Xeno-Nucleic Acid (XNA) 2'-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor-Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding.

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2'-fluoro-arabinonucleic acid (FANA). The best selected ~79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~10-20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, respectively. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.

A multi-target antisense approach against PDE4 and PDE7 reduces smoke-induced lung inflammation in mice.

BACKGROUND: Recent development in the field of COPD has focused on strategies aimed at reducing the underlying inflammation through selective inhibition of the phosphodiesterase type IV (PDE4) isoform. Although the anti-inflammatory and bronchodilator activity of selective PDE4 inhibitors has been well documented, their low therapeutic ratio and dose-dependent systemic side effects have limited their clinical utility. This study examined the effect of 2'-deoxy-2'-Fluoro-beta-D-Arabinonucleic Acid (FANA)-containing antisense oligonucleotides (AON) targeting the mRNA for the PDE4B/4D and 7A subtypes on lung inflammatory markers, both in vitro and in vivo. METHODS: Normal human bronchial epithelial (NHBE) cells were transfected with FANA AON against PDE4B/4D and 7A alone or in combination. mRNA levels for target PDE subtypes, as well as secretion of pro-inflammatory chemokines were then measured following cell stimulation. Mice were treated with combined PDE4B/4D and 7A AON via endo-tracheal delivery, or with roflumilast via oral delivery, and exposed to cigarette smoke for one week. Target mRNA inhibition, as well as influx of inflammatory cells and mediators were measured in lung lavages. A two-week smoke exposure protocol was also used to test the longer term potency of PDE4B/4D and 7A AONs. RESULTS: In NHBE cells, PDE4B/4D and 7A AONs dose-dependently and specifically inhibited expression of their respective target mRNA. When used in combination, PDE4B/4D and 7A AONs significantly abrogated the cytokine-induced secretion of IL-8 and MCP-1 to near baseline levels. In mice treated with combined PDE4B/4D and 7A AONs and exposed to cigarette smoke, significant protection against the smoke-induced recruitment of neutrophils and production of KC and pro-MMP-9 was obtained, which was correlated with inhibition of target mRNA in cells from lung lavages. In this model, PDE AONs exerted more potent and broader anti-inflammatory effects against smoke-induced lung inflammation than roflumilast. Moreover, the protective effect of PDE4B/4D and 7A AON was maintained when a once-weekly treatment schedule was used. CONCLUSION: These results indicate that inhaled AON against PDE4B/4D and 7A have unique effects on biomarkers that are believed to be important in the pathophysiology of COPD, which supports further development as a potential therapy in this disease.

Synthesis and characterization of a novel liver-targeted prodrug of cytosine-1-beta-D-arabinofuranoside monophosphate for the treatment of hepatocellular carcinoma.

Cytotoxic nucleosides have proven to be ineffective for the treatment of hepatocellular carcinoma (HCC) due, in part, to their inadequate conversion to their active nucleoside triphosphates (NTP) in the liver tumor and high conversion in other tissues. These characteristics lead to poor efficacy, high toxicity, and a drug class associated with an unacceptable therapeutic index. Cyclic 1-aryl-1,3-propanyl phosphate prodrugs selectively release the monophosphate of a nucleoside (NMP) into CYP3A4-expressing cells, such as hepatocytes, while leaving the prodrug intact in plasma and extrahepatic tissues. This prodrug strategy was applied to the monophosphate of the well-known cytotoxic nucleoside cytosine-1-beta-D-arabinofuranoside (cytarabine, araC). Compound 19S (MB07133), in mice, achieves good liver targeting compared to araC, generating >19-fold higher cytarabine triphosphate (araCTP) levels in the liver than levels of araC in the plasma and >12-fold higher araCTP levels in the liver than in the bone marrow, representing a >120-fold and >28-fold improvement, respectively, over araC administration.

Characterization of antisense oligonucleotides comprising 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA): specificity, potency, and duration of activity.

