Discovery of bioactive small molecules for metabolic diseases via phenotype-based approach

Abstract

Phenotype-based approach in drug discovery has emerged as a promising approach to discover new bioactive small molecules. Unlike the conventional target-based approach that selects specific target protein based on an understanding of the molecular mechanism about pathogenesis of certain disease, the phenotype-based approach focuses on phenotypic changes in cells or organisms as a final outcome of perturbation in interconnected multiple pathways. Compared with the target-based approach, the main strength of the phenotype-based approach is that it has a high potential for discovering new therapeutic target that its biological role in managing disease is unrevealed. In this context, the phenotype-based approach is most powerful when applied to disease in which the therapeutic target is not thoroughly defined. In this study, I describe the systematic studies to discover bioactive small molecules for metabolic diseases via phenotype-based approach. In chapter 2, I discuss the construction of phenotypic screening for monitoring cellular glucose uptake to discover bioactive small molecules for hyperglycemia and Type 2 Diabetes Mellitus (T2DM). In this chapter, I explored the biophysical properties of the fluorescence glucose bioprobe, GB2-Cy3. Based on the understanding of biophysical properties of GB2-Cy3, I developed the first image-based high-throughput screening (HTS) system to monitor the glucose uptake in living cells with robustness and accuracy. From the screening of compound library in living cells, I could successfully discover the new glucose uptake enhancers has anti-diabetic potential. In addition, I investigated an impact of molecular charge of fluorescence glucose bioprobes on their GLUT-specific cellular uptake. I synthesized charge derivatives of GB2-Cy3, and demonstrated that zwitterionic and anionic charge diminish GLUT-specificity of fluorescent glucose bioprobes. In conclusion, I demonstrated the superiority of GB2-Cy3 as a GLUT-specific fluorescent glucose bioprobe. Moreover, I validated the in vivo applicability of GB2-Cy3 in zebra fish larvae in vivo system. This in vivo imaging system of GB2-Cy3 will be an important method to validate the therapeutic potential of glucose uptake enhancers in the anti-diabetic drug discovery process. In chapter 3, I describe an approach using fluorescence imaging for monitoring dynamic GLUT4 translocation in living cells. I developed a novel imaging system, EGFP-GLUT4-SNAP, based on genetically encoded SNAP tag and EGFP into GLUT4. EGFP-GLUT4-SNAP is a novel imaging system for monitoring GLUT4 trafficking in a real-time and quantitative manner. This novel approach enables the determination of the quantity and kinetics of GLUT4 translocation to the PM. Using this novel GLUT4 monitoring system, I could reveal the insulin action on the regulation of intracellular distribution of GLUT4. I anticipate that this approach allows us to understand the mechanism of GLUT4 regulation, and to study mechanism-of-action of chemical compounds which increase cellular glucose uptake by regulating GLUT4 trafficking. In chapter 4, discovery of novel anti-obesity small molecule, named as SB1501, via phenotype-based approach is described. I performed image-based phenotypic screening by monitoring cellular lipid droplets using fluorescent lipid droplets bioprobe, SF44. From the screening of small molecule library, I discovered SB1501 that reduces cellular lipid droplets in adipocytes. Mode-of-action study of SB1501 revealed that SB1501 decreases lipid droplets by inducing PGC-1 regulatory cellular processes involving mitochondrial biogenesis, fatty acid oxidation (FAO) and adaptive thermogenesis in 3T3-L1 adipocytes. Moreover, SB1501 showed in vivo efficacy by reducing the size of adipocytes in adipose tissue, the body weight and the fat mass in db/db mice. In addition, administration of SB1501 ameliorated insulin resistance. Taken together, novel small molecule, SB1501, has therapeutic potential for combating obesity and obesity-associated insulin resistance. In conclusion, I suggest the utility of image-based phenotype-based approach for discovering novel anti-obesity agents. In summary, this study is consisted of 1) exploration of fluorescence bioprobe for monitoring the cellular phenotype such as cellular glucose uptake and lipid droplet organelles, 2) construction of image-based phenotypic screening system using fluorescence bioprobe, 3) discovery of bioactive compounds via practical HTS, and 4) following mechanism-of-action study of bioactive compounds. Based on these studies, I suggest that phenotype-based approach is a promising approach to discover novel bioactive small molecules. In the present study, it is of major interest to discover new bioactive small molecules for metabolic disease, because a crucial therapeutic target of metabolic diseases has not been thoroughly understood. Although this dissertation focus on the metabolic disease, I anticipate that this approach will shed light on the diseases in which the therapeutic target is not thoroughly defined.