title: 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.
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.
authors: Dipartimento di Scienze del Farmaco, Università degli Studi di Pavia, Viale Taramelli 12, 27100 Pavia, Italy.
authors: Giulia
authors: G
authors: Cattaneo
authors: Dipartimento di Scienze del Farmaco, Università degli Studi di Pavia, Viale Taramelli 12, 27100 Pavia, Italy; ISTM-CNR, Via Golgi 19, 20133 Milano, Italy.
authors: Daniela
authors: D
authors: Ubiali
authors: Dipartimento di Scienze del Farmaco, Università degli Studi di Pavia, Viale Taramelli 12, 27100 Pavia, Italy.
authors: Enrica
authors: E
authors: Calleri
authors: Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy.
authors: Marco
authors: M
authors: Rabuffetti
authors: Department Pharmazie-Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität, Butenandtstr. 5-13, 81377 München, Germany.
authors: Georg C
authors: GC
authors: Höfner
authors: Department Pharmazie-Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität, Butenandtstr. 5-13, 81377 München, Germany.
authors: Klaus T
authors: KT
authors: Wanner
authors: Departamento de Química Orgânica, Instituto de Química, Universidade Federal Fluminense, Outeiro de São João Batista s/n, 24020-141 Niterói, Rio de Janeiro, Brazil.
authors: Marcela C
authors: MC
authors: De Moraes
authors: 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.
authors: Leonardo K B
authors: LKB
authors: Martinelli
authors: 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.
authors: Diógenes Santiago
authors: DS
authors: Santos
authors: Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy; ISTM-CNR, Via Golgi 19, 20133 Milano, Italy.
authors: Giovanna
authors: G
authors: Speranza
authors: Dipartimento di Scienze del Farmaco, Università degli Studi di Pavia, Viale Taramelli 12, 27100 Pavia, Italy. Electronic address: gabriella.massolini@unipv.it.
authors: Gabriella
authors: G
authors: Massolini
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004791:Enzyme Inhibitors; D011683:Purine-Nucleoside Phosphorylase
keywords: 8-Substituted purine ribonucleosides; 96-Well enzymatic assay; Enzyme inhibition; Human purine nucleoside phosphorylase; LC-ESI-MS/MS assay; Mycobacterium tuberculosis purine nucleoside phosphorylase