Chain termination and inhibition of mammalian poly(A) polymerase by modified ATP analogues

We report the inhibition of mammalian polyadenylation by the triphosphate derivatives of adenosine analogues, 8-chloroadenosine (8-Cl-Ado) and 8-aminoadenosine (8-amino-Ado), which are under preclinical and clinical investigations for the treatment of hematological malignancies. The nucleotide substrate specificity of bovine poly(A) polymerase (PAP) towards C8-modified ATP analogues was examined using primer extension assays. Radiolabeled RNA primers were incubated with bovine PAP, and in the absence of ATP, no primer extension was observed with 8-Cl-ATP, whereas 8-amino-ATP resulted in chain termination. The effects of modified ATP analogues on ATP-dependent poly(A)-tail synthesis by bovine PAP also were determined, and incubation with analogue triphosphate resulted in significant reduction of poly(A)-tail length. To model the biochemical consequences of 8-Cl-Ado incorporation into RNA, a synthetic RNA primer containing a 3'-terminal 8-Cl-AMP residue was evaluated, and polyadenylation of the primer by bovine PAP with ATP was blocked completely. To explain these experimental observations and probe the possible structural mechanisms, molecular modeling was employed to examine the interactions between PAP and various ATP analogues. Molecular docking demonstrated that C8-modifications of ATP led to increased distance between the 3'-hydroxyl group of the RNA oligonucleotide terminus and the alpha-phosphate of ATP that render the molecules in an unfavorable position for incorporation into RNA. Similarly, C8-substitution with a chlorine or amino group at the 3'-terminal residue of RNA also inhibits further chain elongation by PAP. In conclusion, modified ATP analogues may exert their biological effects through polyadenylation inhibition, and thus may provide an RNA-directed mechanism of action for 8-Cl-Ado and 8-amino-Ado. (c) 2009 Elsevier Inc. All rights reserved.