Guanosine in a single stranded region of anticodon stem-loop tRNA models is prone to oxidatively generated damage resulting in dehydroguanidinohydantoin and spiroiminodihydantoin lesions.

Oxidation of RNA hairpin models corresponding to anticodon stem-loop (ASL) of transfer RNA led to RNA damage consisting solely of a unique loop guanine oxidation. Manganese porphyrin/oxone treatment of RNA resulted in dehydroguanidinohydantoin (DGh; major) and/or spiroiminodihydantoin (Sp) lesions. Ribose damage was not observed. This two-electron transfer oxidation reaction allowed the identification of guanine oxidation products for further study of RNA species carrying a unique lesion at a single G to investigate their biological impact.

Mutagenicity of secondary oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate).

8-Oxo-7,8-dihydroguanine (8-hydroxyguanine) is oxidized more easily than normal nucleobases, which can produce spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). These secondary oxidation products of 8-oxo-7,8-dihydroguanine are highly mutagenic when formed within DNA. To evaluate the mutagenicity of the corresponding oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate) in the nucleotide pool, Escherichia coli cells deficient in the mutT gene were treated with H(2)O(2), and the induced mutations were analyzed. Moreover, the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh were also introduced into competent E. coli cells. The H(2)O(2) treatment of mutT E. coli cells resulted in increase of G:C → T:A and A:T → T:A mutations. However, the incorporation of exogenous Sp and Gh 2'-deoxyribonucleotides did not significantly increase the mutation frequency. These results suggested that the oxidation product(s) of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate induces G:C → T:A and A:T → T:A mutations, and that the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh exhibit quite weak mutagenicity, in contrast to the bases in DNA.

Chronic cardiotoxicity assessment of BMS-986094, a guanosine nucleotide analogue, using human iPS cell-derived cardiomyocytes.

Predicting drug-induced side effects in the cardiovascular system is very important because it can lead to the discontinuation of new drugs/candidates or the withdrawal of marketed drugs. Although chronic assessment of cardiac contractility is an important issue in safety pharmacology, an in vitro evaluation system has not been fully developed. We previously developed an imaging-based contractility assay system to detect acute cardiotoxicity using human iPS cell-derived cardiomyocytes (hiPSC-CMs). To extend the system to chronic toxicity assessment, we examined the effects of the anti-hepatitis C virus (HCV) drug candidate BMS-986094, a guanosine nucleotide analogue, which was withdrawn from phase 2 clinical trials because of unexpected contractility toxicities. Additionally, we examined sofosbuvir, another nucleotide analogue inhibitor of HCV that has been approved as an anti-HCV drug. Motion imaging analysis revealed the difference in cardiotoxicity between the cardiotoxic BMS-986094 and the less toxic sofosbuvir in hiPSC-CMs, with a minimum of 4 days of treatment. In addition, we found that BMS-986094-induced contractility impairment was mediated by a decrease in calcium transient. These data suggest that chronic treatment improves the predictive power for the cardiotoxicity of anti-HCV drugs. Thus, hiPSC-CMs can be a useful tool to assess drug-induced chronic cardiotoxicity in non-clinical settings.

Design, synthesis and evaluation of a novel double pro-drug: INX-08189. A new clinical candidate for hepatitis C virus.

We herein report a novel double pro-drug approach applied to the anti-HCV agent 2'-beta-C-methyl guanosine. A phosphoramidate ProTide motif and a 6-O-methoxy base pro-drug moiety are combined to generate lipophilic prodrugs of the monophosphate of the guanine nucleoside. Modification of the ester and amino acid moieties lead to a compound INX-08189 that exhibits 10nM potency in the HCV genotype 1b subgenomic replicon, thus being 500 times more potent than the parent nucleoside. The potency of the lead compound INX-08189 was shown to be consistent with intracellular 2'-C-methyl guanosine triphosphate levels in primary human hepatocytes. The separated diastereomers of INX-08189 were shown to have similar activity in the replicon assay and were also shown to be similar substrates for enzyme processing. INX-08189 has completed investigational new drug enabling studies and has been progressed into human clinical trials for the treatment of chronic HCV infection.

