title: Solvent Selection for Insoluble Ligands, a Challenge for Biological Assay Development: A TNF-α/SPD304 Study.
abstract: 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-α 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-α/SPD304 system, and a K d of 5.36 ± 0.21 μM was determined. The results of this study could be used for the future screening of potential TNF-α inhibitors, while the protocols developed in this work could be applied to other proteins.
authors.affiliation: The Centre for Research and Technology of Thessaly (CE.RE.TE.TH.) , 3rd km Karditsa-Mitropolis, Karditsa 43100, Greece.
authors.forename: Christos P
authors.initials: CP
authors.lastname: Papaneophytou
authors.affiliation: The Centre for Research and Technology of Thessaly (CE.RE.TE.TH.) , 3rd km Karditsa-Mitropolis, Karditsa 43100, Greece ; Veterinary School, University of Thessaly , Trikalon 224, Karditsa 43100, Greece.
authors.forename: Anthi K
authors.initials: AK
authors.lastname: Mettou
authors.affiliation: Laboratory of Genetics, Department of Agricultural Biotechnology, Agricultural University of Athens , Athens 11855, Greece ; Biomedical Science Research Center 'Alexander Fleming' , Vari 16672, Greece.
authors.forename: Vagelis
authors.initials: V
authors.lastname: Rinotas
authors.affiliation: Laboratory of Genetics, Department of Agricultural Biotechnology, Agricultural University of Athens , Athens 11855, Greece ; Biomedical Science Research Center 'Alexander Fleming' , Vari 16672, Greece.
authors.forename: Eleni
authors.initials: E
authors.lastname: Douni
authors.affiliation: The Centre for Research and Technology of Thessaly (CE.RE.TE.TH.) , 3rd km Karditsa-Mitropolis, Karditsa 43100, Greece ; Veterinary School, University of Thessaly , Trikalon 224, Karditsa 43100, Greece.
authors.forename: George A
authors.initials: GA
authors.lastname: Kontopidis
keywords: aqueous solubility; fluorescence binding assay; insoluble ligands; protein stability; solvent selection