Antisense oligonucleotides (AON) are being developed for a wide array of therapeutic applications. Significant improvements in their serum stability, target affinity, and safety profile have been achieved with the development of chemically modified oligonucleotides. Here, we compared 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA)-containing AONs with phosphorothioate oligodeoxynucleotides (PS-DNA), 2'-O-methyl-RNA/DNA chimeras and short interfering RNAs (siRNA) with respect to their target knockdown efficacy, duration of action and resistance to nuclease degradation. Results show that two different configurations of FANA/DNA chimeras (altimers and gapmers) were found to have potent antisense activity. Specific target inhibition was observed with both FANA configurations with an estimated EC50 value comparable to that of an siRNA but 20-to 100-fold lower than the other commonly used AONs. Moreover, the FANA/DNA chimeras showed increased serum stability that was correlated with sustained antisense activity for up to 4 days. Taken together, these results indicate that chimeric FANA/DNA AONs are promising new tools for therapeutic gene silencing when increased potency and duration of action are required.

Modulation of arabinosylnucleoside metabolism by arabinosylnucleotides in human leukemia cells.

Previous studies have indicated that deoxycytidine kinase (dCK) is requisite and rate limiting in the phosphorylation of 1-beta-D-arabinofuranosylcytosine (ara-C) and 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on the pathway to their respective cytotoxic 5'-triphosphates. In K562 cells, the rate of triphosphate accumulation was maximal during incubation with 10 microM ara-C (35 microM/h) and 300 microM F-ara-A (102 microM/h). Under these conditions, accumulation of cellular ara-CTP plateaued at about 110 microM after 3 h, whereas in separate cultures, F-ara-ATP continued to accumulate at a linear rate to cellular concentrations greater than 500 microM after 5 h. Other laboratories have demonstrated that dCK activity in cell-free extracts was inhibited by ara-CTP. To determine whether ara-CTP exhibited the same activity in whole cells, K562 cells were preincubated with ara-C to accumulate 110 microM ara-CTP. After washing into medium containing F-ara-A, the rate of F-ara-ATP accumulation was significantly decreased (37 microM/h). However, cells loaded with F-ara-ATP exhibited an increased rate of ara-CTP accumulation (110 microM/h) that resulted in cellular ara-CTP concentrations in excess of 400 microM after 5 h. This stimulation was proportional to the cellular concentration of F-ara-ATP, achieving a maximum effect between 75 and 100 microM. Phosphorylation of ara-C by cell-free extracts supplemented with physiological levels of ribo- and deoxyribonucleoside 5'-triphosphates was stimulated by addition of F-ara-ATP. The decreased rate of accumulation of products of dCK in intact cells containing 110 microM ara-CTP suggests that this active triphosphate may limit its own synthesis and phosphorylation of other substrates. In contrast, stimulation of the accumulation of ara-CTP in cells containing F-ara-ATP suggests new possibilities for the design of combination chemotherapy regimens.

1-beta-D-arabinofuranosylcytosine nucleotide inhibition of sialic acid metabolism in WI-38 cells.

Because cytidine nucleotides have been demonstrated to affect activity of sialytransferases of both normal and malignant cells, we have investigated the effects of nucleotides of 1-beta-D-arabinofuranosylcytosine (ara-C) [1-beta-D-arabinofuranosylcytosine 5'-monophosphate and 1-beta-D-arabinofuranosycytosine 5'-triphosphate (ara-CTP)] on synthesis of sialoglycoproteins. Normal human diploid fibroblasts (WI-38 cells) were used in culture at confluency, when fewer than 1% of the cells were synthesizing DNA. 1-beta-D-Arabinofuranosylcytosine 5'-monophosphate was inhibitory to both sialytransferase activity of the intact cell and total cell homogenate transferase activity. The enzymes which synthesize and degrade the substrate of sialyltransferases, cytidine 5'-monophosphate-N-acetylneuraminic acid (CMP-AcNeu), were also tested for inhibition by nucleotides of ara-C. Synthesis of CMP-AcNeu was competitively inhibited by ara-CTP; however, formation of CMP-AcNeu when ara-CTP was supplied as substrate could not be detected. Hydrolysis of CMP-AcNeu was inhibited more severely by cytidine 5'-triphosphate than by ara-CTP or 1-beta-D-arabinofuranosylcytosine 5'-monophosphate. Confluent cultures of WI-38 cells exposed to ara-C have decreased amounts of glycoprotein sialic acid, suggesting that ara-C nucleotides may reach sufficient intracellular concentrations to affect the enzyme systems described.

