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Genetic deficiency of purine nucleoside phosphorylase (PNP; EC 2.4.2.1) activity leads to a severe selective disorder of T-cell function. Therefore, potent inhibitors of mammalian PNP are expected to act as selective immunosuppressive agents against, for example, T-cell cancers and some autoimmune diseases. | 9-Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow-binding picomolar inhibitors of trimeric purine nucleoside phosphorylase Katarzyna Breer1 Ljubica Glavas-Obrovac2 Mirjana Suver2 Sadao Hikishima3 Mariko Hashimoto3 Tsutomu Yokomatsu3 Beata Wielgus-Kutrowska1 Lucyna Magnowska1 and Agnieszka Bzowska1 1 Department of Biophysics Institute of ExperimentalPhysics Warsaw University Poland 2 University HospitalOsijek and Schoolof Medicine University of J. J. Strossmayer in Osijek Croatia 3 Schoolof Pharmacy Tokyo University of Pharmacy and Life Science Japan Keywords 9-deazaguanine multisubstrate analogue inhibitors purine nucleoside phosphorylase slow-binding inhibitors tight-binding inhibitors Correspondence A. Bzowska Department of Biophysics Institute of ExperimentalPhysics Warsaw University Zwirki i Wigury 93 02-089 Warsaw Poland Fax 48 22 554 0771 Tel 48 22 554 0789 E-mail abzowska@biogeo.uw.edu.pl Received 4 October 2009 revised 14 January 2010 accepted 29 January 2010 doi 10.1111 j.1742-4658.2010.07598.x Genetic deficiency of purine nucleoside phosphorylase PNP EC 2.4.2.1 activity leads to a severe selective disorder of T-cell function. Therefore potent inhibitors of mammalian PNP are expected to act as selective immunosuppressive agents against for example T-cell cancers and some autoimmune diseases. 9- 5 5 -difluoro-5 -phosphonopentyl -9-deazaguanine DFPP-DG was found to be a slow- and tight-binding inhibitor of mammalian PNP. The inhibition constant at equilibrium 1 mM phosphate concentration with calf spleen PNP was shown to be Keq 85 13 pM pH 7.0 25 C whereas the apparent inhibition constant determined by classical methods was two orders of magnitude higher Ki pp 4.4 0.6 nM . The rate constant for formation of the enzyme inhibitor reversible complex is 8.4 0.5 X 105 M 1-s 1 which is a value that is too low to be diffusion-controlled. The picomolar binding of DFPP-DG was confirmed by fluorimet-ric titration which led
