Substituted nucleosides as potential inhibitors of purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP)

Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) from Mycobacterium tuberculosis (MtPNP) has been identified as a target for the development of specific inhibitors with potential antimycobacterial activity. Although MtPNP and the human enzyme (HsPNP) belong to the same family, slight differences in transition states[1] and structural features[2] may be exploited to achieve specific inhibition. To this aim seven 8-halo-, 8-alkylamino- and 8-alkoxypurine ribonucleosides (Figure 1) were synthesized by either direct regioselective halogenation of the corresponding substrate or halogen substitution with a nucleophile on the 8-bromo intermediate. All products were prepared in very good yield (58-89%) and isolated in high purity. These nucleoside analogues were then submitted to a new 96-well LC-ESI-MS/MS method to assess their inhibition activity towards MtPNP and HsPNP, by monitoring the phosphorolysis of inosine to hypoxanthine. The enzymatic assay was performed with an overall time of about 15 min/plate for sample processing and 2 min/sample for LC-MS analysis. Validation of the quantification method met FDA criteria. Preliminary experimental data showed that the activity exerted towards MtPNP was negligible in most cases, with only 1 and 3 resulting to be weak inhibitors (at a μM scale). Although the inhibition results were not remarkable and further investigations are currently in progress, a safe and rapid screening assay towards MtPNP and HsPNP was developed. References: [1] Lewandowicz, A. et al. Biochemistry 2003, 42, 6057-6066. [2] a) Caceres, R. A. et al. Biochimie 2012, 94, 155-165; b) Ducati, R. G. et al. Bioorg. Med. Chem. 2010, 18, 4769-4774.