formycins and 9-deazainosine

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway, which provides an alternative to the de novo pathway for the biosynthesis of purine nucleotides. PNP catalyzes the reversible phosphorolysis of 2'-deoxypurine ribonucleosides to the free bases and 2-deoxyribose 1-phosphate. Absence of PNP activity in humans is associated with specific T-cell immune suppression. Its key role in these two processes has made PNP an important drug design target. We have investigated the structural details of the PNP-catalyzed reaction by determining the structures of bovine PNP complexes with various substrates and substrate analogues. The preparation of phosphate-free crystals of PNP has allowed us to analyze several novel complexes, including the ternary complex of PNP, purine base, and ribose 1-phosphate and of the completely unbound PNP. These results provide an atomic view for the catalytic mechanism for PNP proposed by M. D. Erion et al. [(1997) Biochemistry 36, 11735-11748], in which an oxocarbenium intermediate is stabilized by phosphate and the negative charge on the purine base is stabilized by active site residues. The bovine PNP structure reveals several new details of substrate and inhibitor binding, including two phosphate-induced conformational changes involving residues 33-36 and 56-69 and a previously undetected role for His64 in phosphate binding. In addition, a well-ordered water molecule is found in the PNP active site when purine base or nucleoside is also present. In contrast to human PNP, only one phosphate binding site was observed. Although binary complexes were observed for nucleoside, purine base, or phosphate, ribose 1-phosphate binding occurs only in the presence of purine base.

Topics: Acyclovir; Animals; Arsenates; Binding Sites; Cattle; Crystallography, X-Ray; Formycins; Guanine; Humans; Hypoxanthine; Inosine; Ligands; Macromolecular Substances; Models, Molecular; Phosphates; Purine-Nucleoside Phosphorylase; Ribosemonophosphates; Spleen; Structure-Activity Relationship; Substrate Specificity; Sulfates

Kinetoplastid hemoflagellates are sensitive to growth inhibition by various purine analogs. In this study the activities of 9-deazainosine (9-DINO), formycin B, and sinefungin were compared in experimental murine Trypanosoma brucei subsp. brucei infections, both singly and in combination with the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (DFMO, eflornithine). Used singly, all of the purine analogs were able to suppress an acute T. brucei subsp. brucei infection. 9-DINO and formycin B were the most active. None of the purine analogs was curative when used singly against a strain causing chronic central nervous system infection. 9-DINO was highly effective when used in combination with DFMO in curing this central nervous system infection and another more stringent experimental infection. Neither sinefungin nor formycin B was active in combination with DFMO in curing the central nervous system experimental infection. 9-DINO was metabolized to phosphorylated derivatives of 9-deazaadenosine and 9-deazaguanosine by bloodstream trypomastigotes, but not by murine erythrocyte suspensions or kidney or liver homogenates--a potential rationale for the selectivity of the analog. These studies indicate that 9-DINO is a potent, nontoxic purine analog which, in combination with DFMO, is capable of late-stage cures of African trypanosomiasis.

Topics: Animals; Antimetabolites; Biotransformation; Drug Synergism; Eflornithine; Formycins; Inosine; Mice; Ornithine Decarboxylase; Purines; Time Factors; Trypanosoma brucei brucei; Trypanosomiasis, African

The dose of orally administered 9-deazainosine calculated to suppress 50% of Leishmania donovani amastigotes in hamster livers was 19 mg/kg (body weight) per day; 96 to 99% of Leishmania organisms were eliminated from the liver and spleen of squirrel monkeys by 50 mg/kg per day. Because these activities were greater than that of the experimental clinical agent allopurinol and comparable to that of the classical agent parenteral pentavalent antimony, 9-deazainosine should be considered for clinical development for visceral leishmaniasis.

Topics: Administration, Oral; Allopurinol; Animals; Cricetinae; Formycins; Guanosine; Injections, Intramuscular; Inosine; Leishmania donovani; Leishmaniasis, Visceral; Liver; Male; Meglumine; Meglumine Antimoniate; Mesocricetus; Organometallic Compounds; Saimiri; Spleen

We have developed a simple and reproducible system for infecting a human macrophage cell line (U937) with stationary-phase Leishmania donovani promastigotes. Four days after infection, greater than 90% of the promastigotes had transformed to amastigotes. The antileishmanial agents allopurinol riboside, formycin B, 9-deazainosine, and sodium stibogluconate effectively inhibited the growth of L. donovani amastigotes in this cell line. To study the capability of amastigotes in the U937 cell line to carry out biochemical reactions that could be monitored experimentally, we incubated the cells with radiolabeled 9-deazainosine. This purine analogue underwent metabolism in the amastigote phase similar to that occurring in the promastigote phase. This cell line should be useful for studies of parasite maturation and differentiation, parasite-human interactions, and antiparasitic drugs.

Topics: Allopurinol; Animals; Antimony Sodium Gluconate; Antiprotozoal Agents; Cell Line; Formycins; Humans; Inosine; Leishmania donovani; Macrophages; Ribonucleosides

Three analogs of inosine, formycin B, allopurinol ribonucleoside, and 9-deazainosine, were tested for their ability to suppress proliferation of Pneumocystis carinii in culture with WI-38 cells. The organism was inhibited by 9-deazainosine at 10 micrograms/ml, and there was some inhibition at 1 microgram/ml. Formycin B was effective only at 40 micrograms/ml. Allopurinol ribonucleoside had little effect.

Topics: Allopurinol; Animals; Antiprotozoal Agents; Cells, Cultured; Drug Combinations; Formycins; Inosine; Microbial Sensitivity Tests; Pneumocystis; Rats; Ribonucleosides; Sulfamethoxazole; Trimethoprim; Trimethoprim, Sulfamethoxazole Drug Combination