The system will be unavailable due to maintenance on Thursday July 19 from 7:00-8:30 am ET.
Functionalizing Branched Peptides with Unnatural Amino Acids Toward Targeting HIV-1 RRE RNA and Microbials
Wynn, Jessica Elaine
MetadataShow full item record
The interaction of the protein Rev with Rev Response Element (RRE) RNA is critical to the HIV-1 life cycle as this complex is required for the export of singly-spliced and unspliced mRNAs from the nucleus to the cytoplasm. Disruption of this interaction is considered to be a powerful strategy towards the development of HIV-1 therapeutics. Therefore, we have developed several branched peptide libraries containing unnatural amino acids to target the high-affinity binding site of RRE RNA (RRE IIB), with the idea that branching in peptides can provide multivalent contacts with folded RNA structures and boost binding affinity and selectivity for the target. Unnatural amino acids were incorporated into the library design to encourage non-canonical interactions with the RNA and to improve proteolytic stability. The on-bead high-throughput screening of our first branched peptide library (46,656 sequences) against HIV-1 RRE RNA generated hit peptides with binding affinities in the low micromolar range. We demonstrated that branching in the peptide is required for efficient binding and selectivity towards the RNA, and that the peptides bind a large surface area of RRE IIB. Introduction of boronic acids into branched peptides boosted selectivity of the peptides for RRE IIB, and proved to be a novel and tunable mode of binding towards RNA. Additionally, we revealed that these branched peptide boronic acids (BPBAs) were cell permeable and non-toxic. One BPBA (BPBA3) bound RRE IIB selectively and was able to inhibit HIV-1 replication in vitro, revealing enzymatic cleavage of the RNA upon binding. A second generation BPBA library that introduced acridinyl lysine as an intercalator (4,096 sequences) was screened against RRE IIB. Several hit compounds bound in the low nanomolar regime, and a significant number of compounds inhibited HIV-1 replication in vitro. These BPBAs were also found to severely inhibit the microbial growth of bacteria and fungus, with MICs as low as 1 µg/mL against Staphylococcus aureus, Candida albicans, and Escherichia coli. These compounds were also found to significantly inhibit biofilm formation and growth, and were non-hemolytic. High-throughput screening of a third generation BPBA library containing all unnatural amino acids (46,656 sequences) revealed several hits that bound RRE IIB RNA in the nanomolar range. Sequence motifs present in the hit peptides suggested that the location and composition of amino acids within the branched peptide structure were important for recognizing the RNA target. In particular, lead compounds 2C5 and 4B3 demonstrated selectivity towards RRE, and footprinting experiments combined with SHAPE experiments revealed different interactions of the peptides with the RNA Toxicity assays revealed no impact on cell viability for the majority of hit sequences tested up to 100 µM, and several compounds also demonstrated inhibition of HIV-1 replication.
- Doctoral Dissertations