Acyclonucleoside analogue inhibitors of mammalian purine nucleoside phosphorylase

Bzowska, Agnieszka; Kulikowska, Ewa; Shugar, David; Chen, Bing-yi; Lindborg, B; Johansson, Nils‐Gunnar

doi:10.1016/0006-2952(91)90117-n

Cited by 32 publications

(16 citation statements)

References 46 publications

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“…1). This is in line with the sequence reported for a bovine PNP (origin not specified and no details given), which exhibits 87% indentity with the human PNP and conservation of all amino acids in the catalytic region [35]; and is consistent with the similar physico-chemical and kinetic properties of the calf and human enzymes [12,20,26] and their similar responses to inhibitors [8].…”

Section: Discussionsupporting

confidence: 73%

“…The high cellular level of this ubiquitous enzyme, and its propensity to cleave and inactivate chemotherapeutically active nucleoside analogues, has led to an intensive search for potent inhibitors of the enzyme from various sources [1][2][3][4][5][6][7][8][9]. Furthermore, patients with PNP deficiency exhibit marked T-cell immunodeficiency [10,11], and it is considered that PNP inhibitors could be useful in treatment of T-cell proliferative diseases, in suppression of host versus graft response in organ transplantation, and in treatment of T-cell-mediated autoimmune diseases [10][11][12].…”

Section: Introductionmentioning

confidence: 99%

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Calf spleen purine nucleoside phosphorylase: purification, sequence and crystal structure of its complex with an N(7)‐acycloguanosine inhibitor

et al. 1995

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Calf spleen pnrine nucleoside phosphorylase was purified to homogeneity and its amino acid sequence was determined. The complex of the enzyme with an N(7)-acycloguanosine inhibitor crystallized in the cubic space group P213, with unit cell dimension a = 94.02 A and one monomer in the asymmetric crystal unit. The biologically active trimer is formed by the crystallographic three-fold axis. The structure was solved by molecular replacement methods, using the model of the human erythrocyte enzyme, and refined at a resolution of 2.9 A to an R-factor of 0.21. The orientation of the inhibitor at the active site is examined in relation to the catalytic activity of the enzyme in the phosphorolysis of N(7)-fl-o-purine nucleosides.

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Section: Discussionsupporting

confidence: 73%

Section: Introductionmentioning

confidence: 99%

Calf spleen purine nucleoside phosphorylase: purification, sequence and crystal structure of its complex with an N(7)‐acycloguanosine inhibitor

et al. 1995

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“…2), which demonstrates competitive inhibition, with a Kl'l' of 2100 pM, as compared to the value of 1860 pM from Table 3. are inhibitors of the calf spleen enzyme [30], and formycins A and B, both specific inhibitors of the enzyme from E. coli [5].…”

Section: Methodsmentioning

confidence: 99%

Nicotinamide Riboside, an Unusual, Non‐Typical, Substrate of Purified Purine‐Nucleoside Phosphorylases

Wielgus‐Kutrowska

Kulikowska

Wierzchowski

et al. 1997

European Journal of Biochemistry

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Nicotinamide 1 -1l-D-riboside (Nir), the cationic, reducible moiety of the coenzyme NAD', has been confirmed as an unusual substrate for purified purine-nucleoside phosphorylase (PNP) from a mammalian source (calf spleen). It is also a substrate of the enzyme from Escherichia coli. The K,, values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1-2 orders of magnitude higher than for the natural substrate inosine, but the V,,,,>, values were comparable, 96% and 35% that for Ino. The pseudo first-order rate constants, V,,,,,IK,,,, were 1 .I % and 2.5 o/o for the calf spleen and E. coli enzymes. The aglycon, nicotinamide, was neither a substrate nor an inhibitor of PNP.Nir was a weak inhibitor of inosine phosphorolysis catalyzed by both enzymes, with K, values close to the K,,, for its phosphorolysis, consistent with simple competitive inhibition; this was further confirmed by Dixon plots. Phosphorolysis of the fluorescent positively charged substrate 7-methylguanosine was also inhibited in a competitive manner by both Ino and Nir. Phosphorolysis of Nir by both enzymes was inhibited competitively by several specific inhibitors of calf spleen and E. coli PNP, with K, values similar to those for inhibition of other natural substrates.The pH dependence of the kinetic constants for the phosphorolysis of Nir and of a variety of other substrates, was extensively investigated, particularly in the alkaline pH range, where Nir exhibited abnormally high substrate activity relative to the reduced reaction rates of both enzymes towards other anionic or neutral substrates.The overall results are discussed in relation to present concepts regarding binding and phosphorolysis of substrates by PNP based on crystallographic data of enzyme-inhibitor complexes, and current studies on enzymatic and nonenzymatic mechanisms of the cleavage of the Nir glycosidic bond.Keywords; nicotinamide riboside ; purine-nucleoside phosphorylase; substratehnhibitor properties ; enzyme kinetics: pH dependence.The ubiquitous enzyme purine-nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine nucleosides such as guanosine and inosine and their deoxy counterparts in mammals [I], and additionally adenosine in some lower organisms such as Escherichiu coli and Salmonella typhimurium 121, as follows:Considerable attention is currently devoted to elucidation of the reaction mechanism, and development of potent specific inhibitors of the enzymes from various sources 13-51. Potential clinical applications of inhibitors involve treatment of T-cell leukemias, suppression of the host-versus-graft response in organ transplantations, and potentiation of the chemotherapeutic activity of PNP-cleavable nucleoside analogues 13, 61.Some non-natural purine-nucleoside analogues have been shown to be substrates, e.g. 7-alkyl congeners of guanosine and inosine [7], 7-P-D-and 3-P-D-purine ribosides [8, 91. while 8-azapurine analogues [lo], as well as the mesoionic anti-influenza agent 1,3,4-thiadiazol-2-ylcyanamide [ 11 1 undergo irreversible ribosy...

