Development of multiplexed microarray binding assays for high-throughput drug discovery.

The ability to combine primary hit identification assays with target profiling would significantly streamline the current drug discovery process. Working towards this end, we report here the development of a microarray-based ligand binding assay that supports multiplexed analysis of G protein-coupled receptor systems in a 96-well microplate format that is compatible with the equipment and infrastructure typical of high-throughput screening laboratories. A prototype microarray was generated by pin-printing seven different receptors within the wells of a specially coated glass-bottom microplate and assaying with a cocktail of fluorescent ligands. Development of the multiplexed system included optimization of methods for depositing receptor membrane proteins and establishing a generic set of assay conditions that simultaneously satisfied the pharmacology requirements of all of the receptor systems included on the array. The multiplexed system is shown to produce valid pharmacological results as evidenced by its ability to report K(i) values for receptor-specific fluorescent ligands and rank ordered potencies for diagnostic displacing compounds comparable to values generated by conventional simplexed assays. Moreover, the results of a 40-compound mini-screen confirmed that the assay accurately identifies valid hits. The results suggest the assay may be immediately suitable for routine profiling tasks and demonstrate the potential of the format for high-throughput multiplexed drug discovery.

Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function.

UNLABELLED: We identified a previously unknown integrin, alpha(9)beta(1), on OCLs and their precursors. Antibody to alpha(9) inhibited OCL formation in human marrow cultures, and OCLs from alpha(9) knockout mice had a defect in actin ring reorganization and an impaired bone resorption capacity. INTRODUCTION: Integrins play important roles in osteoclast (OCL) formation and function. Mature OCLs mainly express alpha(v)beta(3) integrin, a heterodimer adhesion receptor that has been implicated in osteoclastic bone resorption. We identified ADAM8, a disintegrin and metalloproteinase, as a novel stimulator of OCL differentiation and showed that the disintegrin domain of ADAM8 mediated its effects on OCL formation. Because the disintegrin domain of ADAM8 does not bind Arg-Gly-Asp (RGD) sequences, we determined which integrin bound ADAM8 and characterized its role in OCL formation and activity. MATERIALS AND METHODS: Chinese hamster ovary cells (CHO) expressing different integrin subunits were tested for their capacity to bind the disintegrin domain of ADAM8. Mouse or human bone marrow cells and purified OCL precursors were tested for alpha(9)beta(1) integrin expression by Western blot, immunocytochemistry, and real-time RT-PCR. A monoclonal antibody to human alpha(9) was used to block alpha(9)beta(1) on OCL precursors stimulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or RANKL. Vertebrae of 7-day-old alpha(9)(-/-) mice and wildtype (WT) littermates were compared using bone histomorphometry and 3D microCT analysis. RESULTS: Alpha(9) integrin was expressed by mouse and human bone marrow-derived OCLs and their precursors. Importantly, the anti-alpha(9) antibody inhibited human OCL formation stimulated by 1alpha,25(OH)(2)D(3) or RANKL dose-dependently. Furthermore, analysis of OCLs formed in marrow cultures from alpha(9)(-/-) mice showed that the OCLs formed were more contracted and formed significantly less bone resorption pits on dentin slices. Histologic analysis of alpha(9)(-/-) vertebrae showed thickened trabecular regions and retained cartilage within vertebral bodies of alpha(9)(-/-) mice. 3D microCT analysis of alpha(9)(-/-) vertebrae also showed a significant increase in trabecular bone volume/total tissue volume and a tendency for decreased trabecular separation compared with WT mice. CONCLUSIONS: These results support a previously unknown role for alpha(9)beta(1) integrin in OCL formation and function.

The muscle integrin binding protein (MIBP) interacts with alpha7beta1 integrin and regulates cell adhesion and laminin matrix deposition.

Integrins are alphabeta transmembrane receptors that function in key cellular processes, including cell adhesion, differentiation, and extracellular matrix deposition through interactions with extracellular, membrane, and cytoplasmic proteins. We previously identified and cloned a muscle beta1 integrin cytoplasmic binding protein termed MIBP and found that the expression level of MIBP is critical in the decision-making process of terminal myogenic differentiation. We report here that MIBP interacts with the alpha7beta1 integrin but not the alpha5beta1 integrin in C2C12 myoblasts, suggesting an important role of integrin alpha chains in the regulation of the beta1-MIBP interaction. Furthermore, consistent with its selective binding activity toward the alpha7beta1 laminin receptor, we have found that overexpression of MIBP in C2C12 myoblasts resulted in a significant reduction of cell adhesion to laminin and inhibition of laminin matrix deposition. By contrast, neither cell adhesion to fibronectin nor fibronectin matrix deposition was significantly altered in cells overexpressing MIBP. Finally, we show that both the protein level and tyrosine phosphorylation of paxillin, a key signaling molecule involved in the cellular control of myogenic differentiation, are increased by MIBP. These results suggest that MIBP functions in the control of myogenic differentiation by regulating alpha7beta1 integrin-mediated cell interactions with laminin matrix and intracellular signaling through paxillin.