Inhibitory effects of 9-beta-D-arabinofuranosylguanine 5'-triphosphate and 9-beta-D-arabinofuranosyladenine 5'-triphosphate on DNA polymerases from murine cells and oncornavirus.

The effects of the newly synthesized compound 9-beta-D-arabinofuranosylguanine 5'-triphosphate (ara-GTP) on the activity of DNA polymerases from mouse cells and oncornavirus were compared with those of 9-beta-D-arabinofuranosyladenine 5'-triphosphate. Ara-GTP did not replace deoxyguanosine 5'- triphosphate as substrate for these DNA polymerases but inhibited the activities of DNA polymerase alpha, beta, and gamma and viral DNA polymerase. DNA polymerase alpha was more sensitive than DNA polymerases beta and gamma and viral DNA polymerase to inhibition by ara-GTP. The inhibitions by ara-GTP and 9-beta-D-arabinofuranosyladenine 5'-triphosphate were due to competition or partial competition 5'-triphosphate were due to competition or partial competition with deoxynucleoside triphosphate with the same base. The inhibition constant (Ki) and the mode of inhibition of nucleotide incorporation varied depending on the combination of inhibitor, substrate(s), and enzyme species.

Interaction of 9-beta-D-arabinofuranosyladenine, 9-beta-D-arabinofuranosyladenine 5'-monophosphate, and 9-beta-D-arabinofuranosyladenine 5'-triphosphate with S-adenosylhomocysteinase.

9-beta-D-Arabinofuranosyladenine (ara-A), 9-beta-D-arabinofuranosyladenine 5'-monophosphate, and 9-beta-D-arabinofuranosyladenine 5'-triphosphate competitively inhibit both the synthesis and hydrolysis of S-adenosylhomocysteine catalyzed by S-adenosylhomocysteinase [S-adenosylhomocysteine hydrolase (EC 3.3.1.1)] from mouse liver, and the inhibitor constants were 5.0 X 10(-6), 1.1 X 10(-4), and 1.0 X 10(-3) M, respectively. A time-dependent inactivation of the enzyme was observed when the enzyme was preincubation with ara-A, 9-beta-D-arabinofuranosyladenine 5'-monophosphate, or 9-beta-D-arabinofuranosyladenine 5'-triphosphate. ara-A was the most potent inactivator. The inactivation with ara-A was less pronounced in the presence of adenosine, S-adenosylhomocysteine, adenine, adenosine 5'-monophosphate, or adenosine 5'-diphosphate, showed first-order kinetics, saturability, and irreversibility. The rate of inactivation was half-maximal at 5 X 10(-6) M ara-A, and the rate constant of inactivation was 0.43 min-1 at saturating concentrations of ara-A. ara-A was tightly but not covalently bound to the enzyme. ara-A bound to the enzyme was not available for deamination to 9-beta-D-arabinofuranosylhypoxanthine catalyzed by the enzyme adenosine deaminase.

Effects of deoxyadenosine triphosphate and 9-beta-D-arabinofuranosyl-adenine 5'-triphosphate on human ribonucleotide reductase from Molt-4F cells and the concept of "self-potentiation".

