9-Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow-binding picomolar inhibitors of trimeric purine nucleoside phosphorylase.

Abstract

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 = 85 +/- 13 pm (pH 7.0, 25 degrees C), whereas the apparent inhibition constant determined by classical methods was two orders of magnitude higher ( = 4.4 +/- 0.6 nm). The rate constant for formation of the enzyme/inhibitor reversible complex is (8.4 +/- 0.5) x 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 pm (68% confidence interval is 147-389 pm). 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) x 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 nm) and = 237 pm (68% confidence interval is 123-401 pm), 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.

• Authors: Katarzyna Breer, Ljubica Glavas-Obrovac, Mirjana Suver, Sadao Hikishima, Mariko Hashimoto, Tsutomu Yokomatsu, Beata Wielgus-Kutrowska, Lucyna Magnowska, Agnieszka Bzowska
• Journal: The FEBS journal (FEBS J) Vol. 277 Issue 7 Pg. 1747-60 (Apr 2010) ISSN: 1742-4658 [Electronic] England
• PMID: 20193043 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Organophosphonates
• Clodronic Acid
• difluoromethylene diphosphonate
• Guanine
• 9-deazaguanine
• Purine-Nucleoside Phosphorylase

Topics

• Biochemistry (methods)
• Cell Line, Tumor
• Cell Membrane (metabolism)
• Clodronic Acid (analogs & derivatives, chemistry)
• Endocytosis
• Guanine (analogs & derivatives, chemistry)
• Humans
• Jurkat Cells
• Kinetics
• Lymphocytes (metabolism)
• Models, Chemical
• Organophosphonates (chemistry)
• Permeability
• Protein Binding
• Purine-Nucleoside Phosphorylase (adverse effects, chemistry)