Potential therapeutics for multiple myeloma and leukemia: The effects of C8-modified adenosine analogs on nucleic acid biochemistry.

摘要

C8-Modified adenosine analogs, 8-chloroadenosine (8-Cl-Ado), 8-aminoadenosine, and 8-azidoadenosine, are potential new treatments for multiple myeloma and leukemia. Treatment of cells with these analogs produced an accumulation of the corresponding triphosphate derivatives as the active metabolites. Further analysis indicated that 8-Cl-Ado produced a decrease in global RNA levels, and was incorporated into cellular RNA.; An 8-Cl-Ado phosphoramidite and controlled-pore glass support derivative were prepared and used to synthesize RNA containing 8-Cl-AMP at internal and 3'-terminal sites, respectively. Circular dichroism spectroscopy indicated that 8-Cl-AMP modified RNA duplexes formed A-form helices, and UV thermal denaturation analysis revealed that 8-Cl-AMP residues decreased duplex stability by ∼5 kcal/mole, which is similar to a U:U mismatch.; To examine its effects on DNA synthesis, 8-chloro-2'-deoxyadenosine (8-Cl-dAdo) was incorporated into DNA oligonucleotides, which then were assayed with Klenow fragment of DNA Polymerase I (KF-). Single nucleotide insertion assays using KF- inserted TTP opposite 8-Cl-dAdo template sites, but with decreased efficiency relative to deoxyadenosine. The triphosphate analog, 8-Cl-dATP, also was incorporated opposite thymidine approximately two-fold less efficiently than dATP. Running-start primer extensions with KF- resulted in polymerase pausing at 8-Cl-dAdo template sites during DNA synthesis.; The effects of 8-modified adenosine analogs were also examined for RNA transcription and polyadenylation. Our results suggest that 8-modified analogs inhibit polyadenylation by yeast poly(A) polymerase. 8-Amino-ATP, 8-azido-ATP and 8-aza-ATP all produced chain termination, and no primer extension was observed with 8-Br-ATP and 8-Cl-ATP. ATP-dependent poly(A)-tail synthesis was also examined, and C8 modified ATP analogs all reduced poly(A)-tail length to varying degrees. The consequences of 8-Cl-Ado incorporation into RNA were modeled using a synthetic RNA primer containing a 3'-terminal 8-Cl-AMP, which was assayed for polyadenylation. 3'-Terminal 8-Cl-AMP sites abolished extension by yeast poly(A) polymerase entirely. 2-D NMR spectroscopy experiments on 8-Cl-AMP and 8-amino-AMP were used to examine the potential structural mechanism of inhibition. The results suggest that C-8 substitution may shift the ribose conformation equilibrium to favor C2 '-endo over C3'- endo.