic and preclinical evaluation as potential therapeutic agents for sickle cell anemia.


Assuntos
Anemia Falciforme/metabolismo , Inibidores Enzimáticos/farmacologia , Hemoglobina Fetal/metabolismo , Globinas/metabolismo , Inibidores de Histona Desacetilases , Transcrição Genética/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Globinas/genética , Histona Desacetilases/metabolismo , Humanos , Células K562 , RNA Mensageiro/metabolismo
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