Design, Synthesis, Biological Evaluation, and Crystallographic Study of Novel Purine Nucleoside Phosphorylase Inhibitors.

Purine nucleoside phosphorylase (PNP) is a well-known molecular target with potential therapeutic applications in the treatment of T-cell malignancies and/or bacterial/parasitic infections. Here, we report the design, development of synthetic methodology, and biological evaluation of a series of 30 novel PNP inhibitors based on acyclic nucleoside phosphonates bearing a 9-deazahypoxanthine nucleobase. The strongest inhibitors exhibited IC50 values as low as 19 nM (human PNP) and 4 nM (Mycobacterium tuberculosis (Mt) PNP) and highly selective cytotoxicity toward various T-lymphoblastic cell lines with CC50 values as low as 9 nM. No cytotoxic effect was observed on other cancer cell lines (HeLa S3, HL60, HepG2) or primary PBMCs for up to 10 µM. We report the first example of the PNP inhibitor exhibiting over 60-fold selectivity for the pathogenic enzyme (MtPNP) over hPNP. The results are supported by a crystallographic study of eight enzyme-inhibitor complexes and by ADMET profiling in vitro and in vivo.

Identification of N-methyl-D-aspartate receptor antagonists using the rat postnatal mixed cortical and hippocampal neurons.

The goal of this study was to evaluate mixed cortical and hippocampal primary rat postnatal neuronal culture as in vitro tool for identification of N-methyl-D-aspartate receptor (NMDAR) antagonists and to find out, whether this model is comparable with other commonly used primary rat neuronal models differing in their origin (pure cortical vs. mixed cortical and hippocampal) and differentiation state (embryonal vs. postnatal). Induced pluripotent stem cell (iPSC) - derived human glutamatergic neurons have been included in this study as well. First, the cultures were characterized by their neuron/astrocyte composition, the mRNA expression of NR2B/NR2A NMDAR subunit ratios, and the expression of glutamate transporters (GLT1, GLAST). Then, selected endogenous steroids and synthetic neuroactive steroids that have been previously identified as negative allosteric modulators of recombinant GluN1/GluN2B NMDA receptors, were evaluated for their ability to prevent an NMDA or glutamate-induced Ca2+ influx (acute effect) and excitotoxicity over 24 h. Though the neuroprotective potential against excitotoxic stimuli varied among the models studied, postnatal mixed cortical and hippocampal culture proved to be a convenient and robust tool for NMDAR antagonist screening. The most widely used embryonal (E18) cultures offered higher cell yields but at the expense of a higher sensitivity to compounds' cytotoxicity. iPSC-derived neurons were not found to be superior to rat cultures for screening purposes.