Abstract
Analogs of (A2'p)2A core and ppp(A2'p)2A were chemically synthesized and their susceptibility to phosphodiesterase degradation and ability to either activate an endonuclease or to inhibit cell growth were determined. The absence of the internal 3'-OH groups ((3'dA2'p)2A) resulted in a 5-fold increase in stability, but also in a 10-fold decrease in activity, as measured by (a) activation of an endonuclease in cell-free extracts and inhibition of protein synthesis in intact cells by the 5'-triphosphate species and (b) inhibition of DNA synthesis in synchronized cells by the core analogs. An uncharged derivative of this analog containing two methylphosphotriesters, although significantly more stable, was even less active. Additional deletion of the terminal 3'-OH ((3'd A2'p)23'dA) resulted in a further 6-fold increase in stability (30-fold overall increase in stability), as well as approximately a 2-fold increase in ability to inhibit cell growth, as compared to the natural 2'5' A core. The analog lacking a terminal 2'-OH as well as lacking the internal 3'-OH group ((3'dA2'p)22'dA) showed an overall 15-fold increased stability, yet showed very little activity in inhibiting cell growth. The most stable (120-fold increased overall stability) as well as most active analog was a xyloadenosine analog of 2'5' A core, (xyloA2'p)2xyloA. These results show that modification of the 3'-terminal OH appears to be most important in increasing 2'-5' A core stability as well as biological activity. However, the mechanism of cell growth inhibition by these 2'-5' A core analogs may involve pathways different from those utilized by the 2'-5' A-dependent endonuclease.
Highlights
Ically synthesized and their susceptibility to phospho- triphosphate oligomers result in activationof a latent cellular diesterase degradation andability to either activatean endonuclease that degrades viral mRNA [4, 5, 11, 28,29,30] as endonuclease orto inhibit cell growth were deter- well as some cellular RNAs [4,31,32]
Treatment of cells with pp~(A2‘p)~A ahlasso been shown to result in subsequent inhibition of both RNA and DNA synthesis [16]
Martin et al [33]have shown that two or three 5”terminal terminal 3’-OH ((3’d A2’~)~3’dA) resulted in a fu6r-theprhosphates are necessary for activity of 2 ‘ 4 A in cell-free fold increase in stability (30-fold overall increase in extracts; the 5’-monophosphate as well as the dephosphorylstability), as well as approximately a 2-fold increase in ated “core” [12] were inactive
Summary
Chemicals-Cytidine 3’,5’-[5-”P]bisphosphate, 2-3 X IO6 Ci/mol, was from Amersham; [methyl-3H]thymidine, [2,8-3H]ATP. [methyl3H]dTTP, [4,5-3H]leucine, [5,6-3H]UTP, and Aquassure were from New England Nuclear; (A2’p)2A,poly[1]-poly(C)Sepharose, and T4RNA ligase (lot 880-11) were from P-L Biochemicals; (A3’p),A was from Boehringer Mannheim; (2’dA3’p)22’dAwas a generous gift from Genentech, Inc., S. Calcium Phosphate Co-precipitation Assay-Murine L, cells were seeded in Costar 24 well plates at 1.5 X lo5cells/well in Eagle’s minimal essential medium containing 10%fetal calf serum, and grown overnight before use. Solutions were aspirated off, cells were labeled for 90 min with [”HILeu (1 pCi/0.5 d/well), and trichloroacetic acid-precipitable radioactivity was determined as described by Hovanessian and Wood [16]. 2’-5‘A Analogs addition of fresh medium containing 10%serum, and a t this time, the interferon or the2’-5‘ A/core compounds to be tested were added to triplicate wells (24-wellplates) or quadruplet wells (96-wellplates). Reference Units, based on National Institutes of Health mouse interferon reference reagent G002-094-511
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
