Investigation of small molecule - SL1 RNA interactions and implications in drug design targeted at HIV-1 genomic dimer maturation.

摘要

Like all retroviruses, the HIV-1 virus contains two copies of the genome as a RNA dimer. The study of genomic dimerization has been facilitated by the use of short RNA oligonucleotides containing the dimer initiation site (DIS) that dimerize spontaneously in vitro. In HIV-1, DIS maps to the first stem-loop (SL1) of the packaging signal. On the basis of the palindromic nature of the apical loop of SL1, a kissing loop model has been proposed. First, a metastable kissing dimer is formed via a loop-loop interaction and then converted into a more stable extended dimer by the viral nucleocapsid protein (NCp7). This dimerization process is believed to mimic the in vivo RNA maturation. The formation of the mature dimer is correlated with viral infectivity. During experimental screening of potential inhibitors that can block dimer maturation, we unexpectedly discovered a small molecule activator, Lys-Ala-7-amido-4-methylcoumarin (KA-AMC), that facilitates this process instead. We wish to understand the mechanism of action of KA-AMC and have the following specific aims: (1) determine the structure-activity relationship (SAR) of the family of the activators with native gel shift assay, (2) determine the binding affinity of the activators to the SL1 RNA utilizing the fluorescence emission of AMC analogs, and (3) structurally characterize activator -- SL1 RNA interactions using nuclear magnetic resonance (NMR). Results from the SAR studies show that the O1 in the coumarin scaffold is essential for activity, while the binding affinity can be further improved by the inclusion of guanidine group and the addition of positive charges. NMR studies suggest KA-AMC stacks with aromatic bases of the RNA while the side chain interacts via hydrogen bonding and electrostatic interactions and binds preferentially to the SL1 RNA. Although KA-AMC is not an inhibitor for dimer maturation, it can still be developed as an antiretroviral agent in one of two ways. The more obvious way is to use our growing knowledge of KA-AMC analogues binding features to design a compound that would bind and inhibit dimer maturation rather than promote dimer maturation. The less obvious, but potentially more effective, way is by inducing early maturation of the genomic dimer prior to budding.