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Analysis of the structure-anti-HIV activity correlations of halo dideoxynucleosides (ddN's) in the public domain was accomplished through computer substructure searching, retrieval and sorting of in vitro anti-HIV data. In the survey, selectivity index (ratio of cytotoxicity to the potency in inhibiting HIV replication in vitro) was used to rank compounds in congeneric groups. Factors contributing to the anti-HIV activity, e.g. the nature and location of the halogen on the sugar or the base and its stereochemical configuration, could not be generalized for all the halogenated ddN's. Conclusions were drawn for specific classes within the pyrimidine and purine series, with compounds further divided into halo substitutions at the sugar 2',3',4'-positions or in the pyrimidine or purine ring systems. At the 3'-position, only a fluoro substitution enhanced the activity of the dideoxypyrimidine nucleosides. A 2'-ara fluoro substituent increased the activity of purine ddN's but decreased activity of pyrimidine ddN's. Halogenation of the side chain in acyclic adenine and 2,6-diaminopurine nucleosides improved their anti-HIV activity. Halo substitution at the 6- or 2'-ara position of selected purine ddN's resulted in compounds with increased lipophilicity, chemical stability and retention of anti-HIV activity. The number of halodideoxynucleosides tested as anti-HIV reagents suggested that this class of compounds is well studied.

Antisera elicited by immunization of rabbits with 4'-aminomethyl-trioxsalen (AMT)-modified poly(A,U) complexed with methylated bovine serum albumin was characterized in competition radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). AMT-poly(A,U) was over 10,000-fold more reactive than unmodified poly(A,U) or AMT alone. The antiserum cross-reacted to varying extents with AMT-modified-RNA's and -DNA's. The presence of AMT-uridine usually assured strong reactivity. The amino group of AMT contributed to antibody binding to a small degree. Binding was not significantly affected by high ionic strength, suggesting that binding does not involve ion pair formation. Murine encephalomyocarditis virus replicative intermediates, as well as cellular RNA and DNA were modified by psoralen in intact cells, suggesting that EMCV RNA and cellular RNA's in intact cells possess detectable stretches of base pairs. The antibodies described here will be useful in studying the secondary and tertiary structure of RNA's in vitro and in intact cells.

5'-O-Monophosphoryladenylyl(2' goes to 5')adenylyl-(2' goes to 5')adenosine [(2' goes to 5')(pA)3] antagonizes the protein synthesis inhibitory effects of 5'-O-triphosphoryladenylyl-(2' goes to 5')adenylyl(2' goes to 5')adenosine by preventing activation of the (2' goes to 5')oligo(a)-activated ribonuclease which degrades mRNA. The oligoribonucleotide (2' goes to 5')(pA)3 also antagonizes the translational inhibitory capacity of poly(I).poly(C) in extracts of interferon-treated L cells, suggesting that (2' goes to 5')oligo(A) is the primary mediator of the protein synthesis inhibitory effects of poly(I).poly(C) in this cell-free system.

A competition immunoenzymometric assay for 2',5'-oligoadenylate was developed and employed to measure the interferon-inducible enzyme 2',5'-oligoadenylate synthetase in cell extracts. Microtiter plates coated with a novel conjugate of p5'A2'p5'A2'p5'A and N-(2-aminoethyl)-carbamylmethylated-Ficoll (AECM-Ficoll) bound rabbit polyclonal or mouse monoclonal antibody directed against 2',5'-oligoadenylate. Binding was inhibited by soluble 2',5'-oligoadenylate. Estimates of 2',5'-oligoadenylate concentrations based on inhibition of antibody binding compared favorably with those obtained using a protein synthesis inhibition assay. Low concentrations of 2',5'-oligoadenylate synthesized in vitro by extracts of human peripheral mononuclear cells were conveniently estimated using less than or equal to 10(6) cells. Virtually identical results were obtained when either total extract or synthetase bound to poly(I) . poly(C)-agarose was used for the in vitro incubation. When peripheral mononuclear cells were incubated in vitro with interferon, the normally low levels of 2',5'-oligo(A) synthetase rose dramatically. The assay was employed to measure synthetase levels in peripheral mononuclear cells isolated from patients with systemic lupus erythematosus. Some of these patients were found to have elevated levels of 2',5'-oligoadenylate synthetase.

