Discovery of a Novel Inhibitor of Human Purine Nucleoside Phosphorylase by a Simple Hydrophilic Interaction Liquid Chromatography Enzymatic Assay.
Marco RabuffettiFrancesca RinaldiAlessandra Lo BiancoGiovanna SperanzaDaniela UbialiMarcela Cristina de MoraesLuiz Claudio Rodrigues Pereira da SilvaGabriella MassoliniEnrica CalleriAntonio LavecchiaPublished in: ChemMedChem (2021)
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.
Keyphrases
- liquid chromatography
- endothelial cells
- mass spectrometry
- high throughput
- induced pluripotent stem cells
- high resolution mass spectrometry
- hydrogen peroxide
- small molecule
- bone marrow
- oxidative stress
- induced apoptosis
- risk assessment
- cell proliferation
- radiation therapy
- molecular dynamics
- climate change
- dna methylation
- neoadjuvant chemotherapy
- combination therapy
- real time pcr
- replacement therapy
- single cell