Development,validation and application of a 96-well enzymatic assay based on LC-ESI-MS/MS quantification for the screening of selective inhibitors against Mycobacterium tuberculosis purine nucleoside phosphorylase |
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Authors: | Giulia Cattaneo Daniela Ubiali Enrica Calleri Marco Rabuffetti Georg C Höfner Klaus T Wanner Marcela C De Moraes Leonardo KB Martinelli Diógenes Santiago Santos Giovanna Speranza Gabriella Massolini |
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Institution: | 1. Dipartimento di Scienze del Farmaco, Università degli Studi di Pavia, Viale Taramelli 12, 27100 Pavia, Italy;2. Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy;3. Department Pharmazie-Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität, Butenandtstr. 5-13, 81377 München, Germany;4. Departamento de Química Orgânica, Instituto de Química, Universidade Federal Fluminense, Outeiro de São João Batista s/n, 24020-141 Niterói, Rio de Janeiro, Brazil;5. ISTM-CNR, Via Golgi 19, 20133 Milano, Italy;6. Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Av. Ipiranga 6681/92, 90619-900 Porto Alegre, Rio Grande do Sul, Brazil |
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Abstract: | Mycobacterium tuberculosis (Mtb) purine nucleoside phosphorylase (PNP, EC 2.4.2.1) has been identified as a target for the development of specific inhibitors with potential antimycobacterial activity. We hereby described the development and validation of a new 96-well LC-ESI-MS/MS method to assess the inhibition activity of nucleoside analogues towards MtbPNP and the human PNP (HsPNP). Enzyme activity was determined by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx). The enzymatic assay (v = 0.5 mL, enzyme<0.2 μg/well, T = 37 °C) 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 the criteria of the CDER guidance of FDA. Kinetic parameters were in agreement with those reported in literature (HsPNP KM = 0.150 ± 0.020 mM vs 0.133 ± 0.015 mM; MtbPNP KM = 0.060 ± 0.009 mM vs 0.040 ± 0.003 mM for Ino), thus demonstrating the reliability of the newly developed enzymatic assay. Preliminary inhibition assays confirmed the effects reported for Acyclovir (Acv) and Formycin A (FA) against HsPNP and MtbPNP. The validated enzymatic assay was applied to the evaluation of a set of 8-halo-, 8-amino-, 8-O-alkyl-substituted purine ribonucleosides synthesized on purpose as potential inhibitors against MtbPNP. The assayed 8-substituted ribonucleosides did not exert a significant inhibitory effect against the tested enzymes up to 1 mM. |
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Keywords: | Mycobacterium tuberculosis purine nucleoside phosphorylase Human purine nucleoside phosphorylase Enzyme inhibition LC-ESI-MS/MS assay 96-Well enzymatic assay 8-Substituted purine ribonucleosides |
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