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Updated: Jul 29, 2026
A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example
Published on: May 29, 2012
Nucleoside hydrolase immobilized on magnetic particles as a tool for onflow screening and characterization of
Pamella Christina Ortega de Oliveira1, Millena Santana Ceroullo1, Mayane Barbosa Dos Santos2
1BioCrom, Organic Chemistry Department, Chemistry Institute, Fluminense Federal University, Niterói, RJ, Brazil.
Abstract:
Nucleoside Hydrolases (NH) are considered a target for the development of new antiprotozoal agents. The development of new and automated screening assays for the identification of NH inhibitors can accelerate the first stages of the drug discovery process. In this work, NH from Leishmania donovani (LdNH) was covalently immobilized onto magnetic particles (LdNH-MPs) and trapped by magnets into a TFE tube to yield an immobilized enzyme reactor (IMER). For an automated assay, the LdNH-MP-IMER was connected in-line to an analytical column in an HPLC-DAD system to monitor the enzyme activity through quantification of the product hypoxanthine. Kinetic studies provided a KM value of 2079 ± 87 µmol.L-1 for the inosine substrate. Validation of the LdNH-MP-IMER for onflow screening purposes was performed with a library containing 12 quinolone ribonucleosides. Among them, three were identified as new competitive LdNH inhibitors, with Ki values between 83.5 and 169.4 µmol.L-1. This novel in-line screening assay has proven to be reliable, fast, low cost, and applicable to large libraries of compounds.
