RESUMO
The therapeutic and toxic effects of drugs are often generated through effects on distinct cell types in the body. Selective delivery of drugs to specific cells or cell lineages would, therefore, have major advantages, in particular, the potential to significantly improve the therapeutic window of an agent. Cells of the monocyte-macrophage lineage represent an important target for many therapeutic agents because of their central involvement in a wide range of diseases including inflammation, cancer, atherosclerosis, and diabetes. We have developed a versatile chemistry platform that is designed to enhance the potency and delivery of small-molecule drugs to intracellular molecular targets. One facet of the technology involves the selective delivery of drugs to cells of the monocyte-macrophage lineage, using the intracellular carboxylesterase, human carboxylesterase-1 (hCE-1), which is expressed predominantly in these cells. Here, we demonstrate selective delivery of many types of intracellularly targeted small molecules to monocytes and macrophages by attaching a small esterase-sensitive chemical motif (ESM) that is selectively hydrolyzed within these cells to a charged, pharmacologically active drug. ESM versions of histone deacetylase (HDAC) inhibitors, for example, are extremely potent anticytokine and antiarthritic agents with a wider therapeutic window than conventional HDAC inhibitors. In human blood, effects on monocytes (hCE-1-positive) are seen at concentrations 1000-fold lower than those that affect other cell types (hCE-1-negative). Chemical conjugates of this type, by limiting effects on other cells, could find widespread applicability in the treatment of human diseases where monocyte-macrophages play a key role in disease pathology.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Esterases/antagonistas & inibidores , Esterases/química , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Aminoácidos/química , Animais , Anisomicina/farmacologia , Artrite/imunologia , Carboxilesterase/antagonistas & inibidores , Carboxilesterase/química , Carboxilesterase/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocinas/biossíntese , Citocinas/sangue , Citocinas/genética , Inibidores Enzimáticos/farmacologia , Esterases/genética , Ésteres/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/sangue , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
A novel series of HDAC inhibitors demonstrating class I subtype selectivity and good oral bioavailability is described. The compounds are potent enzyme inhibitors (IC50 values less than 100 nM), and improved activity in cell proliferation assays was achieved by modulation of polar surface area (PSA) through the introduction of novel linking groups. Employing oral pharmacokinetic studies in mice, comparing drug levels in spleen to plasma, we selected compounds that were tested for efficacy in human tumor xenograft studies based on their potential to distribute into tumor. One compound, 21r (CHR-3996), showed good oral activity in these models, including dose-related activity in a LoVo xenograft. In addition 21r showed good activity in combination with other anticancer agents in in vitro studies. On the basis of these results, 21r was nominated for clinical development.
Assuntos
Antineoplásicos/síntese química , Compostos Azabicíclicos/síntese química , Inibidores de Histona Desacetilases/síntese química , Pirimidinas/síntese química , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Compostos Azabicíclicos/farmacocinética , Compostos Azabicíclicos/farmacologia , Linhagem Celular Tumoral , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Inibidores de Histona Desacetilases/farmacocinética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Técnicas In Vitro , Camundongos , Camundongos Nus , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Transplante de Neoplasias , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Distribuição Tecidual , Transplante HeterólogoRESUMO
CHR-2797 is a novel metalloenzyme inhibitor that is converted into a pharmacologically active acid product (CHR-79888) inside cells. CHR-79888 is a potent inhibitor of a number of intracellular aminopeptidases, including leucine aminopeptidase. CHR-2797 exerts antiproliferative effects against a range of tumor cell lines in vitro and in vivo and shows selectivity for transformed over nontransformed cells. Its antiproliferative effects are at least 300 times more potent than the prototypical aminopeptidase inhibitor, bestatin. However, the mechanism by which inhibition of these enzymes leads to proliferative changes is not understood. Gene expression microarrays were used to profile changes in mRNA expression levels in the human promyelocytic leukemia cell line HL-60 treated with CHR-2797. This analysis showed that CHR-2797 treatment induced a transcriptional response indicative of amino acid depletion, the amino acid deprivation response, which involves up-regulation of amino acid synthetic genes, transporters, and tRNA synthetases. These changes were confirmed in other leukemic cell lines sensitive to the antiproliferative effects of CHR-2797. Furthermore, CHR-2797 treatment inhibited phosphorylation of mTOR substrates and reduced protein synthesis in HL-60 cells, both also indicative of amino acid depletion. Treatment with CHR-2797 led to an increase in the concentration of intracellular small peptides, the substrates of aminopeptidases. It is suggested that aminopeptidase inhibitors, such as CHR-2797 and bestatin, deplete sensitive tumor cells of amino acids by blocking protein recycling, and this generates an antiproliferative effect. CHR-2797 is orally bioavailable and currently undergoing phase II clinical investigation in the treatment of myeloid leukemia.
Assuntos
Aminoácidos/metabolismo , Aminopeptidases/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glicina/análogos & derivados , Ácidos Hidroxâmicos/farmacologia , Aminopeptidases/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Eletroforese em Gel de Poliacrilamida , Fator de Iniciação 2 em Eucariotos/metabolismo , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Glicina/farmacologia , Células HL-60/efeitos dos fármacos , Células HL-60/enzimologia , Células HL-60/patologia , Humanos , Immunoblotting , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fragmentos de Peptídeos/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Inibidores da Síntese de Proteínas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR , Tiofenos/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The role of NMR in structural genomics is outlined, with particular emphasis on using protein domains as targets. Strategies for domain expression, characterization, and labeling are presented.