Nucleoside hydrolase immobilized on magnetic particles as a tool for onflow screening and characterization of inhibitors

Pamella Christina Ortega de Oliveira; Millena Santana Ceroullo; Mayane Barbosa Dos Santos; Pedro Rodrigues Coelho Medeiros; Bruno Clemente Brandão Marques; Luzineide Wanderley Tinoco; Maria Cecília Bastos Vieira de Souza; Fernanda da Costa Santos Boechat; Marcela Cristina de Moraes

doi:10.1016/j.jpba.2023.115589

Nucleoside hydrolase immobilized on magnetic particles as a tool for onflow screening and characterization of inhibitors

J Pharm Biomed Anal. 2023 Oct 25:235:115589. doi: 10.1016/j.jpba.2023.115589. Epub 2023 Jul 20.

Authors

Pamella Christina Ortega de Oliveira¹, Millena Santana Ceroullo¹, Mayane Barbosa Dos Santos², Pedro Rodrigues Coelho Medeiros¹, Bruno Clemente Brandão Marques³, Luzineide Wanderley Tinoco³, Maria Cecília Bastos Vieira de Souza², Fernanda da Costa Santos Boechat², Marcela Cristina de Moraes⁴

Affiliations

¹ BioCrom, Organic Chemistry Department, Chemistry Institute, Fluminense Federal University, Niterói, RJ, Brazil.
² LNHC, Organic Chemistry Department, Chemistry Institute, Fluminense Federal University, Niterói, RJ, Brazil.
³ Laboratory for Analysis and Development of Enzyme Inhibitors, Natural Products Research Institute, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
⁴ BioCrom, Organic Chemistry Department, Chemistry Institute, Fluminense Federal University, Niterói, RJ, Brazil. Electronic address: mcmoraes@id.uff.br.

PMID: 37531732
DOI: 10.1016/j.jpba.2023.115589

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 K_M 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 K_i 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.

Keywords: Immobilized enzymes; Inhibitors; Liquid Chromatography; Ribonucleosides; Screening assays.

MeSH terms

Chromatography, High Pressure Liquid
Enzymes, Immobilized* / chemistry
Kinetics
Magnetic Phenomena
N-Glycosyl Hydrolases*

Substances

N-Glycosyl Hydrolases
Enzymes, Immobilized