2',5'-Oligoadenylate synthetase was induced 3-2000-fold in spleen, liver, kidney and brain of NIH Swiss mice injected intravenously with 2-200 micrograms of the misaligned dsRNA, poly(I).poly(C12,U). Levels of 2',5'-oligoadenylates extracted from these tissues were also elevated, although the amount of 2',5'-oligoadenylates extracted did not correlate directly with the amount of enzyme present. These results suggest that double-stranded portions of the misaligned polymer survived intracellularly and activated the 2',5'-oligoadenylate synthetase, and that the level of dsRNA may contribute to the control of 2',5'-oligoadenylate metabolism.

Raman spectra of model compounds and of 2',5'-oligoadenylates in D2O were utilized to assign the Raman bands of 2',5'-oligoadenylates. The Raman spectra of A2'pA2'pA, pA2'pA2'pA, and pppA2'pA2'pA contained features that were similar to those of adenosine, adenosine 5'-monophosphate (AMP), and adenosine 5'-triphosphate, respectively. When AMP and pA2'pA2'pA were titrated from pH 2 to 9, the normalized Raman intensity of their ionized (980 cm-1) and protonated (1080 cm-1) phosphate bands revealed similar pKa's for the 5'-monophosphates. The Raman spectrum of pA2'pA2'pA was altered slightly by elevations in temperature, but not in a manner supporting the postulate that 2-5A possesses intermolecular base stacking. Major differences in the Raman spectrum of 2',5'- and 3',5'-oligoadenylates were observed in the 600-1200-cm-1 portion of the spectrum that arises predominately from ribose and phosphate vibrational modes. Phosphodiester backbone modes in A3'pA3'pA and pA3'pA3'pA produced a broad band at 802 cm-1 with a shoulder at 820 cm-1, whereas all 2',5'-oligoadenylates contained a major phosphodiester band at 823 cm-1 with a shoulder at 802 cm-1. The backbone mode of pppA2'pA2'pA contained the sharpest band at 823 cm-1, suggesting that the phosphodiester backbone may be more restrained in the biologically active, 5'-triphosphorylated molecule. The Raman band assignments for 2',5'-oligoadenylates provide a foundation for using Raman spectroscopy to explore the mechanism of binding of 2',5'-oligoadenylates to proteins.

A rapid and convenient assay for adenylyl(2′ → 5′)adenosine(A2′p5′A) or adenylyl(3′ → 5′)adenosine(A3′p5′A) phosphodiesterase activities is described. The dinucleotides A3′p5′A and A2′p5′A were labeled to a high specific activity by means of a catalytic-exchange procedure. Degradation studies of each of these labeled dinucleotides showed an asymmetrical distribution of label between the two adenine bases. Enzymatic degradation of [ 3 H]A3′p5′A or [ 3 H]A2′p5′A could be quantitated by first digesting the reaction products with bacterial alkaline phosphatase and then adding a slurry of DEAE-Sephadex. Under conditions described, adenosine did not adsorb to the resin, whereas dinucleotides as well as AMP did adsorb. As a consequence, when liquid scintillation fluid was added to the DEAE-Sephadex reaction mixture slurry, the radioactivity of the dinucleotides and AMP was severely quenched. This permitted a direct estimation of the amount of adenosine liberated during the phosphodiesterase degradation and subsequent alkaline phosphatase digestion. This method was applied to the measurement of A2′p5′A degrading activities in extracts of mouse L cells. Extracts from control mouse L cells were as active in degrading A2′p5′A as extracts from interferon pretreated cells.

A number of oligonucleotides have been synthesized and evaluated as antagonists of the protein synthesis inhibitory effects of 2-5A in order to gain information on the oligonucleotide structural features involved in binding to the 2-5A-activated endoribonuclease. The base moieties, the ribose-phosphate backbone and the 5′-monophosphate group of p5′A2′p5′A2′p5′A are strategic regions for binding to the endonuclease; however, rather extensive modification of the 2′-terminal ribose unit is possible without adversely affecting interaction with the endonuclease. Analogues of p5′A2′p5′A2′p5′A, p5′A2′p5′ A2′p5′A2′p5′A or ppp5′A2′p5′A2′p5′A2′p5′A, in which the 2′-terminal ribose has been modified to an N-hexylmorpholine ring have significantly enhanced activity as antagonists of 2-5A action or as inhibitors of protein synthesis. Such analogues are much more resistant to degradation by the phosphodiesterase activity found in L cell extracts. These analogues of p5′A2′p5′A2′p5′A are also effective antagonists of the inhibition of protein synthesis caused by dsRNA in extracts of interferon-treated L cells, and their use demonstrates that 2-5A is the primary mediator of inhibition caused by dsRNA. In accord with this finding, the synthetic dsRNA, poly(A). poly(dUfl), an activator of the protein P 1 kinase but not the 2-5A synthetase, was a potent inhibitor of protein synthesis in rabbit reticulocyte lysates, but was without discernable activity in extracts of interferon-treated L cells. Thus, phosphorylation of eIF-2α and/or protein does not appear to be a sufficient condition for protein synthesis inhibition in extracts of interferon-treated L cells.

