Structural and chemical studies of RNA as anti-cancer target and signaling molecule

Title from PDF of title page, viewed on January 6, 2012Dissertation advisor: Simon H. FriedmanVitaIncludes bibliographic references (p. 163-183)Thesis (Ph.D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2011Ribonucleic acids have been found to have a major role in causation and propagation of diseases. Targeting such therapeutically important RNA can be a method of treatment of various diseases. Two such therapeutically important RNA discussed in this dissertation are telomerase and extracellular microRNA. Telomerase is a ribonucleoprotein whose role has been implicated in cancer. Telomerase catalyses telomere extension by using a portion of its RNA as a template. During this process a RNA/DNA heteroduplex is formed. We have attempted to inhibit telomerase by targeting this heteroduplex using nucleic acid binders such as intercalators. In this dissertation, we sought to understand the mechanism of telomerase inhibition by intercalators, using a structural approach. The affinity of binding of ethidium bromide to the RNA/DNA heteroduplex was examined by photocrosslinking ethidium azide to the telomerase RNA and using a primer extension assay. Ethidium azide had a greater affinity for the RNA/DNA heteroduplex compared to the telomerase RNA alone. Designing specific inhibitors using ethidium bromide as the scaffold proved to be challenge due to the off target binding to other nucleic acids. Thus, the next part of the dissertation dealt with identifying weak inhibitors of telomerase and then increasing the affinity for the target enzyme by developing interactions with the protein portion of the enzyme. Naproxen was used as a scaffold to introduce functionalities which would interact with the inner surfaces of the protein portion of the enzyme. Peptide libraries using naproxen as the scaffold having varying lengths and motifs were designed. The synthesized derivatives of naproxen were tested for their ability to inhibit telomerase in order to obtain a molecule which was a better inhibitor of telomerase than naproxen alone. The final portion of the dissertation deals with identifying and targeting extracellular microRNA released from cancerous cells. MicroRNA are small (22-25 nucleotides) RNA duplex which regulate gene expression by cleaving the target mRNA or by blocking protein formation. We hypothesized that microRNA released in the extracellular fluid behave as signaling molecules. Extracellular microRNA were observed in the media of culturing carcinoma cells. Thus identifying and targeting these extracellular species can be another strategy in cancer therapy.Introduction and literature review -- Determining the mechanism of telomerase inhibition by ethidium azide -- Designing of peptide libraries with a weak intercalator as the scaffold -- Design of naproxen based libraries with varying lengths and point of attachment of naproxen -- Introduction: micro RNA processing, role in diseases and detection techniques -- Experiment to observe and identify small RNA in the extracellular media of culturing cells -- Design of media to generically capture extracellular micro RNA -- Designing of a disc capable of specifically capturing extracellular micro RNA from the media of culturing cells -- Summary and conclusion

Surface-Matrix Screening Identifies Semi-specific Interactions that Improve Potency of a Near Pan-reactive HIV-1-Neutralizing Antibody

SUMMARY Highly effective HIV-1-neutralizing antibodies could have utility in the prevention or treatment of HIV-1 infection. To improve the potency of 10E8, an antibody capable of near pan-HIV-1 neutralization, we engineered 10E8-surface mutants and screened for improved neutralization. Variants with the largest functional enhancements involved the addition of hydrophobic or positively charged residues, which were positioned to interact with viral membrane lipids or viral glycan-sialic acids, respectively. In both cases, the site of improvement was spatially separated from the region of antibody mediating molecular contact with the protein component of the antigen, thereby improving peripheral semi-specific interactions while maintaining unmodified dominant contacts responsible for broad recognition. The optimized 10E8 antibody, with mutations to phenylalanine and arginine, retained the extraordinary breadth of 10E8 but with ~10-fold increased potency. We propose surface-matrix screening as a general method to improve antibodies, with improved semi-specific interactions between antibody and antigen enabling increased potency without compromising breadth

Thermoresponsive Polymer Nanoparticles Co-deliver RSV F Trimers with a TLR-7/8 Adjuvant

Structure-based vaccine design has been used to develop immunogens that display conserved neutralization sites on pathogens such as HIV-1, respiratory syncytial virus (RSV), and influenza. Improving the immunogenicity of these designed immunogens with adjuvants will require formulations that do not alter protein antigenicity. Here, we show that nanoparticle-forming thermoresponsive polymers (TRP) allow for co-delivery of RSV fusion (F) protein trimers with Toll-like receptor 7 and 8 agonists (TLR-7/8a) to enhance protective immunity. Although primary amine conjugation of TLR-7/8a to F trimers severely disrupted the recognition of critical neutralizing epitopes, F trimers site-selectively coupled to TRP nanoparticles retained appropriate antigenicity and elicited high titers of prefusion-specific, T_H1 isotype anti-RSV F antibodies following vaccination. Moreover, coupling F trimers to TRP delivering TLR-7/8a resulted in ∼3-fold higher binding and neutralizing antibody titers than soluble F trimers admixed with TLR-7/8a and conferred protection from intranasal RSV challenge. Overall, these data show that TRP nanoparticles may provide a broadly applicable platform for eliciting neutralizing antibodies to structure-dependent epitopes on RSV, influenza, HIV-1, or other pathogens