Biomedical subjects

R W Sobol

Publications and source records attributed to R W Sobol.

25 records · Page 2Linked to original sources

PubMed1990

Phosphorothioate analogues of (2'-5')(A)4: agonist and antagonist activities in intact cells.

Metabolically stable phosphorothioate tetramer analogues of (2'-5')(A)n with Rp and/or Sp chirality in the 2'-5'-phosphodiester linkages constitute a new class of antiviral agents since they mimic the effects of interferons. Three of the diastereomeric 5'-monophosphates (i.e., pRpRpRp, pSpRpRp, and pRpSpSp) bind to and activate RNase L from extracts of HeLa cells. However, the pSpSpSp (2'-5')-(A)4-phosphorothioate is unique in that it binds to, but cannot activate, RNase L to cleave rRNA. When microinjected into the cytoplasm of HeLa cells followed by virus infection, the pRpRpRp, pSpRpRp, and pRpSpSp (2'-5')(A)4-phosphorothioates demonstrate antiviral activity, as does (2'-5')(A)4ox-red, an active (2'-5')(A)n analogue. When microinjected simultaneously with (2'-5')(A)nox-red, an active the pSpSpSp (2'-5')(A)4-phosphorothioate inhibits activation of RNase L in HeLa cells, thereby blocking direct protection of vesicular stomatitis virus. The agonist and antagonist properties of pRpRpRp and pSpSpSp, respectively, are transient probably as a consequence of the hydrolysis of the 5'-monophosphate and formation of the less active (2'-5')(A)4-phosphorothioate cores. The possible use of these (2'-5')(A)4-phosphorothioates as tools for dissecting the biological significance of the (2'-5')(A)n system or in antiviral chemotherapy is discussed.

Antiviral Agents↗

PubMed1989

Phosphorothioate and cordycepin analogues of 2',5'-oligoadenylate: inhibition of human immunodeficiency virus type 1 reverse transcriptase and infection in vitro.

Natural antiviral activity can be mediated by the interferon-induced synthesis of 2',5'-oligoadenylates (2-5As) and subsequent RNase L activation by these molecules. Analogues of 2-5A that are biologically active and metabolically stable were synthesized and analyzed for antiviral activity against the human immunodeficiency virus type 1 (HIV-1). Replacement of the 3' hydroxyl group of the adenosine moieties of 2-5A with hydrogen atoms (i.e., cordycepin analogues of 2-5A) converted authentic 2-5A trimer into anti-HIV-1 agents in vitro. These cordycepin analogues of 2-5A also inhibited partially purified HIV-1 reverse transcriptase. Introduction of chirality into the 2',5'-phosphodiester internucleotide linkages or 5'-phosphate moieties of the 2-5A molecule (i.e., phosphorothioate analogues of 2-5A) converted authentic 2-5A into more potent inhibitors of HIV-1 reverse transcriptase. However, these phosphorothioate 2-5As demonstrated little or no anti-HIV-1 activity in vitro. Thus, some analogues of 2-5A may form a class of anti-HIV-1 drugs with possible pleiotropic activities that include activation of latent RNase L and inhibition of reverse transcription.

Adenine Nucleotides↗

PubMed1988

2- and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins.

The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)

2',5'-Oligoadenylate Synthetase↗

PubMed1988

2- and 8-azido photoaffinity probes. 2. Studies on the binding process of 2-5A synthetase by photosensitive ATP analogues.

The photoaffinity probes [gamma-32P]2-azidoATP (2-N3ATP) and [alpha-32P]8-azido-ATP (8-N3ATP) were used to investigate the binding of ATP to highly purified 2-5A synthetase. 2-N3ATP and 8-N3ATP are substrates for 2-5A synthetase [Suhadolnik, R.J., Karikó, K., Sobol, R.W., Jr., Li, S.W., Reichenbach, N.L., & Haley, B.E., preceding paper]. In this study we show that 2- and 8-N3ATP are competitive inhibitors of the enzymatic conversion of ATP to 2-5A. Ultraviolet irradiation results in the photoinsertion of 2-N3ATP and 8-N3ATP into the enzyme. The covalent photoinsertion of [alpha-32P]8-N3ATP into the 2-5A synthetase is proportional to the inactivation of the enzyme as UV irradiation is increased. Photolabeling of 2-5A synthetase is saturated at 1.5 mM 2-N3ATP and 2.0 mM 8-N3ATP. Computer analysis of the curvilinear Scatchard plots of the 2-5A synthetase suggests the presence of high-affinity and low-affinity binding sites that may correspond to the acceptor and the 2'-adenylation sites of the enzyme. The competition of nucleotides for the covalent photoinsertion of 8-N3ATP into the binding site(s) of the synthetase was as follows: ATP greater than 2'dATP = 3'dATP greater than CTP greater than ITP greater than AMP greater than NAD+ greater than UTP greater than UMP greater than CMP. Photoinsertion of 8-N3ATP into 2-5A synthetase increases with the addition of poly(rI).poly(rC).(ABSTRACT TRUNCATED AT 250 WORDS)

2',5'-Oligoadenylate Synthetase↗

PubMed1987

Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatically synthesized 2',5'-phosphorothioate dimer and trimer: unequivocal structural assignment and activation of 2',5'-oligoadenylate-dependent endoribonuclease.

