Robert E. Parks

Purine Nucleoside Phosphorylase

It has long been appreciated that purine nucleoside phosphorylase (PNP; purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) may play a role in cancer chemotherapy by catalyzing the degradation of potentially cytotoxic purine deoxynucleoside analogs, e.g., 2′-deoxy-6-thioguanosine. More recently, the identification of an immunodeficiency disorder associated with a deficiency in PNP (Giblett et al., 1975) has drawn attention to this enzyme as a possible target for the design of novel immunosuppressive agents. In contrast to the severe combined immunodeficiency disease seen with adenosine deaminase deficiency, where defects occur in both cellular and humoral immunity, patients with PNP deficiency lack cellular, but not humoral, immunity. Also, these individuals excrete the more soluble nucleosides of hypoxanthine and guanine rather than the relatively insoluble end product of purine metabolism, uric acid. Thus it has been proposed that a potent inhibitor of PNP might serve as a biochemical modifier in chemotherapy with purine nucleoside analogs, as a selective immunosuppressive agent, and perhaps in the treatment of secondary gout (Parks et al., 1981; Stoeckler et al., 1980a, 1982; Kazmers et al., 1981).

Tight-binding inhibitors—II: Non-steady state nature of inhibition of milk xanthine oxidase by allopurinol and alloxanthine and of human erythrocytic adenosine deaminase by coformycin

Abstract The non-steady state nature of the inhibition of milk xanthine oxidase by allopurinol and alloxanthine was demonstrated, and the kinetic data presented are consistent with the known mechanisms of inhibitions by these inhibitors. With the use of human erythrocytic adenosine deaminase and its tight-binding inhibitor, coformycin, it was demonstrated that the classical methods of enzyme kinetics based on the steady state assumptions are grossly inadequate for determining the inhibition mechanisms or inhibition constants for tight-binding inhibitors. The application of the Ackermann-Potter plot, I50. the Easson-Stedman plot (or Henderson plot), and the rates of association and dissociation of enzyme-inhibititor complex were presented and their usefulness was evaluated. The molar equivalency and the catalytic number of human erythrocytic adenosine deaminase were estimated at about 1.0 × 10−10 mole/unit and 1.0 × 104 min−1 respectively. It was also demonstrated that the Ki, value of coformycin for this enzyme does not exceed 1.2 × 10−10 M, and that the second-order rate constant for the association of the enzyme with coformycin is approximately 2 × 106 M−1 sec−1. The biphasic nature of the dissociation of the deaminase-coformycin complex (EI complex) indicates that the EI complex undergoes a slow conformational change. The implications of these new kinetic approaches for the study of tight-binding inhibitors, including transition-state analogs, were discussed.

5′-methylthioadenosine phosphorylase—I: Substrate activity of 5′-deoxyadenosine with the enzyme from Sarcoma 180 cells

Abstract 5′-Deoxy-5′-methylthioadenosine phosphorylase (MTA phosphorylase), an enzyme involved in the salvage of adenine moieties from 5′-deoxy-5′-methylthioadenosine (MTA) produced primarily during polyamine biosynthesis, is present in Sarcoma 180 cells (0.0026 ± 0.0002 μ M units/mg cytosol protein). 5′-Deoxyadenosine (5′-dAdo), an adenosine analog previously thought not to be metabolizable, has been shown [D. Hunting and J.F. Henderson, Biochem . Pharmac . 27 , 2163 (1978)] to have a number of biochemical effects on Ehrlich ascites cells. We have now found that 5′-dAdo is a substrate for the MTA phosphorylase from Sarcoma 180 cells, yielding free adenine and 5-deoxyribose-1-phosphate. The reaction was reversible and totally dependent upon phosphate. Evidence that MTA phosphorylase is responsible for 5′-dAdo phosphorylase activity includes the following: (1) Sarcoma 180 MTA phosphorylase preparations did not show additive rates of adenine production in the presence of saturating concentrations of both 5′-dAdo and MTA; (2) double-reciprocal plots of the rates of adenine formation from 5′-dAdo by Sarcoma 180 enzyme preparations in the presence of MTA displayed a pattern characteristic of alternative, competing substrates; (3) the rate of depletion of 5′-dAdo by Sarcoma 180 preparations was inhibited by the presence of MTA; (4) the K i value of a competitive inhibitor of Sarcoma 180 MTA phosphorylase, 5′-deoxy-5′-chloroformycin, was the same when either MTA or 5′-dAdo was employed as substrate; and (5) the apparent K m values of phosphate for both MTA and 5′-dAdo phosphorylase activities were identical (3.5mM). The K m of Sarcoma 180 MTA phosphorylase for MTA is 4 μM; the K m for 5′-dAdo is 23 μM ( V max relative to MTA = 180 per cent). Incubation of Sarcoma 180 cells with either 5′-dAdo or MTA caused profound elevations of adenine nucleotides, as well as an inhibition of 5-phosphoribosyl-l-pyrophosphate (PRPP) accumulation. The reaction of 5′-dAdo with MTA phosphorylase to yield free adenine, which is then salvaged to adenine nucleotides, can account for many of the previously reported biochemical effects of 5′-dAdo, such as inhibitions of PRPP accumulation, purine de novo synthesis, and glycolysis that have previously been attributed to the unmetabolized nucleoside. The other product of this reaction, 5-deoxyribose-l-phosphate, may also contribute to these effects.