Deoxyadenosine triphosphate (dATP) acted as a noncompetitive inhibitor with respect to the specific nucleoside triphosphate activator for the reduction of all four common ribonucleoside diphosphates catalyzed by the reductase derived from human Molt-4F (T-type lymphoblast) cells. The inhibition constant of dATP for different ribonucleotide reduction reactions was different, indicating that the binding of the nucleoside triphosphate activator or substrate could modify the binding affinity of dATP to the enzyme. dATP also acted as a noncompetitive inhibitor with respect to cytidine diphosphate (CDP) for reductase-catalyzed CDP reduction. 9-beta-D-Arabinofuranosyl-adenine 5'-triphosphate acted as a competitive inhibitor with respect to either adenosine triphosphate or guanosine triphosphate for CDP or for adenosine diphosphate reduction, respectively. The inhibition constant was 15 microM for CDP reduction and 4 microM for adenosine diphosphate reduction. 1-beta-D-Arabinofuranosyladenine 5'-triphosphate could not substitute for adenosine triphosphate or guanosine triphosphate as the activator for CDP or adenosine diphosphate reduction, respectively. The effects of 9-beta-D-arabinofuranosylcytosine 5'-triphosphate and 5-iodo-2'-deoxyuridine 5'-triphosphate on ribonucleotide reductase were also included for comparison. The "self-potentiation" mechanism of the action of 9-beta-D-arabinofuranosyladenine and 5-iodo-2'-deoxyuridine is discussed.

Altered sensitivity to 1-beta-D-arabinofuranosylcytosine 5'-triphosphate of DNA polymerase alpha from leukemic blasts of acute lymphoblastic leukemia.

DNA polymerase alpha from the leukemic cells of acute lymphoblastic leukemia (ALL) was found to be more resistant to the inhibition by 1-beta-D-arabinofuranosylcytosine 5'-triphosphate than that from acute myeloblastic leukemia (AML). Apparent Ki values for 1-beta-D-arabinofuranosylcytosine 5'-triphosphate of DNA polymerase alpha from eight patients with ALL [26.7 +/- 7.1 (S.D.) microM] were 5 times higher than those from nine patients with AML [5.2 +/- 1.3 microM]. In contrast, apparent Km values for a normal substrate deoxycytidine 5'-triphosphate of DNA polymerase alpha preparations from either AML and ALL were almost identical (9.4 to 10.9 microM). Likewise, apparent Ki values for another arabinoside analog, 9-beta-D-arabinofuranosyladenine 5'-triphosphate, of DNA polymerase alpha from blasts of seven patients with ALL (16.9 +/- 6.9 microM) were significantly higher than those from patients with AML (3.8 +/- 0.5 microM). These results indicate that DNA polymerase alpha from ALL blast cells has a decreased affinity to the arabinoside analogs of deoxynucleotide triphosphate. The sensitivity of DNA polymerase alpha of blast cells to 1-beta-D-arabinofuranosylcytosine 5'-triphosphate may be one of the determinants of the clinical response to 1-beta-D-arabinofuranosylcytosine treatment.

Modulatory activity of 2',2'-difluorodeoxycytidine on the phosphorylation and cytotoxicity of arabinosyl nucleosides.

This investigation analyzed the metabolism of 2',2'-difluorodeoxycytidine (dFdC) in K562 human leukemia cells and evaluated it as a biochemical modulator for the phosphorylation of several arabinosyl nucleosides. The rate of accumulation of dFdC triphosphate was linear up to 3 h and maximal during incubation with 10 microM dFdC (92 microM/h). Deoxynucleotides analyzed at this time showed a decrease in dCTP, dATP, and dGTP levels, indicating an inhibitory role of dFdC nucleotides in ribonucleotide reduction. We evaluated the hypothesis that dFdC-mediated deoxyribonucleoside triphosphate perturbation enhances the phosphorylation of substrates that use deoxycytidine kinase or deoxyguanosine kinase, because these enzymes are inhibited by dCTP or dGTP, respectively. When the activity of these nucleoside kinases was rate limiting to triphosphate formation, the accumulation of triphosphates of deoxycytidine, 1-beta-D-arabinofuranosylcytosine, and 1-beta-D-arabinofuranosylguanine was potentiated in cells pretreated with dFdC. In contrast, the phosphorylation of 9-beta-D-arabinofuranosyladenine was not affected, since it is mainly phosphorylated by adenosine kinase, which is not influenced by deoxyribonucleoside triphosphates. Treatment of cells with dFdC followed by 1-beta-D-arabinofuranosylcytosine resulted in greater cytotoxicity than sum effects of each drug alone. The data indicate that an enhanced cytotoxicity could be obtained by administering dFdC as a modulator followed by 1-beta-D-arabinofuranosylcytosine or 1-beta-D-arabinofuranosylguanine in optimal sequence, suggesting that these results should be considered in the design of combination clinical protocols.