Mutagenicity of 8-nitroguanosine, a product of nitrative nucleoside modification by reactive nitrogen oxides, in mammalian cells.

8-Nitroguanosine is a nitratively modified nucleoside that is formed endogeneously under inflammatory conditions dependent on nitric oxide production, particularly associated with cancer risks. Here, we investigated the mutagenic potential of 8-nitroguanosine in mammalian cells. Treatment with 8-nitroguanosine (10-1000 microM) for 1h significantly increased (by 6-8 times) the mutation frequency of the xanthine-guanine phosphoribosyltransferase (gpt) gene in AS52 cells without cytotoxic effects. 8-Nitroguanosine treatment induced a G-to-T transversion in gpt gene at position 86. It also significantly increased levels of abasic sites in DNA. These observations suggest that formation of 8-nitroguanosine may contribute to the pathogenesis of inflammation-associated carcinogenesis.

MTH1, an oxidized purine nucleoside triphosphatase, prevents the cytotoxicity and neurotoxicity of oxidized purine nucleotides.

In human and rodent cells, MTH1, an oxidized purine nucleoside triphosphatase, efficiently hydrolyzes oxidized dGTP, GTP, dATP and ATP such as 2'-deoxy-8-oxoguanosine triphosphate (8-oxo-dGTP) and 2'-deoxy-2-hydroxyadenosine triphosphate (2-OH-dATP) in nucleotide pools, thus avoiding their incorporation into DNA or RNA. MTH1 is expressed in postmitotic neurons as well as in proliferative tissues, and it is localized both in the mitochondria and nucleus, thus suggesting that MTH1 plays an important role in the prevention of the mutagenicity and cytotoxicity of such oxidized purines as 8-oxoG which are known to accumulate in the cellular genome. Our recent studies with MTH1-deficient mice or cells revealed that MTH1 efficiently minimizes accumulation of 8-oxoG in both nuclear and mitochondrial DNA in the mouse brain as well as in cultured cells, thus contributing to the protection of the brain from oxidative stress.

2'-deoxyguanosine toxicity for B and mature T lymphoid cell lines is mediated by guanine ribonucleotide accumulation.

Inherited deficiency of the enzyme purine nucleoside phosphorylase (PNP) results in selective and severe T lymphocyte depletion which is mediated by its substrate, 2'-deoxyguanosine. This observation provides a rationale for the use of PNP inhibitors as selective T cell immunosuppressive agents. We have studied the relative effects of the PNP inhibitor 8-aminoguanosine on the metabolism and growth of lymphoid cell lines of T and B cell origin. We have found that 2'-deoxyguanosine toxicity for T lymphoblasts is markedly potentiated by 8-aminoguanosine and is mediated by the accumulation of deoxyguanosine triphosphate. In contrast, the growth of T4+ mature T cell lines and B lymphoblast cell lines is inhibited by somewhat higher concentrations of 2'-deoxyguanosine (ID50 20 and 18 microM, respectively) in the presence of 8-aminoguanosine without an increase in deoxyguanosine triphosphate levels. Cytotoxicity correlates instead with a three- to fivefold increase in guanosine triphosphate (GTP) levels after 24 h. Accumulation of GTP and growth inhibition also result from exposure to guanosine, but not to guanine at equimolar concentrations. B lymphoblasts which are deficient in the purine salvage enzyme hypoxanthine guanine phosphoribosyltransferase are completely resistant to 2'-deoxyguanosine or guanosine concentrations up to 800 microM and do not demonstrate an increase in GTP levels. Growth inhibition and GTP accumulation are prevented by hypoxanthine or adenine, but not by 2'-deoxycytidine. 8-Aminoguanosine appears to effectively inhibit extracellular PNP activity; thus, it prolongs the extracellular half-life of 2'-deoxyguanosine and guanosine, but does not completely inhibit intracellular PNP activity in these lymphoid cells. As a result, 2'-deoxyguanosine and guanosine are phosphorolyzed and actively salvaged within the cell, accounting for the accumulation of GTP. Partial inhibition of PNP activity in vivo, therefore, may lead to nonselective cellular toxicity by a mechanism independent of dGTP accumulation.

