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

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 K i and IC 50 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.

8-Aminoinosine and 8-Aminohypoxanthine Inhibit Purine Nucleoside Phosphorylase and Exert Diuretic and Natriuretic Activity

“…1A], a value similar to that reported by Chern et al (1993). While this work was in progress, Rabuffetti et al (2021) reported that 8-aminoinosine is a competitive inhibitor of rhPNPase, with a Ki of 48 mmol/L. Indeed, we also observed that 8-aminoinosine reduced the activity of rhPNPase (with inosine as substrate) in a manner consistent with competitive inhibition (Fig.…”

“…Independent of our studies, Rabuffetti et al (2021) also observed that 8-aminoinosine is an inhibitor of PNPase, and their kinetic analysis suggested that 8-aminoinosine is a competitive inhibitor of this enzyme. Our results indicate that 8-aminoinosine’s ability to block PNPase activity likely involves two mechanisms.…”

Design and Synthesis of New Modified Flexible Purine Bases as Potential Inhibitors of Human PNP

“…Human purine nucleoside phosphorylase ( h PNP) belongs to a family of enzymes involved in the purine salvage pathway of nucleoside biosynthesis, which promotes the utilization of purine bases [ 1 ]. Its primary role is to catalyze the cleavage of inosine, 2’-deoxyinosine, guanosine and 2’-deoxyguanosine (dG) to the corresponding base and sugar-1-phosphate via reversible phosphorolysis.…”

“…In addition, h PNP inhibitors are used in organ transplant surgeries to provide an immunodeficiency status during and after the procedure. In some cases, h PNP inhibitors are also able to enhance the activity of antiviral and antitumor nucleoside drugs [ 1 , 4 ] by inhibiting the premature metabolism of the nucleoside drug. All of these reasons explain the great interest in studying the structure of human purine nucleoside phosphorylase and the continued search for more effective inhibitors.…”

“…Several immucillins have successfully completed or are currently in various stages of clinical trials, including Immucillin-H (Forodesine ® , Mundesine ® ), which was approved in Japan for treating relapsed or refractory peripheral T-cell lymphoma [ 7 ], and DADMe-immucillin H (DADMe-Immucillin-H, BCX4208, Ulodesine), which passed the second stage of clinical trials in the treatment of gout [ 8 ]. High levels of inhibitory activity were demonstrated by acyclic analogues of immucillins, namely DATMe-Immucillin-H and SerMe-Immucillin-H [ 1 ]. It should also be noted that many of the known h PNP inhibitors are derivatives of deazapurine bases.…”