Inhibition of nuclear envelope nucleoside triphosphatase-regulated nucleocytoplasmic messenger RNA translocation by 9-beta-D-arabinofuranosyladenine 5'-triphosphate in rodent cells.

Nucleocytoplasmic translocation of polyadenylated messenger RNA is an energy-dependent process which is regulated by a nuclear envelope nucleoside triphosphatase; this enzyme was found to be stimulated by the 3'-terminal polyadenylic acid [poly(A)] tail of messenger RNA (Bernd, A., Schröder, H. C., Zahn, R. K., and Müller, W. E. G. Eur. J. Biochem., 129: 43-49, 1982). RNA efflux from isolated mouse lymphoma (L5178Y) cell nuclei is strongly reduced if 9-beta-D-arabinofuranosyladenine 5'-triphosphate (ara-ATP) is present in the transport medium. Half-maximal inhibition of RNA efflux occurs with 120 microM ara-ATP. Most likely, the inhibitory effect of ara-ATP is caused by inhibition of nuclear envelope nucleoside triphosphatase; this enzyme was found to be highly sensitive to inhibition by this antibiotic. The inhibition type of the nucleoside triphosphatase of rat liver nuclear ghosts is competitive with respect to ATP; the Ki:Km ratio was determined to be 0.27. Besides nucleoside triphosphatase, nuclear envelopes contain a protein phosphokinase modulating the affinity of pore complex laminae to poly(A). This enzyme was also found to be strongly inhibited by ara-ATP in a competitive way with respect to ATP (Ki:Km, 0.056) and could therefore also contribute to the overall inhibition of RNA transport. The polyadenylation of endogenous RNA by poly(A) polymerase(s) in intact rat liver nuclei as well as in nuclear matrices isolated from the same source was found to be markedly suppressed in the presence of ara-ATP. The inhibitions of both poly(A) polymerase activities (contained in whole nuclei or nuclear matrix bound) are of the competitive type with respect to ATP. In in vitro assays, nuclear envelope nucleoside triphosphatase is inhibited by microtubule protein. Of the 2 ATP-dependent enzyme activities associated with microtubule protein (cyclic adenosine 3':5'-monophosphate-dependent protein kinase and adenosine triphosphatase), only the kinase was slightly affected by ara-ATP. Cellular uptake of adenosine 5'-monophosphate and perhaps 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) is facilitated by a cellular membrane-bound 5'-nucleotidase. Our studies revealed that neither cleavage of ara-AMP nor inhibition of the enzyme activity by ara-AMP occurs. 9-beta-D-Arabinofuranosyladenine and ara-AMP represent neither direct mutagens nor premutagens as determined by the Salmonella-mammalian microsome mutagenicity test.

Correlation of cytotoxicity with total intracellular exposure to 9-beta-D-arabinofuranosyladenine 5'-triphosphate.

The mechanism by which 9-beta-D-arabinofuranosyladenine produces cell death has been studied extensively, but the details remain controversial. The results presented here describe an evaluation of 9-beta-D-arabinofuranosyladenine-induced cytotoxicity in terms of the product of the total amount of the active 5'-triphosphate metabolite, 9-beta-D-arabinofuranosyladenine 5'-triphosphate (ara-ATP), which accumulated in the cells, and the duration of the exposure expressed in units of ara-ATP microM-hr. It was demonstrated that a strong correlation exists between these parameters which was not affected by the rate of accumulation of ara-ATP. In addition, inhibition of 9-beta-D-arabinofuranosyladenine deamination by 2'-deoxycoformycin did not alter the relationship between cell death and total intracellular exposure to ara-ATP. The consistency of this relationship both within and between experiments indicates that the quantitation of the total cellular exposure to ara-ATP is useful in predicting cytotoxicity.

