Discovery of Inhibitors of a Protein?RNA (Lin28-let-7) Interaction and Antimalarial Agents Through the Development of High-Throughput Screening

Abstract

One of the greatest challenges in the field of chemical biology is the identification of novel bioactive small molecules. These molecules can serve as specific bioprobes for the mechanistic studies of biological systems or therapeutic candidates with unique modes of action. There are two main approaches for identifying bioactive small molecules. In the target-based approach, a target biomacromolecule that plays crucial roles in a disease model is selected. High-throughput biochemical assays identify small molecules that modulate the function of the disease-related target. Finally, the desired phenotypic changes induced by the active small molecules are investigated. In the phenotype-based approach, a phenotype related to a disease model is selected, and small molecules that induce the changes in the phenotype are selected. Target identification process can reveal the mechanism of the action of chemical modulators. Efficient discovery of bioactive small molecules would be possible by combining advantages of each method. Regardless of the type of high- throughput screening, however, the successful discovery of new small molecule modulators has been significantly influenced by the molecular diversity of small molecule screening libraries. Therefore, a new strategy to construct a small molecule library is presented in chapter 1 with the purpose of modulating new therapeutic targets such as protein?protein interactions. The library members contained two distinct hetereocycles connected in a single molecule with stereodivergent linkers. Pyrimidine-, pyrazole-, or pyrazolopyrimidine-based carbohybrids were constructed through condensations of the key intermediates, 2-C-formyl glycals, with various dinucleophiles. Fused-triazole scaffolds were obtained through intramolecular 1,3-dipolar cycloadditions after selective functionalization of the carbohybrid polyol moieties with azide and alkyne functionalities. Overall, this synthetic method affords two distinct privileged substructures in a single molecule, connected by stereodivergent diol linkers derived from abundant natural chiral sources, carbohydrates. The resulting vicinal diols in the linker were further modified to achieve unique connectivities between the two privileged structures for maximized three-dimensional shape diversity. The diversity was verified through principal moment inertial analysis and the structure alignment of tenergy-minimized compounds. In chapter 2, a target-based approach is exemplified in an attempt to identify inhibitors of a protein?miRNA (Lin28?let-7) interaction, an important therapeutic target. MicroRNAs (miRNAs) regulate gene expression by targeting most protein- coding transcripts. As a result, miRNAs are involved in every important cellular process in animal. Dysregulation of miRNA biogenesis is associated with many human diseases. Let-7 miRNA family is well known for its tumor suppression function, and it is down-regulated in many cancers. Lin28 protein is abnormally expressed in many cancer cells, and the protein bind primary and precursor let-7 miRNAs to inhibit their maturation. In this study, FRET- based high throughput screening system was constructed in order to identify inhibitors of the Lin28?let-7 interaction. Unnatural amino acid mutagenesis and bioorthogonal chemistry enabled site-specific fluorescent labeling of Lin28, resulting in a highly robust and reliable Lin28?let-7 binding assays. High-throughput screening using the assay identified flavone-based natural products as inhibitors of the protein?miRNA interaction. Biophysical studies revealed that inhibitors specifically bind Lin28 to block the access of let-7 miRNAs. In chapter 3, discovery of 2-aminopyrimidine-based antimalarial agents using a high-content phenotype-based screening is presented. New antimalarial agents that exhibit multistage activities against drug-resistant strains of malaria parasites represent good starting points for developing next- generation antimalarial therapies. In order to discover such agents, an image- based parasitological screening method for defining drug effects on different asexual life cycle stages of Plasmodium falciparum was developed. High- throughput screening of the in house chemical library using this image-based approach led to the identification of carbohybrid-based 2-aminopyrimidine compounds with fast-acting growth inhibitory activities against three laboratory strains of multidrug-resistant P. falciparum. Structure?activity relationship study led to the identification of two derivatives (8aA and 11aA) as the most promising antimalarial candidates, targeting all major blood stages of multidrug-resistant P. falciparum parasites.