Potentiation of cytotoxicity of 1-beta-D-arabinofuranosylcytosine for K562 human leukemic cells by cadeguomycin.

The treatment of K562 human myeloblastic leukemia cells and YAC-1 murine lymphoma cells with cadeguomycin at concentrations over 0.6 microM significantly enhanced the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C). The degree of potentiation depended upon the antibiotic concentration. The treatment with 75 microM cadeguomycin for 18 h increased cellular uptake of [3H]ara-C into K562 cells and formation of ara-C nucleotides, as well as incorporation into nucleic acids. The level of the diphosphate of ara-C plus the triphosphate of ara-C was approximately 10 times higher in the cadeguomycin-treated cells than in the untreated cells by 30 min of incubation with [3H]ara-C. The extracts of 15 microM cadeguomycin-treated K562 cells showed increased activity of formation of ara-C nucleotides, resulting in 4- to 5-fold higher formation of the di- and triphosphates of ara-C than the control cell extracts. Cadeguomycin did not significantly change the level of ribonucleotide and deoxyribonucleotide pool in K562 cells. The mechanism of potentiation of ara-C by cadeguomycin was discussed.

Cytological effects of sulfur and selenium purine analogues on two transplantable hepatomas and on normal renewing cells in mice.

The effects of 50% lethal doses of three purine analogues, 6-methylmercaptopurine riboside ( MMPR ), 6-thioguanosine (TGR), and 6- selenoguanosine ( SeGR ), on mitotic activity in a slow-growing ( SS1H ) and a fast-growing ( BH3 ) transplantable hepatocellular adenoma in C3H/ StW and BUB mice, respectively, were analyzed statistically. No significant difference in response was found between the two benign hepatomas. MMPR alone effectively reduced mitotic activity in the tumors and did so as efficiently on the first day of treatment as on subsequent days of daily i.p. administrations for up to 10 days. TGR alone and SeGR alone were ineffective in reducing the mitotic index significantly below that of controls. When either TGR or SeGR was injected simultaneously with MMPR , the effect on tumor mitosis resembled that of MMPR alone. The reactions of normal cells of the hosts to these agents were analyzed quantitatively in duodenal epithelium with respect to mitotic activity and to the number of cells present in the crypts. Differences between the two strains of mice were small and, for the most part, not significant. MMPR produced a slight but not significant reduction in duodenal mitotic activity and cell number. TGR alone induced significant decreases in both after 3 and 5 days of treatment. SeGR alone had no effect on the duodena . The effects of a combination of SeGR with MMPR on the duodena differed only slightly from MMPR or SeGR alone, but TGR plus MMPR produced greater inhibition of mitosis than did either administered alone. Our results suggest that MMPR may be a promising chemotherapeutic agent against some types of solid hepatocellular tumors in vivo because it can inhibit mitosis in these tumors effectively, rapidly, and continuously, while its inhibitory effect on normal replicating cells of the host intestine occurs more slowly and only with long-sustained treatment.

Effects of 8-aminoguanosine on the toxicity of guanosine and deoxyguanosine for malignant and normal lymphoid cells.

The toxicity of guanosine and deoxyguanosine in the presence or absence of the purine nucleoside phosphorylase inhibitor, 8-aminoguanosine, for malignant lymphoid cell lines and mitogen-stimulated peripheral blood lymphocytes has been studied. Deoxyguanosine inhibited the proliferation of lymphoid cells more strongly than guanosine. Addition of 100 microM 8-aminoguanosine neither enhanced nor diminished the toxicity of guanosine to the lymphoid cells. Only the toxicity of deoxyguanosine for the leukemic T cell line, MOLT 4, and the leukemic nonBnonT cell line, KM-3, was enhanced by the addition of 100 microM 8-aminoguanosine. These data suggest a possible role of purine nucleoside phosphorylase inhibitors in the treatment of lymphoproliferative disorders of the T-acute lymphoblastic leukemia (ALL) as well as the nonBnonT-ALL subclass.