Clinical pharmacology of 1-beta-D-arabinofuranosylcytosine-5'-stearylphosphate, an orally administered long-acting derivative of low-dose 1-beta-D-arabinofuranosylcytosine.

1-beta-D-Arabinofuranosylcytosine-5'-stearylphosphate (cytarabine ocfosfate, stearyl-ara-CMP) is a newly synthesized 5'-alkylphosphate derivative of 1-beta-D-arabinofuranosylcytosine (ara-C), which is lipophilic, resistant to inactivation by deamination, and orally active. Pharmacology of this drug was studied in patients with hematological malignancies. The concentrations of stearyl-ara-CMP, ara-C (its active metabolite), and 1-beta-D-arabinofuranosyluracil (ara-U, its inactive metabolite) were determined by radioimmunoassay. When six patients received a single p.o. dose of the drug (500 mg/m2), stearyl-ara-CMP, ara-C, and ara-U could be detected in the plasma for at least 72 h afterwards. The plasma disappearance curve of stearyl-ara-CMP corresponded to a one-compartment open model with first-order absorption kinetics. The peak plasma level (Cmax) was 322 +/- 218 nM, and the predicted time to reach Cmax (Tmax) was 6.5 +/- 4.5 h, while the elimination half-life (t1/2) was very long (32.0 +/- 8.4 h). The plasma ara-C level increased slowly to a Cmax of 26.3 +/- 12.7 nM (Tmax, 13.3 +/- 4.7 h) after stearyl-ara-CMP administration. This level was quite low compared with that achieved by low-dose s.c. ara-C therapy, but ara-C persisted longer in the plasma in the former case, and the area under the curve was similar for both regimens. For ara-U, the Cmax, Tmax, and t1/2 were 483 +/- 315 nM, 23.6 +/- 4.0 h, and 19.6 +/- 5.3 h, respectively. No stearyl-ara-CMP was detected in the urine, and only 8.0% of the administered dose was excreted as ara-C and ara-U within 72 h. The stearyl-ara-CMP concentration in the cerebrospinal fluid was below the limit of detection in three patients without meningeal involvement at 6 h. During clinical use of stearyl-ara-CMP, macrocytic anemia was observed, and some patients also developed megaloblastic change of their erythroblasts, suggesting a mild and persistent cytostatic effect. In conclusion, p.o. therapy with stearyl-ara-CMP achieved prolonged maintenance of the plasma drug level. Thus, the drug released a very low dose of ara-C over a long period in plasma and tissues and had a prolonged mild antineoplastic effect in patients with hematological malignancies.

Differential effects of cordycepin triphosphate and 9 beta-D-arabinofuranosyladenine triphosphate on tRNA and 5 S RNA synthesis in isolated nuclei.

Although cordycepin 5'-triphosphate (3'-dATP), at low concentrations, preferentially inhibits chromatin-associated poly(A) synthesis in isolated nuclei, higher levels of the inhibitor prevent both rRNA (RNA polymerase I activity) and hnRNA (RNA polymerase II activity) synthesis in vitro (Rose, K.M., Bell, L.E. and Jacob, S.T. (1977) Nature 267, 178-180). The present studies demonstrate that this nucleotide can also inhibit tRNA and 5 S RNA synthesis (RNA polymerase III activity). At 50-200 microgram/ml, 3'-dATP inhibits incorporation of [3H]UTP into tRNA and 5 S RNA by approximately 65%, whereas the syntheses of these RNAs were completely blocked when [3H]GTP was used as the substrate. These data suggest the formation of poly(U) in the tRNA and 5 S RNA regions, which is resistant to 3'-dATP. In contrast, another ATP analog, Ara-ATP, which selectively inhibits poly(A) synthesis, does not block tRNA and 5 S RNA synthesis in isolated nuclei. The production of these RNA species in isolated nuclei is also insensitive to Ara-CTP and 2'-dATP. These data suggest that 3'-dATP exerts general inhibitory effects on RNA synthesis and further substantiate the conclusion that Ara-ATP is a selective inhibitor of the polyadenylation reaction in vitro.

