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9‐Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow‐binding picomolar inhibitors of trimeric purine nucleoside phosphorylase
Author(s) -
Breer Katarzyna,
GlavašObrovac Ljubica,
Suver Mirjana,
Hikishima Sadao,
Hashimoto Mariko,
Yokomatsu Tsutomu,
WielgusKutrowska Beata,
Magnowska Lucyna,
Bzowska Agnieszka
Publication year - 2010
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2010.07598.x
Subject(s) - purine nucleoside phosphorylase , chemistry , dissociation constant , stereochemistry , conformational change , enzyme , reaction rate constant , nucleoside , purine , biochemistry , kinetics , receptor , physics , quantum mechanics
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 m m phosphate concentration) with calf spleen PNP was shown to be = 85 ± 13 p m (pH 7.0, 25 °C), whereas the apparent inhibition constant determined by classical methods was two orders of magnitude higher ( = 4.4 ± 0.6 n m ). The rate constant for formation of the enzyme/inhibitor reversible complex is (8.4 ± 0.5) × 10 5 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 fluorimetric titration, which led to a dissociation constant of 254 p m (68% confidence interval is 147–389 p m ). Stopped‐flow experiments, together with the above data, are most consistent with a two‐step binding mechanism: E + I ↔ (EI) ↔ (EI)*. The rate constants for reversible enzyme/inhibitor complex formation (EI), and for the conformational change (EI) ↔ (EI)*, are k on1 = (17.46 ± 0.05) × 10 5 m −1 ·s −1 , k off1 = (0.021 ± 0.003) s −1 , k on2 = (1.22 ± 0.08) s −1 and k off2 = (0.024 ± 0.005) s −1 , respectively. This leads to inhibition constants for the first (EI) and second (EI)* complexes of K i = 12.1 nM (68% confidence interval is 8.7–15.5 n m ) and = 237 p m (68% confidence interval is 123–401 p m ), respectively. At a concentration of 10 −4 m , DFPP‐DG exhibits weak, but statistically significant, inhibition of the growth of cell lines sensible to inhibition of PNP activity, such as human adult T‐cell leukaemia and lymphoma (Jurkat, HuT78 and CCRF‐CEM). Similar inhibitory activities of the tested compound were noted on the growth of lymphocytes collected from patients with Hashimoto’s thyroiditis and Graves’ disease. The observed weak cytotoxicity may be a result of poor membrane permeability.