ABSTRACT
Ribosomal frameshifting, a process whereby a translating ribosome is diverted from one reading frame to another on a contiguous mRNA, is an important regulatory mechanism in biology and an opportunity for therapeutic intervention in several human diseases. In HIV, ribosomal frameshifting controls the ratio of Gag and Gag-Pol, two polyproteins critical to the HIV life cycle. We have previously reported compounds able to selectively bind an RNA stemloop within the Gag-Pol mRNA; these compounds alter the production of Gag-Pol in a manner consistent with increased frameshifting. Importantly, they also display antiretroviral activity in human T-cells. Here, we describe new compounds with significantly reduced molecular weight, but with substantially maintained affinity and anti-HIV activity. These results suggest that development of more "ligand efficient" enhancers of ribosomal frameshifting is an achievable goal.
Subject(s)
Frameshift Mutation/genetics , HIV-1/genetics , RNA, Viral/metabolism , Humans , LigandsABSTRACT
Dynamic Combinatorial Chemistry (DCC) has proven to be a reliable method for identifying hit compounds for target nucleic acid (DNA and RNA) sequences. Typically, these hit compounds are subjected to a lengthy process of optimization via traditional medicinal chemistry. Here, we examine the potential of DCC to also generate and test variations on a hit compound as a method for probing the binding site of an RNA-targeted compound. Specifically, we demonstrate that addition of linker dithiols to a disulfide library containing a known binder to the HIV-1 frameshift-stimulatory RNA (a critical regulator of the HIV life cycle) can yield a mixture of new bridged structures incorporating the dithiol, depending on dithiol structure. Equilibration of this library with the HIV FSS RNA resulted in selection of the original disulfide in preference to bridged structures, suggesting incorporation of the bridge is not compatible with this particular binding site. Application of this strategy to other RNA targets should allow for rapidly profiling the affinity of modified compounds.
Subject(s)
Oligopeptides/chemistry , RNA, Viral/chemistry , Combinatorial Chemistry Techniques , Disulfides/chemical synthesis , Disulfides/chemistry , HIV-1/genetics , Oligopeptides/chemical synthesis , Sulfhydryl Compounds/chemical synthesis , Sulfhydryl Compounds/chemistryABSTRACT
The ever-growing number of RNA species that are recognized as having a role in human disease is driving a demand for novel molecular probes and therapeutics. Producing sequence-selective RNA-binding molecules remains a substantial challenge, however. One approach that has been successful in producing molecules with high affinity and specificity for disease-relevant RNAs is the use of dynamic combinatorial chemistry, a fragment-based method in which fragments combine reversibly in the presence of the target. We describe methods for the design, synthesis, and screening of dynamic combinatorial libraries targeting RNA.