O6-(alkyl/aralkyl)guanosine and 2'-deoxyguanosine derivatives: synthesis and ability to enhance chloroethylnitrosourea antitumor action.

A series of O6-(alkyl/aralkyl)guanosines and 2'-deoxyguanosine analogs extended to peracetyl and N2-acetyl derivatives, potentially water soluble, was synthesized. Each was associated with N'-(2-chloroethyl)-N-[2-(methylsulfonyl)ethyl]-N'-nitrosourea for in vitro evaluation on M4Beu melanoma cells of their ability to enhance the cytotoxic effect of this chloroethylnitrosourea, which is frequently reduced by repairs performed by O6-alkylguanine-DNA-alkyltransferase. Structure-activity analysis revealed that (i) benzyl and 4-halobenzyl are the O6-substituents required to afford a significant activity, (ii) 2'-deoxyguanosine derivatives demonstrate greater potency than guanosine analogs, (iii) acetylation, especially at the N2 position, generally results in compounds with moderate ability but may prevent incorporation of such nucleosides into DNA. Accordingly, O6-(4-iodobenzyl)-N2-acetylguanosine (3b) and O6-benzylperacetyl-2'-deoxyguanosine (2a), as well as O6-benzyl-N2-acetylguanosine (1b) and O6-benzyl-N2-acetyl-2'-deoxyguanosine (2b), by far the most water soluble, exhibit a good profile for further in vivo trials by the intravenous route.

Dissimilar actions of 6-mercaptopurine and 6-thioguanine in Chinese hamster ovary cells.

The actions of 6-thioguanine (TG) and 6-mercaptopurine (MP) were compared in Chinese hamster ovary (CHO) cells. Several differences were noted between these two agents. TG caused a greater maximal loss of clonogenicity, leaving about one log fewer survivors than did MP, although the cells killed by MP appeared to succumb much more rapidly than those killed by TG. MP-treated populations experienced a G1 or G1/S arrest which was quickly reversed upon drug removal, while TG-treated cells were arrested in late S/G2, after some delay. Although TG induced a gross chromosome deformation [unilateral chromatid damage, as described earlier in Maybaum and Mandel, Cancer Res. 43, 3852 (1983)] MP caused little or no such deformation. Addition of 4-amino-5-imidazolecarboxamide (AIC) to MP treatments antagonized MP-induced loss of clonogenicity, while AIC caused a dose-dependent potentiation of TG-induced loss of clonogenicity. The interaction between TG and AIC does not seem to represent an increase in either purine starvation or incorporation of TG into DNA, suggesting that a third mechanism is involved. We suggest that this additional mechanism may possibly be related to the induction of differentiation by TG that has been reported in other systems.

Prophylactic and therapeutic activities of 7-thia-8-oxoguanosine against Punta Toro virus infections in mice.

The biological response modifier 7-thia-8-oxoguanosine was evaluated in mice against the hepatotropic Adames strain of Punta Toro virus. When administered intraperitoneally in divided doses, significant protection from death was conferred at doses of 50 and 100 mg/kg/day given 24 and 17 h pre-virus inoculation, 25-100 mg/kg/day administered 4 h pre- and 3 h post-virus challenge, and 12.5 to 100 mg/kg/day administered 24 and 31 h after virus inoculation. These doses preventing death reduced liver icterus scores, serum alanine aminotransferase and aspartate aminotransferase levels, and liver and serum virus titers relative to placebo controls. Full daily doses administered at 24 h were somewhat less protective to mice than divided daily doses starting at the same time. The initiation of treatment could be delayed as late as 36 h after virus inoculation, resulting in complete protection from mortality at 100 mg/kg/day. This prevention of death occurred despite the acute nature of the infection which resulted in deaths by 96 h in the placebo-treated controls. These results show that 7-thia-8-oxoguanosine has both prophylactic and therapeutic potential as an anti-Phlebovirus agent. Interferon induction appears to be the reason for antiviral activity in this model, since up to 10,000 units of interferon/ml were induced in mice 1 h after treatment with 100 mg 7-thia-8-oxoguanosine per kg, and antibody to interferon alpha/beta administered shortly after treatment with the nucleoside negated the antiviral effect.

