Solvent Selection for Insoluble Ligands, a Challenge for Biological Assay Development: A TNF-alpha/SPD304 Study

Many active compounds may be excluded from biological assays due to their low aqueous solubility. In this study, a simple method for the determination of the solubility of compounds containing aromatic rings is proposed. In addition to DMSO, five organic solvents for screening experiments of TNF-alpha inhibitors were explored. DMSO and PEG3350 were the most suitable for both protein stability and ligand-binding experiments. In addition, glycerol is a promising solvent for the screening of other compounds for which it might provide acceptable solubilization, due to its strong tendency to preserve the protein. Moreover, a fluorescence binding assay was developed using the TNF-alpha/SPD304 system, and a K-d of 5.36 +/- 0.21 mu M was determined. The results of this study could be used for the future screening of potential TNF-alpha inhibitors, while the protocols developed in this work could be applied to other proteins.