In continued studies to elucidate the requirements for binding to and activation of the 2',5'-oligoadenylate-dependent endoribonuclease (RNase L), chirality has been introduced into the 2',5'-oligoadenylate (2-5A, p3An) molecule to give the Rp configuration in the 2',5'-internucleotide backbone and the Sp configuration in the alpha-phosphorus of the pyrophosphoryl moiety of the 5'-terminus. This was accomplished by the enzymatic conversion of (Sp)-ATP alpha S to the 2',5'-phosphorothioate dimer and trimer by the 2-5A synthetase from lysed rabbit reticulocytes. The most striking finding reported here is the ability of the 2',5'-phosphorothioate dimer 5'-triphosphate (i.e., p3A2 alpha S) to bind to and activate RNase L. p3A2 alpha S displaces the p3A4[32P]pCp probe from RNase L with an IC50 of 5 X 10(-7) M, compared to an IC50 of 5 X 10(-9) M for authentic p3A3. Further, p3A2 alpha S activates RNase L to hydrolyze poly(U)-3'-[32P]pCp (20% at 2 X 10(-7) M), whereas authentic p3A2 is unable to activate the enzyme. Similarly, the enzymatically synthesized p3A2 alpha S at 10(-6) M activated RNase L to degrade 18S and 28S rRNA, whereas authentic p3A2 was devoid of activity. p3A3 alpha S was as active as authentic p3A3 in the core--cellulose and rRNA cleavage assays. The absolute structural and configurational assignment of the enzymatically synthesized p3A2 alpha S and p3A3 alpha S was accomplished by high-performance liquid chromatography, charge separation, enzymatic hydrolyses, and comparison to fully characterized chemically synthesized (Rp)- and (Sp)-2', 5'-phosphorothioate dimer and trimer cores.(ABSTRACT TRUNCATED AT 250 WORDS)

2',5'-Oligoadenylate Synthetase↗

PubMed1987

Phosphorothioate analogues of 2',5'-oligoadenylate. Activation of 2',5'-oligoadenylate-dependent endoribonuclease by 2',5'-phosphorothioate cores and 5'-monophosphates.

The preceding paper in this issue described the synthesis and structural elucidation of the phosphorothioate analogues of 2',5'-oligoadenylate (2-5A) dimer and trimer cores [Karikó, K., Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (preceding paper in this issue)]. In this report, the binding and activation processes of 2-5A-dependent endoribonuclease (RNase L) have been examined by using four diastereomeric 2',5'-phosphorothioate trimer core analogues and their 5'-monophosphates. These 2',5'-phosphorothioates have revealed a distinct separation of the structural parameters that govern binding vs activation of RNase L. Radiobinding assays have demonstrated that extensive stereochemical modification of the internucleotide linkages of 2-5A is possible without adversely affecting its ability to bind to RNase L. However, a marked difference was observed in the activation of RNase L by the stereochemically modified 2-5A molecules as determined in core--cellulose and rRNA cleavage assays. Three of the four 2',5'-phosphorothioate trimer cores (with RpRp,SpRp, and RpSp internucleotide linkages) are the first 2-5A core molecules able to activate RNase L. For example, the RpRp, SpRp, and RpSp diastereoisomers activate RNase L to hydrolyze poly(U)-3'-[32P]pCp 65%, 20%, and 15%, respectively, at 5 X 10(-5) M. The SpSp diastereomer cannot activate RNase L. The order of RNase L activation was the same for the core analogues and their 5'-monophosphates (RpRp greater than SpRp greater than RpSp).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗

PubMedDate not supplied

Heterogeneous nuclear RNA from hairy cell leukemia patients activates 2',5'-oligoadenylate synthetase.

Interferon treatment of cells induces double-stranded RNA (dsRNA)-dependent 2',5' oligoadenylate (2-5A) synthetase, an enzyme which has been implicated in the mechanism of growth arrest in tumour cells. Since interferon (IFN) can inhibit the growth of cells that are not infected with virus, natural non-viral dsRNAs should be present in these cells which can activate 2-5A synthetase. If such nuclear dsRNAs are associated with the mechanism of growth control, cells inherently sensitive to growth inhibition by IFN should contain significant levels of 2-5A synthetase-activating dsRNAs. We measured the ability of size fractionated nuclear dsRNAs isolated from patients with hairy cell leukemia (HCL) to activate purified 2-5A synthetase. Peripheral blood mononuclear cells from HCL patients were utilized because of the inherent sensitivity of these patients to IFN treatment. The heterogeneous nuclear RNA fraction from four out of five HCL patients showed high levels of 2-5A synthetase-activating dsRNAs. The 2-5A formed contained biologically active trimers, tetramers, pentamers and hexamers as demonstrated by HPLC analysis and their ability to activate RNase L. In contrast, the nuclear RNA fraction from three out of four healthy controls were unable to activate 2-5A synthetase. These results indicate that natural, nuclear dsRNAs inherently exist in IFN-sensitive cells and imply that these molecules may play a role in the inhibition of cellular growth.

2',5'-Oligoadenylate Synthetase↗