On-flow magnetic particle activity assay for the screening of human purine nucleoside phosphorylase inhibitors

On-flow magnetic particle activity assay for the screening of human purine nucleoside phosphorylase inhibitors

•Human PNP was covalently immobilized on magnetic particles.•Firstly, the activity of the immobilized enzyme was monitored off-line by HPLC-UV.•An on-flow assay was developed to assess the enzyme activity by HPLC-UV.•Both approaches were validated for ligand recognition using a previously identified...

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Bibliographic Details
Published in:Journal of Chromatography A Vol. 1663; p. 462740
Main Authors: Ximenes, I.A.T., Albino, M., Sangregorio, C., Cass, Q.B., de Moraes, M.C.
Format: Journal Article
Language:English
Published: Netherlands Elsevier B.V 25-01-2022
Subjects:
Bioaffinity chromatography
Enzyme Inhibitors - pharmacology
Humans
Immobilized enzyme
Kinetics
Magnetic particles
Magnetic Phenomena
On-flow enzymatic assay
Purine nucleoside phosphorylase
Purine Nucleosides
Purine-Nucleoside Phosphorylase - metabolism
Screening assay
On-flow enzymatic assay
Purine nucleoside phosphorylase
Screening assay
Magnetic particles
Bioaffinity chromatography
Immobilized enzyme
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Summary:•Human PNP was covalently immobilized on magnetic particles.•Firstly, the activity of the immobilized enzyme was monitored off-line by HPLC-UV.•An on-flow assay was developed to assess the enzyme activity by HPLC-UV.•Both approaches were validated for ligand recognition using a previously identified inhibitor.•The on-flow assay demonstrated high throughput due to its automation. Human purine nucleoside phosphorylase (HsPNP) catalyzes reversible phosphorolysis of nucleosides and deoxynucleosides in the purine cascade. HsPNP has been a target on behalf of the development of new leads for the treatment of a variety of T-cell mediated disorders. Several studies on the HsPNP are focused on the identification of effective, safe, and selective inhibitors. Therefore, this study describes the development of direct, simple, reliable, and inexpensive enzymatic assays to screen HsPNP inhibitors. Initially, HsPNP was covalently immobilized on the surface of magnetic particles (MPs). Due to the versatility of the MPs as solid support for enzyme immobilization, two different methods to monitor the enzyme activity are presented. Firstly, the activity of HsPNP-MPs was assessed offline by HPLC-DAD quantifying the formed hypoxanthine. Then, HsPNP-MPs were trapped in a peek tube, furnishing a microreactor which was inserted on-flow in an HPLC-DAD system to monitor the enzyme activity by the hypoxanthine quantification. Kinetic assays provided KMapp values for the inosine substrate of 488.2 ± 49.1 and 1084 ± 111 µM for the offline and on-flow assays, respectively. For the first time, kinetic studies for Pi as substrate using the HsPNP-MPs exhibits a Michaelis-Menten kinetic, yielding KMapp values for offline and on-flow of 521.2 ± 62.9 µM and 601 ± 66.5 µM, respectively. Inhibition studies conducted with a fourth generation immucillin derivative (DI4G) were employed as proof of concept to validate the use of the HsPNP-MPs assays for screening purposes. Additionally, a small library containing 11 compounds was used to assess the selectivity of the developed assays. The results showed that both presented assays can be applied to selectively recognizing and characterizing HsPNP inhibitors. Particularly, the on-flow method exhibited a high throughput and performance because of its automation and represents an easy and practical approach to reuse the HsPNP-MPs. Besides, this novel enzyme activity assay model can be further applied to other biological targets.
ISSN:0021-9673
1873-3778
DOI:10.1016/j.chroma.2021.462740