Publisher Summary This chapter presents an assay for 2',5'-oligoadenylate synthetase that provides reproducibly high yields of (2'-5')-oligo(A) when crude cell extracts are employed, and provides an opportunity for variation of nucleic acid activator. This method is based on the binding of (2'-5')-oligo(A) synthetase from crude extracts to 2',5'-ADP-Sepharose at 4°. After binding, the resin is washed to remove unbound molecules, and the resin bound synthetase is incubated at 30° in the presence of ATP and a nucleic acid activator. The (2'-5')-oligo(A) in the reaction supernatant is then assayed by its ability to inhibit cell free protein synthesis. This method is a convenient and quantitative alternative to the assay of (2'-5')-oligo(A) synthetase from crude extracts on poly(I) . poly(C)-Sepharose. In extracts that contain high levels of inactivating enzymes, both methods may be superior to a solution assay in that both resin assays afford a high degree of purification in the binding step.

The enzyme oligo(2′–5′)adenylate synthetase, when activated by double-stranded RNA, polymerizes ATP into the novel oligonucleotide (2′–5′)ppp(Ap)nA. We describe conditions for assay of this enzyme in crude extracts of a human lymphoblastoid cell line, Namalwa. The production of (2′–5′)ppp(Ap)nA by Namalwa extracts was 3–5 times greater than the production by extracts of interferon pretreated mouse L cells, and 700 fold higher than the production by extracts of untreated mouse L cells. The relatively high level of oligo(2′–5′)adenylate synthetase in Namalwa cells was not attributable solely to their constitutive secretion of low levels of interferon. Analysis of the size distribution of the oligomers formed at different times suggested that the enzyme can add ATP to a free pppApA. Infection by Newcastle disease virus or treatment with interferon raised the apparent synthetase levels only marginally. Experiments that employed antibody to interferon suggested that the interferon must be externalized from the NDV-infected cell to induce maximal synthetase levels.

To define the epitopes involved in binding anti-oligonucleotide antibodies, several hybridomas producing monoclonal antibodies directed against 2',5'-oligoadenylate were established. A solid-phase enzyme-linked immunoassay that employed microtiter wells coated with Ficoll-2',5'-oligoadenylate conjugates proved useful in screening and characterizing hybridoma supernatants. Control experiments demonstrated that the conjugates were irreversibly adsorbed to polystyrene wells under the conditions employed in the assay. Reactivity of monoclonal antibodies with numerous analogues of 2',5'-oligoadenylate was measured by using a competition assay. Several monoclonal antibodies originating from different mice immunized with the same or different immunogens possessed distinctive fine specificities. At least one 2',5'-phosphodiester bond was important in forming each epitope, suggesting that the ribose phosphate backbone is a critical element in defining an antigenic domain of an oligonucleotide. The purine bases were also important, and modification of the bases had varied effects on the extent of antibody recognition. The length of the oligonucleotide and the nature of the termini were also of some importance. In several instances the modification created by linkage of 2',5'-oligoadenylate to carrier protein also contributed to the determinant. The monoclonal antibody most specific for 2',5'-oligoadenylates was relatively insensitive to ionic strength. In contrast, a monoclonal antibody with a 2',5'-oligopurine specificity appeared to bind 2',5'-oligoadenylate through one ion pair, whereas the binding of a monoclonal antibody with a low degree of base specificity appeared to bind through two ion pairs. The results demonstrated that 2',5'-linked oligoadenylate-protein complexes possess at least three distinct oligonucleotide-related antigenic surfaces that can be recognized with high apparent affinity by monoclonal antibodies. A model for the three epitopes is presented.