Pathways of nucleotide metabolism in Schistosoma mansoni—III: Identification of enzymes in cell-free extracts☆

Abstract A survey of purine anabolic and catabolic enzymes has resulted in identification of major pathways in schistosome nucleotide biosynthesis. It is shown that multiple pathways for the incorporation of purine bases and nucleosides exist. The evidence suggests that adenosine phosphoribosyltransferase (APRT) activity is about ten times greater than adenosine kinase activity. Furthermore adenosine is converted to AMP principally via the pathway of adenosine deaminase, followed by conversion of inosine to hypoxanthine. In this sequence hypoxanthine phosphoribosyltransferase (HPRT) activity is rate limiting. On the basis of enzyme activities determined, one can suggest candidates of nucleotide analogs which might be useful chemotherapeutic agents.

Tight binding inhibitors—VI: Interactions of deoxycoformycin and adenosine deaminase in intact human erythrocytes and sarcoma 180 cells

Abstract The inactivation and reactivation of adenosine deaminase (ADA) by deoxycoformycin was studied in intact human erythrocytes and murine Sarcoma 180 cells in vitro . The second-order rate constant ( k 1 ) for the association reaction between deoxycoformycin and intraerythrocytic ADA was calculated to be 5.1 × 10 3 M −1 sec −1 . This is about 300 to 500-fold lower than the k 1 values determined either with hemolyzed human erythrocytes ( k 1 = 1.4 × 10 6 M −1 sec −1 ) or with partially purified human erythrocytic ADA ( k 1 = 2.6 × 10 6 M −1 sec −1 ). In intact erythrocytes only slight reactivation ( EI complex) was detectable over 24 hr, whereas with hemolysates about 50 per cent reactivation of the inhibited ADA was observed in about 25 hr ( k 2 = 7.7 × 10 −6 sec −1 ). The k 1 values with intact and supernatant fractions from homogenized Sarcoma 180 cells were determined to be 1.1 × 10 4 M −1 sec −1 and 4.2 × 10 6 M −1 sec −1 respectively. With intact Sarcoma 180 cells, negligible reactivation of ADA was seen during a 48-hr period. Preliminary studies indicate an important role for the erythrocytic nucleoside transport system on the apparent k 1 values and the rate of inactivation of ADA by deoxycoformycin in intact cells.

Pathways of nucleotide metabolism in schistosoma mansoni—IV: Incorporation of adenosine analogs in vitro☆

Abstract The incorporation in vitro of adenine or adenosine analogs into schistosome nucleotides is demonstrated. Tubercidin, 2-fluoroadenosine and 2-fluoroadenine were all shown to be converted into analog triphosphate nucleotides. Since tubercidin and 2-fluoroadenosine are not substrates for adenosine deaminase or purine nucleoside phosphorylase and are not susceptible to degradation to the free base level, it is assumed that they are converted to nucleotides by reaction with adenosine kinase. The incorporation of 2-fluoroadenine into the nucleotide pools indicates that it serves as a substrate for adenine phosphoribosyltransferase. Tubercidin, added to the culture medium, interferes with the maintenance of normal ATP levels. When the concentration of the analog greatly exceeded that of adenine or adenosine in the medium, virtual shutdown of adenosine triphosphate synthesis followed. It is suggested that stoichiometric competition for enzyme sites may determine the relative amounts of nucleotides formed.

Transport of deoxycoformycin in human erythrocytes: Measurement by adenosine deaminase titration and radioisotope assays

The assay of residual adenosine deaminase (ADA) activity was used as a sensitive measure of the transport of deoxycoformycin (dCF) into human erythrocytes. Contrary to prior reports from this laboratory, the inactivation of intraerythrocytic ADA by dCF was linear rather than log-linear, with time. Linear inactivation rates were also seen when erythrocytes were preloaded with a 5-fold excess of calf intestinal ADA. The uptake of tritium-labeled dCF molecules and the rate of inactivation of ADA molecules showed an approximate 1:1 stoichiometry. The nucleoside transport inhibitors, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR) and dipyridamole, and the permeant, uridine, inhibited dCF transport with Ki values of 35 nM, 45 nM, and 340 microM respectively. The affinity of dCF for the nucleoside transporter was low with a Ki of approximately 10 mM for the inhibition of adenosine influx.

