RESUMO
The beta2 integrin leukocyte function antigen-1 (LFA-1) has an important role in the pathophysiology of inflammatory and autoimmune diseases. Here we report that statin compounds commonly used for the treatment of hypercholesterolemia selectively blocked LFA-1-mediated adhesion and costimulation of lymphocytes. This effect was unrelated to the statins' inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A reductase; instead it occurred via binding to a novel allosteric site within LFA-1. Subsequent optimization of the statins for LFA-1 binding resulted in potent, selective and orally active LFA-1 inhibitors that suppress the inflammatory response in a murine model of peritonitis. Targeting of the statin-binding site of LFA-1 could be used to treat diseases such as psoriasis, rheumatoid arthritis, ischemia/reperfusion injury and transplant rejection.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Adesão Celular/efeitos dos fármacos , Lovastatina/farmacologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Naftalenos/farmacologia , Pravastatina/farmacologia , Linfócitos T/efeitos dos fármacos , Sítio Alostérico , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Modelos Animais de Doenças , Desenho de Fármacos , Humanos , Integrina alfa4beta1 , Integrinas/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/farmacologia , Antígeno de Macrófago 1/metabolismo , Camundongos , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Naftalenos/toxicidade , Peritonite/tratamento farmacológico , Ligação Proteica/efeitos dos fármacos , Receptores de Retorno de Linfócitos/metabolismo , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
The lymphocyte function-associated antigen (LFA-1) belongs to the family of beta2-integrins and plays an important role in T-cell activation and leukocyte migration to sites of inflammation. We report here that lovastatin, a drug clinically used for lowering cholesterol levels, inhibits the interaction of human LFA-1 with its counter-receptor intercellular adhesion molecule-1. Using nuclear magnetic resonance spectroscopy and X-ray crystallography we show that the inhibitor binds to a highly conserved domain of the LFA-1 alpha-chain called the I-domain. The first three-dimensional structure of an integrin inhibitor bound to its receptor reveals atomic details for a hitherto unknown mode of LFA-1 inhibition. It also sheds light into possible mechanisms of LFA-1 mediated signalling and will support the design of novel anti-adhesive and immunosuppressive drugs.
Assuntos
Antígenos CD11/metabolismo , Lovastatina/farmacologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Antígenos CD11/química , Adesão Celular/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Células Tumorais CultivadasRESUMO
The design and synthesis of cyclic mimetics of VCAM-1 protein that reproduce the integrin-binding domain are presented. The unprotected peptide precursor 37-43, Thr-Gln-Ile-Asp-Ser-Pro-Leu, was grafted onto functional templates of type naphthalene, biphenyl and benzyl through the chemoselective formation of C- and N-terminal oximes resulting in a mixture of four isomeric forms due to syn-anti isomerism of the oxime bonds. Some isomers could be monitored by HPLC and identified by NMR. The molecule containing a naphthalene-derived template was found to inhibit the VCAM-1/VLA-4 interaction more efficiently than previously reported for sulfur-bridged cyclic peptides containing similar sequences. The finding confirms the importance of incorporating conformational constraints between the terminal ends of the peptide loop 37-43 in the design of synthetic inhibitors of the VCAM-1/integrin interaction.
Assuntos
Mimetismo Molecular , Molécula 1 de Adesão de Célula Vascular/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
A simple, rapid and reliable method has been developed for assessing the number and viability of cells, as well as cell size, in suspension culture by the use of flow cytometry. Propidium iodide exclusion is used for viability determination and fluorescent beads serve as an internal standard for cell enumeration. The main advantages of this method are its ability to handle a large number of samples with a high degree of precision and its specificity in detecting viable cells quantitatively in a heterogeneous culture of living and dead cells and debris. The method shows only a fraction of the variation found in the haemacytometer/trypan blue counting method due to its very low operator dependence. CHO - Chinese hamster ovary; FCS - Foetal calf serum; FS - Forward scatter light; MTT - 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; NCS - newborn calf serum; PBS - Phosphate buffered saline; PI - Propidium iodide; SS - Side scatter light.
