Lucyna Magnowska

Interactions of Calf Spleen Purine Nucleoside Phosphorylase with Antiviral Acyclic Nucleoside Phosphonate Inhibitors: Kinetics and Emission Studies

Association between calf spleen purine nucleoside phosphorylase and a series of phosphonylalkoxyalkyl derivatives of purine bases was studied by inhibition kinetics and fluorimetric titrations. Dissociation constants, determined by fluorimetric titration in phosphate-free conditions, were lower than inhibition constants in 1 mM phosphate, and inhibition was still weaker in 50 mM phosphate, in accord with the postulated bisubstrate analogue character of this class of inhibitors.

Interactions of potent multisubstrate analogue inhibitors with purine nucleoside phosphorylase from calf spleen: Kinetic and spectrofluorimetric studies

Abstract Dissociation constants and stoichiometry of binding for interaction of trimeric calf spleen purine nucleoside phosphorylase with potent multisubstrate analogue inhibitors were studied by kinetic and spectrofluorimetric methods.

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 °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) × 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 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 kon1 = (17.46 ± 0.05) × 105 m−1·s−1, koff1 = (0.021 ± 0.003) s−1, kon2 = (1.22 ± 0.08) s−1 and koff2 = (0.024 ± 0.005) s−1, respectively. This leads to inhibition constants for the first (EI) and second (EI)* complexes of Ki = 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.

Kinetic model of oxidation catalyzed by xanthine oxidase-the final enzyme in degradation of purine nucleosides and nucleotides.

A new kinetic model is presented for analysis of experimental data of oxidation process catalyzed by milk xanthine oxidase. The kinetics for two substrates, xanthine and its analog 2-chloroadenine, in a broad pH range (5.8–9.0) are best described by an equation which is a rational function of degree 2:3 and 2:2, respectively.

Synthesis of 6-aryloxy- and 6-arylalkoxy-2-chloropurines and their interactions with purine nucleoside phosphorylase from Escherichia coli.

The phase transfer method was applied to perform the nucleophilic substitution of 2,6- dichloropurines by modified arylalkyl alcohol or phenols. Since under these conditions only the 6-halogen is exchanged, this method gives 2-chloro-6-aryloxy- and 2-chloro-6-arylalkoxypurines. 2-Chloro-6-benzylthiopurine was synthesized by alkylation of 2-chloro-6-thiopurine with benzyl bromide. The stereoisomers of 2-chloro-6-(1-phenyl-1-ethoxy)purine were obtained from R- and S-enantiomers of sec.-phenylethylalcohol and 2,6-dichloropurine. All derivatives were tested for inhibition with purified hexameric E. coli purine nucleoside phosphorylase (PNP). For analogues showing IC50 < 10 μm, the type of inhibition and inhibition constants were determined. In all cases the experimental data were best described by the mixed-type inhibition model and the uncompetitive inhibition constant, Kiu, was found to be several-fold lower than the competitive inhibition constant, Kic. This effect seems to be due to the 6-aryloxy- or 6-arylalkoxy substituent, because a natural PNP substrate adenine, as well as 2-chloroadenine, show mixed type inhibition with almost the same inhibition constants Kiu and KiC. The most potent inhibition was observed for 6-benzylthio-2-chloro-, 6-benzyloxy-2-chloro-, 2-chloro-6-(2-phenyl-l-ethoxy), 2-chloro-6-(3-phenyl-l-propoxy)- and 2-chloro-6-ethoxypurines (Kiu = 0.4, 0.6, 1.4, 1.4 and 2.2 μm, respectively). The R-stereoisomer of 2-chloro-6-(1pheny-1-ethoxy)purine has Kiu = 2.0 μm, whereas inhibition of its S counterpart is rather weak (IC50> 12 μm). More rigid (e.g. phenoxy-), non-planar (cyclohexyloxy-), or more bulky (2,4,6-trimethylphenoxy-) substituents at position 6 of the purine base gave less potent inhibitors (IC50 = 26, 56 and >100 μm, respectively). The derivatives are selective inhibitors of hexameric “high-molecular mass” PNPs because no inhibitory activity vs. trimeric Cellulomonas sp. PNP was detected. By establishing the ligand-dependent stabilization pattern of the E. coli PNP it was shown that the new derivatives, similarly as the natural purine bases, are able to form a dead-end ternary complex with the enzyme and orthophosphate. It was also shown that the derivatives are substrates in the reverse synthetic direction catalyzed by E. coli PNP

Simple and universal method to determine dissociation constants for enzyme/ligand complexes

A simple and in principle universal method is proposed for measuring enzyme/ligand dissociation constants. The method is based on measuring enzyme activity remaining after heat treatment in the absence and in the presence of ligands. The method is especially suitable for enzymes interacting with nucleosides, nucleosides and oligonucleotides since for such enzymes convenient spectrophotometric assays are available.

Antiproliferative Activity of Purine Nucleoside Phosphorylase Multisubstrate Analogue Inhibitors Containing Difluoromethylene Phosphonic Acid against Leukaemia and Lymphoma Cells

Potent inhibitors of purine nucleoside phosphorylase (PNP) are expected to act as selective agents against T‐cell tumours. Five compounds with guanine, three with hypoxanthine, and five with 9‐deazaguanine, all connected by a linker with difluoromethylene phosphonic acid, were studied on their inhibitory potential against human and calf PNPs. Antiproliferative activity of these analogues against lymphocytes as well as lymphoma and leukaemia cells has been also investigated. All tested compounds act as multisubstrate analogue inhibitors of PNP with the apparent inhibition constants in the range 5–100 nm, and also show a slight antiproliferative activity. Analogues with 9‐deazaguanine aglycone have better anti‐leukaemic and anti‐lymphoma activities compared to the guanine and hypoxanthine analogues, and applied in the concentration of 100 μm, caused a statistically significant decrease in the cell viability in all human leukaemia and lymphoma cells used. Despite the high PNP inhibitory potential of tested analogues, no differences were observed between the effects on the growth of tumour cells sensible to the inhibition of PNP, such as human adult T‐cell leukaemia and lymphoma cells, and other investigated cells. Obtained poor effects on cell proliferation could be explained probably by a poor ability of tested compounds to penetrate cell membranes.

Synthesis of 2-Chloro-6-aryloxy- and 2-Chloro-6-alkoxyarylpurines and Their Properties in the Purine Nucleoside Phosphorylase (PNP) System

Abstract A series of 2-chloro-6-aryloxy- and 2-chloro-6-alkoxyarylpurines was synthesized and their kinetic properties in the purine nucleoside phosphorylase (PNP) system were determined. All compounds showed inhibitory activity (IC50 in the range 0.5-76 μM) vs. hexameric (“high-molecular weight”) PNP from E. coli. By contrast, no inhibition vs. trimeric Cellulomonas PNP was detected.