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2-Acetylpyridine 5-[(dimethylamino)thiocarbonyl]thiocarbonohydrazone (A1110U) was found to be a potent inactivator of the ribonucleotide reductases (EC 1.17.4.1) encoded by herpes simplex virus types 1 and 2 and by varicella-zoster virus and to be a weaker inactivator of human ribonucleotide reductase. It also markedly potentiated the antiherpetic activity of acyclovir against these viruses in tissue culture. A1110U both decreased the dGTP pool that builds up when infected cells are treated with acyclovir and induced a large increase in the pool of acyclovir triphosphate. The resultant 100-fold increase in the ratio of the concentrations of acyclovir triphosphate to dGTP should facilitate the binding of the fraudulent nucleotide to its target enzyme, herpes virus-encoded DNA polymerase, and could account for the synergy between A1110U and acyclovir. A similar change in the acyclovir triphosphate-to-dGTP ratio was previously reported to be induced by another ribonucleotide reductase inhibitor, 2-acetylpyridine 4-(2-morpholinoethyl)thiosemicarbazone (A723U). However, A1110U is considerably more potent and may have better clinical potential. Synergistic toxic interactions between A1110U and acyclovir were not detected in uninfected cells.

9-([2-Hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) is more potent and has a more prolonged inhibitory effect against Epstein-Barr virus (EBV) in vitro than does acyclovir (ACV). To assess the mechanism of this difference, we first compared the extent of phosphorylation of the two drugs in superinfected Raji cells. BW B759U is phosphorylated to levels 100-fold higher than is ACV. In addition, lower levels of phosphorylation of BW B759U and ACV were observed in uninfected Raji cells. Studies on the kinetics of formation of BW B759U triphosphate in superinfected Raji cells indicated that drug-phosphorylating activity was detected as early as 3 h after superinfection; this activity was steadily maintained for the first 7 h, followed by a burst of activity between 7 and 10 h and a doubling of phosphorylation between 10 and 25 h. During the superinfection cycle, the pool sizes of deoxyribonucleoside and ribonucleoside triphosphates were increased and reached their maxima at 10 h after infection. The maximal amount of triphosphorylated drug in a virus producer cell, P3HR-1 (LS), was obtained at 21 h after drug treatment. During long-term drug treatment, approximately 44 and 77% reduction in EBV genome copies per cell was observed on days 3 and 7, respectively. In a separate experiment, after treatment of P3HR-1 (LS) cells with BW B759U for 36 h, 4.2 pmol of BW B759U triphosphate per 10(6) cells was achieved. After the cells were released into drug-free medium, drug triphosphate was rapidly decreased to 11% of the original level in 1 day. Thereafter, the decrease was slow but steady, down to 0.22 pmol/10(6) P3HR-1 cells by 5 days. We calculated that 0.22 pmol of BW B759U triphosphate per 10(6) cells represents a cellular concentration of 0.22 microM, which is theoretically enough to inhibit EBV replication. This is based upon a comparison with the 50% effective dose of BW B759U (0.05 microM) for inhibition of genome replication and a Ki of 0.08 microM for BW B759U triphosphate inhibition of EBV DNA polymerase.

Certain purine analogs, the pyrazolopyrimidines, are effective chemotherapeutic agents against Leishmania spp. and Trypanosoma cruzi both in vitro and in some clinical models. Heretofore they have not been effective against the African trypanosomes; this suggested that these organisms were not comparable to the other pathogens with respect to their purine metabolism. We have studied the efficacy and metabolism of the pyrazolopyrimidine bases allopurinol and thiopurinol, their respective ribonucleosides, and the C-nucleosides formycin B and 9-deazainosine in Trypanosoma brucei subsp. gambiense and Trypanosoma brucei subsp. rhodesiense. The efficacy of these compounds was dependent on the purine content of the culture medium. The C-nucleosides were the most effective, with 90% effective doses for formycin B and 9-deazainosine of 0.01 and 2 micrograms/ml, respectively. Metabolism was the same in both the bloodstream and culture forms and identical to that reported for Leishmania spp. and T. cruzi. Both agents were phosphorylated to the ribonucleotide and then aminated to produce adenine nucleotide analogs. Growth inhibition studies were performed with three inosine analogs (allopurinol riboside, formycin B, and 9-deazainosine) on trypomastigotes grown in bone marrow tissue culture. Both C-nucleosides eradicated the infection at a concentration of 0.25 micrograms/ml. Unlike formycin B, 9-deazainosine is not known to be aminated by mammalian cells and appears to be relatively nontoxic in three different mammalian tissue culture systems. This nucleoside was very active against all pathogenic leishmaniae and trypanosomes investigated and is worthy of further study.

Previous investigations have suggested that inosine analogs would be good models for the development of chemotherapeutic agents active against pathogenic hemoflagellates. We have systematically modified the five-membered heterocyclic ring of six inosine analogs and tested them for their antiprotozoal activities and toxicity to a mammalian cell line. All six analogs were very active against the three protozoan pathogens Leishmania donovani, Trypanosoma cruzi, and Trypanosoma gambiense. Two of the six, 9-deazainosine and allopurinol ribonucleoside, had very little toxicity for mouse L cells and offer promise as potential chemotherapeutic agents.

