RESUMEN
Mitogen-activated protein kinase phosphatase-1 (MKP-1) has proved to be an attractive target for the development of therapeutics for the treatment of cancer. We report the first example for a successful application of the structure-based virtual screening to identify the novel inhibitors of MKP-1. It is shown that the efficiency of virtual screening can be enhanced significantly by the incorporation of a new solvation energy term in the scoring function. The newly found inhibitors have desirable physicochemical properties as a drug candidate and reveal a moderate potency with IC(50) values ranging from 20 to 50 µM. Therefore, they deserve a consideration for further development by structure-activity relationship studies to optimize the inhibitory activities. Structural features relevant to the stabilization of the inhibitors in the active site of MKP-1 are discussed in detail.
Asunto(s)
Evaluación Preclínica de Medicamentos , Fosfatasa 1 de Especificidad Dual/antagonistas & inhibidores , Fosfatasa 1 de Especificidad Dual/química , Proteína Quinasa 1 Activada por Mitógenos/química , Modelos Moleculares , Sitios de Unión , Dominio Catalítico , Diseño de Fármacos , Pruebas de Enzimas , Inhibidores Enzimáticos/química , Concentración 50 Inhibidora , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Estructura Molecular , Relación Estructura-ActividadRESUMEN
This study was designed to evaluate the additive effects of transforming growth factor-beta3 (TGF-ß3) and hyaluronic acid (HA) on chondrogenic differentiation of human mesenchymal stem cells (hMSCs). The hMSCs were cultured on collagen type I-, HA-, or fibronectin-coated cell culture dishes with or without TGF-ß3 added to the culture medium. Four weeks after cell culture, chondrogenic differentiation of hMSCs was determined by evaluating the expression of cartilage-specific markers using real-time polymerase chain reaction, immunocytochemistry, and Western blot analysis. hMSCs cultured on HA-coated dishes with TGF-ß3 supplementation revealed a prominent increase in collagen type II, aggrecan, and Sox9. When hMSCs were cultured without TGF-ß3 supplementation, only hMSCs cultured on HA-coated dishes showed prominent expression of the cartilage-specific markers. This study shows that chondrogenic differentiation of hMSCs can be enhanced additively by interactions with both a specific cell-adhesion matrix and a soluble growth factor.
Asunto(s)
Condrogénesis , Ácido Hialurónico/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ingeniería de Tejidos/métodos , Factor de Crecimiento Transformador beta3/farmacología , Agrecanos/metabolismo , Animales , Biomarcadores/metabolismo , Cartílago/metabolismo , Células Cultivadas , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Expresión Génica , Humanos , Células Madre Mesenquimatosas/citología , Ratas , Ratas Sprague-Dawley , Factor de Transcripción SOX9/metabolismoRESUMEN
The L1CAM antibody A10-A3 efficiently reduces tumor growth in a nude mouse model. Here, we describe the crystal structure of the Fab fragment of A10-A3 determined at 2.0 angstrom resolution. The A10-A3 antibody H3 loop reveals a characteristic arrangement of exposed aromatic residues that may play an important role in antigen binding. A structure model of the complex between L1CAM Ig1-4 and A10-A3 Fab indicates that the Fab binds to three small loops outside Ig1 and a residue between Ig1 and Ig2, consistent with an epitope mapping result. The data presented here should contribute to the design of high-affinity antibody for therapeutic purposes as well as to the understanding of neural cell remodeling and cancer progression mechanism mediated by L1CAM.
Asunto(s)
Anticuerpos/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Antígenos/inmunología , Molécula L1 de Adhesión de Célula Nerviosa/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Anticuerpos/metabolismo , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Antígenos/química , Antígenos/metabolismo , Sitios de Unión , Cristalización , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Ratones , Modelos Moleculares , Molécula L1 de Adhesión de Célula Nerviosa/química , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Difracción de Rayos XRESUMEN
Long-term release of bone morphogenetic protein-2 (BMP-2) can promote bone regeneration. We developed an injectable system for long-term delivery of BMP-2 by covalently conjugating heparin to fibrinogen. The heparin-conjugated fibrinogen formed an injectable, heparin-conjugated fibrin (HCF) gel when mixed with thrombin. HCF released 89.4 +/- 3.8% of the loaded BMP-2 for 13 days, whereas normal fibrin released 83.7 +/- 7.6% for the initial 3 days. BMP-2 released from HCF significantly increased alkaline phosphatase activity of cultured osteoblasts, whereas BMP-2 released from normal fibrin did not do so, indicating that BMP-2 released from HCF is bioactive and suggesting that long-term delivery of BMP-2 is advantageous over short-term delivery for bone regeneration. HCF, BMP-2-loaded HCF, and BMP-2-loaded normal fibrin containing free heparin were contained in polyester cylindrical tubes and implanted into the hind limb muscle pockets of rats for 8 weeks. Soft X-ray radiography, computed tomography, histomorphometry, calcium assay, and western blot analysis showed that BMP-2-loaded HCF yielded the most extensive bone formation among the groups. Since HCF can deliver BMP-2 over a long term, is an injectable system, and is made of clinically benign materials, this system would have advantages for clinical applications to regenerate bone.