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“…Calf spleen PNP or E. coli PNP enzymes did not exhibit any detectable deamination of RibfAde under the conditions of our experiments. 9-(2-fluoro-3,4-dihydroxybutyl)guanine was synthesized as previously described [6].…”

Section: Methodsmentioning

confidence: 99%

“…We have previously shown that the guanine acyclonucleoside analogue 9-(2-tluoro-3,4-dihydroxybuty1)guanine is a good inhibitor of PNP from various mammalian sources, including that from calf spleen [6], whereas formycin A is a good inhibitor of the enzyme from E. coli, but not from mammalian sources 1211. Both of these inhibitors are competitive with respect to nucleoside substrates (Ino).…”

Section: Effects Of Specific Inhibitorsmentioning

confidence: 99%

Kinetics of Phosphorolysis of 3‐(β‐d‐Ribofuranosyl)Adenine and 3‐(β‐d‐Ribofuranosyl)Hypoxanthine, Non‐Conventional Substrates of Purine‐Nucleoside Phosphorylase

Bzowska

Kulikowska

Poopeiko

et al. 1996

European Journal of Biochemistry

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The properties of two non-conventional substrates of the calf-spleen and Escherichia coli purine nucleoside phosphorylases (PNP), 3-@-~-ribofuranosyl)adenine (RibfAde) and 3-(P-~-ribofuranosyl)hypoxanthine (RibjHyp), are described. In contrast to Ado, RibfAde is a substrate for the mammalian enzyme. With the calf enzyme, the pseudo-first-order rate constants (V,,,,,,/K,,,) for phosphorolysis of RibfAde and RibfHyp are 3% and 13%, respectively, that for phosphorolysis of Ino, while for E. coli PNP the Corresponding values are 22% and 30%, respectively. The Michaelis constants (K,,,) for RibfAde were 800 pM (calf PNP) and 150 pM (E. coli PNP). For Ribflyp, the corresponding K,,, values were 220 pM and 260 pM.Two well-characterized inhibitors of calf spleen PNP [9-(2-fluoro-3,4-dihydroxybutyl)guanine] and E. coli PNP (formycin A) were found to inhibit phosphorolysis of RibfAde and RibjHyp with the same inhibition constants as for Ino. Moreover, the inhibition was competitive, which indicates that phosphorolysis of 3-P-nucleosides occurs at the same active site as for the natural substrate Ino.In particular, the substrate properties of both 3-P-nucleosides are consistent with their binding to the enzyme in the conformation anti to the imidazole ring about the glycosidic bond, which is superimposable on the structure of natural 9-P-nucleosides in the conformation anti to the pyrimidine ring.The results are examined in relation to present concepts regarding the binding of substrates and inhibitors at the active site(s) of these enzymes.Keywords: purine-nucleoside phosphorylase; 3-(P-~-ribofuranosyl)purine; substrate; kinetics; inhibitor.The ubiquitous purine-nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine nucleosides, such as inosine and guanosine in eukaryotes, and adenosine in some (e.g. Esclzerichia coli and Salmonella typhimurium), but not all, procaryotes, as follows: purine nucleoside + P, * purine + ribose-I -phosphate.Because of the well-established relationship between PNP deficiency and certain immunological disorders [I, 21, and the propensity of this enzyme to enzymatically cleave and render inactive a variety of chemotherapeutically active nucleoside analogues [ 3 ] , there is considerable interest in elucidation of the specificity and mechanism of action of these enzymes and in the development of specific inhibitors of the phosphorolytic reaction, particularly of the human enzyme [4-191.We have described elsewhere differences in substrate specificity between the mammalian enzymes and the E. coli enzyme Enzymes. Purine-nucleoside phosphorylase (EC 2.4.2.1); adenosine deaminase (EC 3.5.4.4).[20] and the development of potent inhibitors specific only for the latter [22, 221. There have been a number of reports of unusual substrates for these enzymes. For example, the antiinfluenza agent 1,3,4-thiadiazol-2-ylcyanamide is converted by PNP from various sources to two ribosides, one of which is mesoionic and resistant to phosphorolysis 1231. Other unusual substrates include N7...

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Acyclonucleoside analogue inhibitors of mammalian purine nucleoside phosphorylase

Cited by 32 publications

References 46 publications

Calf spleen purine nucleoside phosphorylase: purification, sequence and crystal structure of its complex with an N(7)‐acycloguanosine inhibitor

Calf spleen purine nucleoside phosphorylase: purification, sequence and crystal structure of its complex with an N(7)‐acycloguanosine inhibitor

Nicotinamide Riboside, an Unusual, Non‐Typical, Substrate of Purified Purine‐Nucleoside Phosphorylases

Kinetics of Phosphorolysis of 3‐(β‐d‐Ribofuranosyl)Adenine and 3‐(β‐d‐Ribofuranosyl)Hypoxanthine, Non‐Conventional Substrates of Purine‐Nucleoside Phosphorylase

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