SELECTIVITY OF F8-ACTINOMYCIN-D FOR RNA-DNA HYBRIDS AND ITS ANTILEUKEMIA ACTIVITY

Although many compounds have been found that bind to DNA in various wa ys and exhibit various biological activities, few compounds that speci fically bind to RNA or RNA:DNA hybrids are known, even though such com pounds are expected to have important biological properties. For examp le, one characteristic function of the retroviruses, which is generall y not found in eukaryotic cells, is the production of an RNA:DNA hybri d in the viral replication phase. If an agent is designed to bind only to an RNA:DNA hybrid, and not to DNA or to RNA, such an agent might b e able to inhibit specifically the RNase H activity of retroviral reve rse transcriptase, and therefore suppress viral replication. Actinomyc in D is known to bind to double-stranded DNA, but not to RNA, because steric hindrance between the 2-amino group of the phenoxazone ring and the 2'-hydroxyl group of RNA prevents intercalation of the compound. However, if the >C-H moiety at the 8-position of the phenoxazone ring is replaced by a >C-F, a possible hydrogen-bond acceptor, this analogu e (8-fluoro-actinomycin D, F8AMD) might be able to bind intercalativel y to an RNA:DNA hybrid by forming an additional hydrogen bond between F8 and the 2'-hydroxyl group of the guanosine ribose. To test this hyp othesis, the crystal structure of d(GAAGCTTC)(2)-F8AMD has been determ ined at 3.0 Angstrom resolution. Based on this crystal structure, a mo del in which F8AMD binds into the hybrid r(GAAGCUUC):d(GAAGCTTC) has b een built using molecular mechanics and dynamic methods. These structu ral studies indicate that F8AMD binds intercalatively to a B-form doub le-stranded DNA whereas the drug intercalates into an RNA:DNA hybrid t aking an A-form conformation. In the RNA:DNA hybrid complex, the F8 at om is located so as to be able to interact to an O2' hydroxyl group wi th either an O-H ... F hydrogen bond or H+... F- electrostatic interac tion. This interaction might stabilize the F8AMD molecule in the RNA:D NA hybrid. A binding study indicates that both actinomycin D (AMD) and F8AMD bind intercalatively not only to double-stranded DNAs, but also to RNA:DNA hybrids. Although the overall binding capacity of F8AMD (k = 4.5 x 10(5) M-1) is reduced slightly in comparison with AMD itself (k = 1.8 x 10(6) M-1), F8AMD tends to bind relatively more favorably t han AMD to the RNA:DNA hybrids. The drugs' effects on RNA synthesis in HeLa cells indicates that the binding capacities of AMD and F8AMD cor relates strongly to their RNA synthesis inhibitory activities. F8AMD r equired a concentration of 78 nM to inhibit RNA polymerase activity in HeLa cells by 50%, whereas AMD reached the same inhibitory lever at 3 0 nM. Surprisingly, F8AMD exhibits unique selectivity against leukemia cells as does another C8-derivatized AMD analogue, N8AMD. F8AMD inhib its 50% of leukemia cell growth at less than 1.0 nM whereas 10- to 130 -fold-higher drug concentrations are required to inhibit the growth of other tumor cell lines by 50%. The GI(50) value of F8AMD for leukemia cells is the lowest among the GI(50) values for all other AMD derivat ives tested. By contrast, AMD is quite potent and kills most cells at less than 50 nM concentration, but it does not show any selectivity fo r certain cell lines. This indicates that AMD should have very limited use as an antitumor agent. It is difficult to rationalize why F8AMD a nd N8AMD show such strong selectivity against leukemia cells. However, this study and our previous study (J. Am. Chem. Sec. 1994, 116, 7971) indicated that F8AMD and N8AMD tended to bind more favorably to RNA:D NA hybrids. Thus, the unique antileukemia selectivity shown by F8AMD a nd N8AMD might be caused by the agents binding to RNA:DNA hybrids rath er than to double-stranded DNA. (C) 1997 Elsevier Science Ltd.