Characteristics of enzymatic DNA methylation in cultured cells of human and hamster origin, and the effect of DNA replication inhibition.

In mammalian cells, inhibitors of DNA replication have been shown to induce chromosomal aberrations, cell death and changes in gene control. Inhibition of DNA synthesis has been reported to induce hypermethylation of mammalian DNA (enzymatic postsynthetic formation of 5-methylcytosine). These 5-methylcytosines in mammalian DNA have variously been suggested to be important in gene control, DNA repair, and control of DNA replication. In establishing the normal characteristics of enzymatic DNA methylation, we have demonstrated that, in asynchronously growing cells of both human and hamster origin, some cytosine methylation is delayed for several hours after strand synthesis and that this delayed methylation is completed before the DNA strand acts as a template for DNA replication in the next S-phase. Further, in testing whether the deleterious effects on mammalian cells of DNA synthesis inhibitors might be mediated via changes in enzymatic DNA methylation, we have found, contrary to some previous findings, no evidence for any change in the level of DNA methylation in DNA strands synthesized during 6 h of treatment of cells of human origin with high concentrations of four different inhibitors of DNA replication or during the 4 h following the 6 h treatment. Almost totally blocking DNA replication had no effect on the small amount of delayed methylation of DNA strands not involved in semi-conservative replication during the time of the experiment. This lack of effect on DNA methylation was obtained when the labelling medium contained normal, undialysed serum. In contrast, if dialysed serum was used in the labelling medium in order to maximize L-[Me-3H]methionine utilization, highly variable, totally irreproducible patterns of apparent DNA hypermethylation were obtained.

DNA repair initiated in chronic lymphocytic leukemia lymphocytes by 4-hydroperoxycyclophosphamide is inhibited by fludarabine and clofarabine.

PURPOSE: Chronic lymphocytic leukemia (CLL) lymphocytes respond to DNA alkylation by excision repair, with the extent of repair increasing as the cells acquire resistance to alkylating agents. Because incorporation of nucleotide analogues into the repair patches elicits death signals in quiescent cells, the increased capacity for excision repair in alkylator-resistant cells could facilitate incorporation of nucleotide analogues. We hypothesized that the mechanism-based interaction of nucleoside analogues with alkylating agents could elicit greater than additive killing of CLL cells. EXPERIMENTAL DESIGN: Lymphocytes from 50 patients with CLL that were not refractory to alkylators were treated in vitro with 4-hydroperoxycyclophosphamide (4-HC) with or without prior incubation with fludarabine nucleoside (F-ara-A) or with clofarabine (Cl-F-ara-A). DNA damage repair kinetics were determined by the single-cell gel electrophoresis (comet) assay. Cytotoxicity was assessed by staining with annexin V. RESULTS: CLL lymphocytes promptly initiated and completed excision repair in response to 4-HC. A 2-h preincubation with 10 microM F-ara-A or 10 microM Cl-F-ara-A inhibited the repair initiated by 4-HC, with inhibition peaking at the intracellular concentrations of 50 microM F-ara-ATP or 5 microM Cl-F-ara-ATP. Combining 4-HC with either F-ara-A or Cl-F-ara-A produced more than additive apoptotic cell death than the sum of each alone. The increase in cytotoxicity was proportional to the initial magnitude of the DNA incision and to the extent of repair inhibition by the nucleoside analogues, suggesting close correlation between the repair inhibition and induction of cell death. CONCLUSIONS: DNA repair, which is active in CLL lymphocytes, may be a biological target for facilitating the incorporation of nucleoside analogues and increasing their cytotoxicity. Thus, the increased repair capacity associated with resistant disease may be manipulated to therapeutic advantage.

Inhibition of DNA polymerase-alpha by ara-CMP in the presence of a regulatory protein extracted from human promyelocytic leukemic cells (HL-60).