Incorporation of the purine moieties of guanosine and inosine analogs into nucleotide pools of human erythrocytes

Abstract The metabolism of several purine ribonucleoside analogs in human erythrocytes was studied. When suspensions of erythrocytes were incubated with 6-thioguanosine (6-TGR) in the presence of dithiothreitol, the 5′-mono, di- and triphosphate ribonucleotides of 6-thioguanosine were synthesized. Although the formation of the monophosphate nucleotide occurred quite early in the incubation period, the di- and triphosphate nucleotides appeared only after more prolonged incubation. When 6-TGR was present at 1.0 mM in the incubation medium, the quantities of nucleotides produced were approximately 0.23 μmole 6-thioGMP, 0.05 μmole 6-thioGDP and 0.17 μmole 6-thioGTP/ml of erythrocytes after 2 hr of incubation. In contrast to the findings with 6-thioguanosine, incubation of erythrocytes with 6-thioinosine (6-mercaptopurine ribonucleoside. 6-MPR) or 6-selenoguanosine (6-SeGR) led to the formation of only the respective 5′-monophosphate ribonucleotides. The triphosphate derivatives were not formed even after incubation of as long as 24 hr. The quantities of the analog ribonucleotides/ml of erythrocytes produced during 2 hr of incubation were 0.66 μmole 6-thioinosine-5′-monophosphate (6-thioIMP)and 0.33 μmole 6-selenoguanosine-5′-monosphate (6-SeGMP). It is proposed that the analog nucleosides are first split by purine nucleoside phosphorylase (PNPasc), liberating the corresponding free base, which then reacts with 5-phosphoribosyl-1-pyrophosphate (PRPP) and the salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) to form analog monophosphate nucleotides.

Effects of adenosine analogs and adenine nucleotides on adenosine 5'-diphosphate-induced rat platelet aggregation.

Abstract Various adenosine analogs and adenine nucleotides have been tested as inhibitors of ADP-induced aggregation of rat platelets. The potent inhibitors of human platelet aggregation, adenosine, 2-fluoroadenosine, 2-chloroadenosine, carbocyclic adenosine and N 6 -phenyl adenosine, had little effect on rat platelet aggregation (0–30 per cent inhibition). The effects of adenosine or its analogs on ADP-induced aggregation of cross-species platelet-rich plasmas (PRPs) (human platelets suspended in rat plasma or rat platelets in human plasma) were similar to those with the native PRPs, indicating that these species differences were due to intrinsic factors in the platelets and not in the plasma. When these analogs were tested in the presence of the cyclic AMP phosphodiesterase inhibitor papaverine, strong inhibiton of rat platelet ADP-induced aggregation was seen. 2′-Deoxyadenosine and 3′-deoxyadenosine were not inhibitory to ADP-induced aggregation of rat PRP even in the presence of papaverine. Adenosine 5′-tetraphosphate strongly inhibited both human and rat platelet aggregation. AMP, like adenosine, did not inhibit rat platelet aggregation but became strongly inhibitory in the presence of papaverine. This inhibitory effect was abolished by preincubating rat PRP with an adenylate cyclase inhibitor, 2′, 5′-dideoxyadenosine or adenosine deaminase. In the later case, however, if the adenosine deaminase inhibitor 2′-deoxycoformycin was included in the incubation mixture, the inhibition by AMP plus papaverine was similar to adenosine plus papaverine. About 50 per cent of [ 14 C]AMP was converted to [ 14 C]adenosine in rat platelet-free plasma or PRP after a 10-min incubation. α,β-Methylene-ADP and β,γ-methylene-ATP (200 μM) inhibited rat platelet aggregation by 50 and 64 per cent, respectively. Cyclic AMP phosphodiesterase of rat and human platelets gave comparable K m , and V max values ( K m 0.53 and 0.21μM and V max 6.0 and 6.7 pmoles/min/10 7 platelets, respectively).

Inhibition of nucleoside transport by nitrobenzylthioformycin analogs

The formycin analogs of nitrobenzylthioinosine and nitrobenzylthioguanosine were synthesized and evaluated as nucleoside transport inhibitors. These analogs have a potential therapeutic advantage over their parent compounds in that their C-nucleosidic linkages prevent them from being degraded to the immunosuppressive agents, 6-mercaptopurine and 6-thioguanine. 7-[(4-Nitrobenzyl)-thio]-3-(beta-D-ribofuranosyl)pyrazolo[4,3- d]pyrimidine (NBTF) and 5-amino-7-[(4-nitrobenzyl)thio]-3-(beta-D- ribofuranosyl)pyrazolo[4,3-d]pyrimidine (NBTGF) were inhibitors of nucleoside transport in human erythrocytes and HL-60 leukemia cells. The IC50 value for nitrobenzylthioinosine, NBTF and NBTGF with 10% erythrocyte suspensions were 18, 18 and 40 nM respectively. Specific binding studies with [3H]NBTF yielded a Kd of 3.4 nM with erythrocytes, approximately 10-fold higher than values reported for nitrobenzylthioinosine. NBTF and nitrobenzylthioinosine bound to HL-60 cells with Kd values of 8.1 and 0.81 nM respectively. The octanol/water partition coefficients of nitrobenzylthioinosine, NBTF and NBTGF were 3.5, 3.2, and 2.8 respectively. NBTF could be expected to be equipotent with nitrobenzylthioinosine in whole blood where inhibitor concentrations of 10(-7) to 10(-6) M are required in order to saturate erythrocytic binding sites; hence, it may exhibit the advantages inherent in a C-nucleoside.