9-Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow-binding picomolar inhibitors of trimeric purine nucleoside phosphorylase.
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.
Breer
Katarzyna
K
Department of Biophysics, Institute of Experimental Physics, Warsaw University, Poland.
Glavas-Obrovac
Ljubica
L
Suver
Mirjana
M
Hikishima
Sadao
S
Hashimoto
Mariko
M
Yokomatsu
Tsutomu
T
Wielgus-Kutrowska
Beata
B
Magnowska
Lucyna
L
Bzowska
Agnieszka
A
D001671:Biochemistry / Q000379:methods; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D004002:Clodronic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D004705:Endocytosis; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism; D008956:Models, Chemical; D063065:Organophosphonates / Q000737:chemistry; D010539:Permeability; D011485:Protein Binding; D011683:Purine-Nucleoside Phosphorylase / Q000009:adverse effects / Q000737:chemistry*
D063065:Organophosphonates; D004002:Clodronic Acid; C041121:difluoromethylene diphosphonate; D006147:Guanine; C076777:9-deazaguanine; D011683:Purine-Nucleoside Phosphorylase