Cytotoxicity of arabinofuranosylcytosine (ara-C) has been related in vitro to the inhibition of the DNA polymerase activities by arabinosylcytosine triphosphate (ara-CTP) and the incorporation of ara-C into the DNA where, acting as a chain terminator, it slows the chain elongation. Induced in vitro cellular resistance to ara-C was shown to be secondary to altered deoxycytidine (dCyd) kinase activity, dCyd deaminase activity, or deoxynucleotides triphosphates (dNTP) pools. Recent studies reported no differences of ara-C metabolism in cells obtained from leukemic patients at diagnosis and at relapse after ara-C therapy, suggesting that unknown cellular biochemical determinants may be involved in acquisition of ara-C resistance. Using dialysed crude extracts of leukemic cells obtained from patients at diagnosis, we observed variable inhibition of their DNA polymerase activities by arabinosylcytosine monophosphate (ara-CMP) at 2 mmol/L (0% to 50% inhibition). In similar conditions, ara-CMP reduced the polymerase activities of human thymus extract by 35% and 55% in extract of HL-60 cells (cultured human promyelocytic cells). The ara-CMP factor responsible for inhibition of DNA polymerase activity was nondialysable, heat labile, proteinase K sensitive, and has an estimated molecular mass of 30 kilodalton by gel filtration. After partial purification, this protein had no DNA polymerase RNA polymerase activities. In presence of the regulator and ara-CMP at 2 mmol/L, we observed no inhibition of the HL-60 3'----5' and 5'----3' exonucleases activities, suggesting the regulator interaction being mainly with the DNA polymerases in presence of ara-CMP. The relevance of the presence or absence of this protein regarding the cell sensitivity to ara-C is under investigation.

Interaction of 1-(5-phospho-beta-D-arabinofuranosyl)-5-substituted-uracils with thymidylate synthetase: mechanism-based inhibition by 1-(5-phospho-beta-D-arabinosyl)-5-fluorouracil.

A number of 1-(5-phospho-beta-D-arabinosyl)-5-substituted-uracils (ara-UMP's) have been examined as inhibitors of dTMP synthetase. As reversible inhibitors, all were substantially less potent than their 2'-deoxyribosyl counterparts. In the presence of 5,10-methylenetetrahydrofolate (CH2-H4folate), ara-FUMP caused a first-order, time-dependent inactivation of the enzyme. At 0 degrees C, kinetic studies indicated a reversible Kd of 3.6 micro M for the ara-FUMP-CH2-H4folate complex, and k = 0.22 min-1 for the subsequent inactivation. Spectral studies of the complex and its behavior toward protein denaturants demonstrate that its structure and stoichiometry are directly analogous to those which have previously been described for FdUMP. The significance of this finding with regard to prodrugs of ara-FU and the potential of ara-FU as a chemotherapeutic agent are discussed.

Synthesis and biological evaluation of neutral derivatives of 5-fluoro-2'-deoxyuridine 5'-phosphate.

5-Fluoro-5'-(2-oxo-1,3,2-oxazaphosphorinan-2-yl)-2'-deoxyuridine (1a) and 5-fluoro-5'-(2-oxo-1,3,2-dioxaphosphorinan-2-yl)-2'-deoxyuridine (1b) were prepared by reaction of 5-fluoro-2'-deoxyuridine (7a) and phosphoryl chloride with 3-amino-1-propanol and 1,3-propanediol, respectively. The thymidine analogues, 1c and 1d, were prepared similarly from thymidine. Compound 1b was synthesized in better yield from 13a and trimethylene phosphate with triphenylphosphine/diethyl azodicarboxylate as a condensing agent. Compounds 1a-d were resistant to degradation by 5'-nucleotidase, alkaline phosphatase, venom phosphodiesterase, and crude snake venom. None of these compounds were significantly biotransformed when incubated with mouse hepatic microsomal preparations in the presence of an NADPH-generating system. When administered intraperitoneally (ip) for 5 consecutive days, 1a was nearly as effective as 5-fluorouracil at prolonging the life spans of BDF1 mice implanted intraperitoneally with leukemia P-388. However, much larger dosages of 1a were required for optimal activity. Compound 1b administered similarly was only marginally effective. Neither 1a nor 1b was active against a P-388 mutant resistant to 5-fluorouracil.