Stimulation of macrophages induces the "respiratory burst" response which is associated with the generation of superoxide (O2-), a drop in cytoplasmic pH, and a pronounced depolarization of the membrane potential. The purpose of the present studies was to determine whether an increase in O2- was temporally related to changes in membrane potential and transmembrane current. Release of O2- at the single cell level was photometrically monitored during phagocytosis of immune complexes while simultaneously measuring whole-cell current. Membrane depolarization and the generation of a non-selective current followed an increase in O2- production with a variable lag time which was correlated with the state of cellular maturation in culture. In the absence of phagocytosis, the exposure of macrophages to O2- generated by a xanthine-xanthine oxidase reaction activated a non-selective current similar to that seen after phagocytosis. These results provide the first demonstration of the relationship between free radical release and the ensuing electrophysiological signaling events which are linked to particle engulfment in phagocytic cells.

Allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine) is an effective agent in vitro against Trypanosoma cruzi. The important forms of this parasite, with respect to the pathogenesis of Chagas' disease in man, are the bloodstream (trypomastigote) and the intracellular forms. Experiments with radiolabeled allopurinol and analysis of the metabolic products of this compound by high-performance liquid chromatography showed that both the bloodstream and the intracellular forms of T. cruzi metabolize allopurinol in the same manner as has been shown for the epimastigotes in vitro. The metabolic pathways for pyrazolopyrimidines in the pathogenic forms were demonstrated with organisms isolated from infected animals and a tissue culture system infected with T. cruzi. Treatment of infected tissue culture with allopurinol eradicated the infection. This investigation implies that allopurinol may be useful in chemotherapy of T. cruzi infections, a supposition which has been borne out in one animal study.

Recent studies have identified a new family of inwardly rectifying K+ channels, members of which are known by the acronyms ROMK1, IRK1, and GIRK1. We have isolated cDNAs encoding the human homologue of ROMK1 from an adult kidney cDNA library. The sequences of the human kidney ROMK1 cDNA clones indicated that they were derived from at least two types of mRNAs, human ROMK1A and human ROMK1B, differing in sequence at their 5' ends. The isolation of the human ROMK1 gene, localized to chromosome band 11q24 by fluorescence in situ hybridization, indicated that the different ROMK1 transcripts were generated by alternative splicing. Human ROMK1A mRNA was predicted to encode a protein of 389 amino acids, having 93% identity with the 391-residue rat ROMK1 protein, and expression studies in Xenopus oocytes indicated that it encoded a Ba(2+)-sensitive inwardly rectifying K+ channel with properties similar to those reported for cloned rat ROMK1. Human ROMK1B mRNA was predicted to encode a protein of 372 amino acids whose sequence was truncated at the amino terminus but otherwise identical to that of the human ROMK1A protein. Translation of human ROMK1B mRNA was predicted to initiate at a codon corresponding to Met-18 of human ROMK1A mRNA. Reverse transcriptase-polymerase chain reaction amplification of human kidney mRNA revealed human ROMK1A and -B transcripts as well as a third type of transcript, human ROMK1C mRNA, which was predicted to encode a protein identical to human ROMK1B. Human ROMK1A, -B, and -C transcripts were identified in kidney, whereas only human ROMK1A mRNA could be detected in pancreatic islets and other tissues in which human ROMK1 was expressed at low levels. Thus, tissue-specific alternative splicing of human ROMK1 mRNA may result in the expression of a family of ROMK1 proteins.

9-[( 2-Hydroxy-1-(hydroxymethyl)ethoxy]-methyl)guanine (BW B759U) is a more potent inhibitor of human cytomegalovirus (HCMV) in vitro than is the related nucleoside analog acyclovir (ACV). BW B759U was selectively activated to the 5'-triphosphate (BW B759U-triphosphate) in cells infected with HCMV to levels at least 10-fold higher than those measured for ACV-triphosphate and up to as much as 100-fold higher than the levels found in uninfected cells. BW B759U-triphosphate accumulated in HCMV-infected cells with time; the rate of this increase was dependent upon the drug dose and virus multiplicity of infection. Enzyme activities that catalyzed the phosphorylation of thymidine and 2'-deoxycytidine increased 3- to 7-fold in extracts of cells early after HCMV infection but thereafter declined. No concomitant increase in the rate of BW B759U phosphorylation was detected under these assay conditions. Maximal rate of accumulation of both BW B759U-triphosphate and ACV-triphosphate after a short exposure to drug occurred in the late phase of the infective cycle, as the titer of extracellular virus reached a peak in untreated cultures, but after the decline of stimulated host deoxypyrimidine kinase activities. Once formed, the BW B759U-triphosphate pool decreased very slowly and thus it persisted for several days in both HCMV-infected and uninfected cells.