Human purine nucleoside phosphorylase (HsPNP) belongs to the purine salvage pathway of nucleic acids. Genetic deficiency of this enzyme triggers apoptosis of activated T-cells due to the accumulation of deoxyguanosine triphosphate (dGTP). Therefore, potential chemotherapeutic applications of human PNP inhibitors include the treatment of T-cell leukemia, autoimmune diseases and transplant tissue rejection. In this report, we present the discovery of novel HsPNP inhibitors by coupling experimental and computational tools. A simple, inexpensive, direct and non-radioactive enzymatic assay coupled to hydrophilic interaction liquid chromatography and UV detection (LC-UV using HILIC as elution mode) was developed for screening HsPNP inhibitors. Enzymatic activity was assessed by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx) by LC-UV. A small library of 6- and 8-substituted nucleosides was synthesized and screened. The inhibition potency of the most promising compound, 8-aminoinosine (4), was quantified through Ki and IC50 determinations. The effect of HsPNP inhibition was also evaluated in vitro through the study of cytotoxicity on human T-cell leukemia cells (CCRF-CEM). Docking studies were also carried out for the most potent compound, allowing further insights into the inhibitor interaction at the HsPNP active site. This study provides both new tools and a new lead for developing novel HsPNP inhibitors.

Discovery of a Novel Inhibitor of Human Purine Nucleoside Phosphorylase by a Simple Hydrophilic Interaction Liquid Chromatography Enzymatic Assay / M. Rabuffetti, F. Rinaldi, A. Lo Bianco, G. Speranza, D. Ubiali, M.C. de Moraes, L.C. Rodrigues Pereira da Silva, G. Massolini, E. Calleri, A. Lavecchia. - In: CHEMMEDCHEM. - ISSN 1860-7179. - 16:8(2021 Apr 20), pp. 1325-1334. [10.1002/cmdc.202000874]

Discovery of a Novel Inhibitor of Human Purine Nucleoside Phosphorylase by a Simple Hydrophilic Interaction Liquid Chromatography Enzymatic Assay

M. Rabuffetti;G. Speranza;
2021

Abstract

Human purine nucleoside phosphorylase (HsPNP) belongs to the purine salvage pathway of nucleic acids. Genetic deficiency of this enzyme triggers apoptosis of activated T-cells due to the accumulation of deoxyguanosine triphosphate (dGTP). Therefore, potential chemotherapeutic applications of human PNP inhibitors include the treatment of T-cell leukemia, autoimmune diseases and transplant tissue rejection. In this report, we present the discovery of novel HsPNP inhibitors by coupling experimental and computational tools. A simple, inexpensive, direct and non-radioactive enzymatic assay coupled to hydrophilic interaction liquid chromatography and UV detection (LC-UV using HILIC as elution mode) was developed for screening HsPNP inhibitors. Enzymatic activity was assessed by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx) by LC-UV. A small library of 6- and 8-substituted nucleosides was synthesized and screened. The inhibition potency of the most promising compound, 8-aminoinosine (4), was quantified through Ki and IC50 determinations. The effect of HsPNP inhibition was also evaluated in vitro through the study of cytotoxicity on human T-cell leukemia cells (CCRF-CEM). Docking studies were also carried out for the most potent compound, allowing further insights into the inhibitor interaction at the HsPNP active site. This study provides both new tools and a new lead for developing novel HsPNP inhibitors.
antitumour activity; drug discovery; enzymatic assay; PNP inhibitors; screening
Settore CHIM/06 - Chimica Organica
Settore CHIM/08 - Chimica Farmaceutica
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/878150
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