6-Methylguanine and 6-methylguanosine inhibit colony-forming ability in a malignant xeroderma pigmentosum cell line but not in other xeroderma pigmentosum and normal human fibroblast strains after treatment with 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl)-urea.

The XP cell strain XP29MA, its malignant counterpart XP29MAmal and a normal human fibroblast strain were tested for colony-forming ability after treatment with HECNU in the presence of m6G, m6Gua, and he7G. In XP29MAmal, inhibition of post-HECNU colony-forming ability was 35% when 0.25 mM of either m6G or m6Gua were present, whereas in XP29MA and the normal fibroblast strain no inhibition was detected. The he7G caused a similar but smaller inhibitory effect in XP29MAmal, but failed to do so in XP29MA. HECNU predominantly exerts its killing effect by alkylating O-6 of DNA-bound guanine and causing DNA interstrand crosslinks. Alkylation of O-6 of guanine can be repaired by 6-methylguanine-DNA methyltransferase. From our experiments we conclude that in XP29MAmal this methyltransferase was inhibited in the presence of the 6-alkylguanines, thus leaving more 2-chloroethylated sites in DNA unrepaired. This results in sensitization in terms of decreased colony-forming ability observed only in the malignant cell line.

Intrathecal injection of cell-permeable analogs of cyclic 3',5'-guanosine monophosphate produces hyperalgesia in mice.

Several recent studies suggest that spinal cord levels of cyclic 3',5'-guanosine monophosphate (cGMP) may participate in the development of hyperalgesia. The purpose of this study was to directly evaluate whether cell permeable analogues of cGMP evoke a thermal hyperalgesia (using a hot-plate assay) when administered intrathecally in mice. Our results indicate that two cell permeable forms of cGMP evoke a dose dependent hyperalgesia when administered intrathecally in mice. Additionally, this hyperalgesia was selective since neither non-cell permeant cGMP nor guanosine had any effect on the latency of paw withdrawal when compared to the vehicle injected controls. These data indicate that cGMP is involved in the facilitation of thermal hyperalgesia at the level of the spinal cord.

Cytotoxic combination of loxoribine with fludarabine and mafosfamide on freshly isolated B-chronic lymphocytic leukemia cells.

Fludarabine has shown a definite clinical activity in B-cell chronic lymphocytic leukemia (B-CLL). Recently it has been demonstrated that loxoribine, a guanine ribonucleotide derivative, is able to increase the cytotoxicity of fludarabine in B-CLL cells, in vitro. We have here extended these findings by testing the activity of loxoribine in combination with fludarabine and mafosfamide. As we have previously demonstrated, loxoribine enhances the activity of fludarabine at all concentrations, while only lower doses of mafosfamide seem to be positively affected by loxoribine. The combination of fludarabine and mafosfamide is synergistic on CLL cells, and the cytotoxic activity is increased by the addition of loxoribine. We have also evaluated the pro-apoptotic activity of each drug, both alone and in combination; these results are concordant with the cytotoxicity data, thus demonstrating that, even though loxoribine is more active in combination with fludarabine than with mafosfamide, the efficacy of the triple combination is higher than that obtained with any other agent alone or in double combination.

Treatment of experimental herpes simplex keratitis with acycloguanosine.

