9-deazaguanine and difluoromethylene-diphosphonate

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.

Topics: Biochemistry; Cell Line, Tumor; Cell Membrane; Clodronic Acid; Endocytosis; Guanine; Humans; Jurkat Cells; Kinetics; Lymphocytes; Models, Chemical; Organophosphonates; Permeability; Protein Binding; Purine-Nucleoside Phosphorylase

9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) was designed as a multi-substrate analogue inhibitor against purine nucleoside phosphorylase (PNP) on the basis of X-ray crystallographic data obtained for a binary complex of 9-(5',5'-difluoro-5'-phosphonopentyl)guanine (DFPP-G) with calf spleen PNP. DFPP-DG and its analogous compounds were adjusted by length of the linker achieved by the Sonogashira-coupling reaction between a 9-deaza-9-iodoguanine derivative and omega-alkynyldifluoromethylene phosphonates as a key reaction. DFPP-DG is a very potent PNP inhibitor with apparent inhibition constants (in the presence of 1 mM phosphate) of 4.4 and 8.1 nM versus calf spleen and human erythrocyte PNPs, respectively. One of its analogues, homo-DFPP-DG, with longer chain linking phosphonate and 9-deazaguanine is even more potent versus human enzyme, with an apparent inhibition constant of 5.3 nM (in the presence of 1mM phosphate).

Topics: Animals; Cattle; Clodronic Acid; Enzyme Inhibitors; Erythrocytes; Guanine; Humans; Models, Molecular; Purine-Nucleoside Phosphorylase; Spleen; Substrate Specificity