Acycloguanosine, a recently developed compound with high inhibitory activity against viruses belonging to the herpes group, has been evaluated in experimental herpes simplex keratitis in rabbits in comparison with trifluorothymidine and preparations of idoxuridine and vidarabine at present in clinical use. All compunds were used in the form of ophthalmic ointments which were applied 5 times a day at intervals of 2 hours. Treatment began on the third day of infection and was continued for 4 days. Complete cure was obtained with acycloguanosine and idoxurdine; trifluorothymidine and vidarabine were considerably less effective. Acycloguanosine was equally effective when given intravenously in the form of its sodium salt, and could be detected in the tear fluid in inhibitory concentrations when given by mouth. The compound was relatively free from toxicity.

Melanoma-cell toxicity of cystemustine combined with O6-benzyl-N2-acetylguanosine.

Cystemustine (N'-(2-chloroethyl)-N-(2-(methylsulphonyl)ethyl)-N'-nitrosourea) is a new chloroethylnitrosourea (CENU) being used in phase II clinical trials of disseminated melanoma. Clinical results show that tumour regression has only been observed in 25% of melanomas treated by CENUs. Tumour resistance to CENU is known to be mainly due to a DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT). The poor remission rate of melanoma with CENUs is attributed to the fact that metastases contain high MGMT levels. Previously, we have shown that O6-benzyl-N2-acetylguanosine (BNAG), an MGMT inhibitor, can be combined with cystemustine by intravenous administration, and increases the antitumour effect of cystemustine in resistant human melanoma. In the work presented here, we investigated the in vitro pharmacological effect of this combination on the DNA of human melanoma cells (M3Dau cells). A quantitative polymerase chain reaction (QPCR) assay was used to measure DNA damage in a fragment (2.7 kb) of the hprt gene. The results show that treatment with BNAG enhances the number of lesions in the DNA of cystemustine-treated resistant malignant melanocytes, which may account for the high tumour-cell toxicity of the combination of cystemustine and BNAG.

Mouse strain differences in induction of micronuclei by base analogues and nucleosides.

The frequency of induced micronucleated polychromatic erythrocytes (MNPCEs) was compared in BALB/c, C57BL/6, and DBA/2 mice after intraperitoneal (i.p.) injection of 5-bromodeoxyuridine (BUdR), 5-fluorodeoxyuridine (FUdR), cytosine arabinoside (Ara-C), 6-mercaptopurine (6-MP), 5-bromouracil (5-BU), thymidine (TdR), uridine (UdR), adenosine (AdR) and guanosine (GdR). The experimental procedure was a single i.p. injection followed by harvest at 30 h. The frequency of MNPCEs was significantly increased in all strains by treatment with BUdR, FUdR, Ara-C and 6-MP compared to vehicle control. TdR and UdR induced MNPCEs slightly in BALB/c mice but showed no effect on C57BL/6 and DBA/2 mice. 5-BU, AdR, and GdR did not increase the frequency of MNPCEs in any mouse strain used. These results suggest that BALB/c mice are more susceptible to induction of MNPCEs by clastogenic base analogues and nucleosides than are C57BL/6 or DBA/2 mice.

Failure to obtain hybridomas between human macrophages and human tumoral U-937 cells is probably due to parental macrophages.

Despite more than 50 attempts and the use of various methods, it has been impossible to establish homologous hybridomas between human mature macrophages and 8-azaguanine-resistant U-937 clones prepared in the laboratory. To rule out the possibility that these clones were unsuitable for the selection of hybrids, a study of their properties was done. It was shown that U-937 wild type cells were able to produce HPRT, whereas 8-azaguanine (8-aza)-resistant clones did not. Curiously, exonic and intronic HPRT sequences were undetectable both in wild type and in 8-aza-resistant cell genomes, under conditions where they were detected in control cells. Chromosome analysis of the clone UM9, one of the most frequently used in fusion experiments, revealed many qualitative and quantitative differences with the U-937 wild type cells. 8-aza-resistant U-937 cells were capable of fusion with human macrophages and gave rise to heterokaryons and probably to synkaryons, which survived for weeks without dividing in hypoxanthine-aminopterin-thymidine medium. The results could be interpreted in terms of the existence of a transacting negative regulatory mechanism of the macrophage genome on the proliferative capacity of homospecific hybridomas.