Phenotype-based discovery of a HeLa-specific cytotoxic molecule that downregulates HPV-mediated signaling pathways via oxidative damage.

Selective bioactive compounds have emerged as major players in chemical biology for their potential in disrupting diverse biological pathways with minimal adverse effects. Using phenotypic screening, we identified an anti-cancer agent, SB2001, with a highly specific cytotoxicity toward HeLa human cervical cancer cells. The subsequent mechanistic study revealed that SB2001 induced apoptotic cell death through restoring p53 function and suppressed the human papillomavirus (HPV)-mediated oncoprotein signaling pathway via oxidative damage in HeLa cells. SB2001 also selectively induced HeLa-specific tumor regression without any adverse effects in an in vivo tumor xenograft model, demonstrating its potential as a promising chemical probe.

SB2301-mediated perturbation of membrane composition in lipid droplets induces lipophagy and lipid droplets ubiquitination.

Lipid droplets (LDs) are involved in various biological events in cells along with their primary role as a storage center for neutral lipids. Excessive accumulation of LDs is highly correlated with various diseases, including metabolic diseases. Therefore, a basic understanding of the molecular mechanism of LD degradation would be beneficial in both academic and industrial research. Lipophagy, a selective autophagy mechanism/LD degradation process, has gained increased attention in the research community. Herein, we sought to elucidate a novel lipophagy mechanism by utilizing the LD-degrading small molecule, SB2301, which activates ubiquitin-mediated lipophagy. Using a label-free target identification method, we revealed that ethanolamine-phosphate cytidylyltransferase 2 (PCYT2) is a potential target protein of SB2301. We also demonstrated that although SB2301 does not modulate PCYT2 function, it induces the cellular translocation of PCYT2 to the LD surface and spatially increases the phosphatidylethanolamine (PE)/phosphatidylcholine (PC) ratio of the LD membrane, causing LD coalescence, leading to the activation of lipophagy process to maintain energy homeostasis.

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli.

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.

One-Pot Synthesis of Nanostructured Ni@Ni(OH)2 and Co-Doped Ni@Ni(OH)2 via Chemical Reduction Method for Supercapacitor Applications.

Crystalline Ni@Ni(OH)2 (cNNH) and Co-doped cNNH were obtained via a simple one-pot hydrothermal synthesis using a modified chemical reduction method. The effect of each reagent on the synthesis of the nanostructures was investigated concerning the presence or absence of each reagent. The detailed morphology shows that both nanostructures consist of a Ni core and Ni(OH)2 shell layer (~5 nm). Co-doping influences the morphology and suppresses the particle agglomeration of cNNH. Co-doped cNNH showed a specific capacitance of 1238 F g-1 at 1 A g-1 and a capacitance retention of 76%, which are significantly higher than those of cNNH. The enhanced performance of the co-doped cNNH is attributed to the reduced path length of the electrons caused by the decrease in the size of the nanostructure and the increased conductivity due to Co ions substituting Ni ions. The reported synthesis method and electrochemical behaviors of cNNH and Co-doped cNNH affirm their potential as electrochemically active materials for supercapacitor applications.

Intravital longitudinal imaging of hepatic lipid droplet accumulation in a murine model for nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a rapidly increasing chronic liver disorder worldwide accompanied by hepatic steatosis, inflammation, fibrosis, and severe liver failure. Unfortunately, an effective treatment strategy for NAFLD has not yet been established, which has been hampered by the limited understanding of the pathophysiological drivers for NAFLD. To examine the unknown cellular and molecular mechanisms in the pathogenesis of NAFLD, there is an increasing need for the direct in vivo observation of hepatic microenvironments over extended periods of time. In this work, using a custom-built intravital imaging system and a novel fluorescent lipid droplet labeling dye, Seoul-Fluor 44 (SF44), we established an intravital imaging method to visualize individual lipid droplets and microvasculature simultaneously in the liver of live mice in vivo. In addition, in the nonalcoholic steatosis and steatohepatitis mouse model induced by a methionine and choline-deficient diet, we longitudinally visualized and quantitatively analyzed the development of lipid droplets in hepatocytes and sinusoid at a subcellular resolution during the progression of NAFLD up to 21 days in vivo.

A high-content screening platform with fluorescent chemical probes for the discovery of first-in-class therapeutics.

Phenotypic screening has emerged as a promising approach to discover novel first-in-class therapeutic agents. Rapid advances in phenotypic screening systems facilitate a high-throughput unbiased evaluation of compound libraries. However, limited sets of phenotypic changes are utilized in high-content screening, which require extensive genetic engineering. Therefore, it is critical to develop new chemical probes that can reflect phenotypic changes in any type of cells, especially primary cells, tissues, and organisms. Herein, we introduce our continuous efforts in the development of fluorescent bioprobes and their application to phenotypic screening. In addition, we emphasize the importance of the phenotype-based approach in conjunction with target identification at an early stage of research to accelerate the discovery of therapeutics with new modes of action.

ß-Turn mimetic-based stabilizers of protein-protein interactions for the study of the non-canonical roles of leucyl-tRNA synthetase.

For the systematic perturbation of protein-protein interactions, we designed and synthesized tetra-substituted hexahydro-4H-pyrazino[2,1-c][1,2,4]triazine-4,7(6H)-diones as ß-turn mimetics. We then devised a new synthetic route to obtain ß-turn mimetic scaffolds via tandem N-acyliminium cyclization and constructed a 162-member library of tetra-substituted pyrazinotriazinediones with an average purity of 90% using a solid-phase parallel synthesis platform. Each library member was subjected to ELISA-based modulator screening for the LRS-RagD interaction, which plays a pivotal role in the nutrient-dependent mTORC1 signalling pathway. Western blot analysis of phosphorylated S6K1 as well as FRET-based imaging confirmed that 5c{3,9} stabilizes the direct interaction between LRS and RagD and activates mTORC1 in live cells under leucine-deprived conditions. Thus, 5c{3,9} can be used as a new research tool for studying the non-canonical role of LRS.

Diversity-oriented synthetic strategy for developing a chemical modulator of protein-protein interaction.

Diversity-oriented synthesis (DOS) can provide a collection of diverse and complex drug-like small molecules, which is critical in the development of new chemical probes for biological research of undruggable targets. However, the design and synthesis of small-molecule libraries with improved biological relevance as well as maximized molecular diversity represent a key challenge. Herein, we employ functional group-pairing strategy for the DOS of a chemical library containing privileged substructures, pyrimidodiazepine or pyrimidine moieties, as chemical navigators towards unexplored bioactive chemical space. To validate the utility of this DOS library, we identify a new small-molecule inhibitor of leucyl-tRNA synthetase-RagD protein-protein interaction, which regulates the amino acid-dependent activation of mechanistic target of rapamycin complex 1 signalling pathway. This work highlights that privileged substructure-based DOS strategy can be a powerful research tool for the construction of drug-like compounds to address challenging biological targets.

Annexin a5 as a new potential biomarker for Cisplatin-induced toxicity in human kidney epithelial cells.

Cisplatin is a member of platinum-containing anti-cancer drugs that causes cross-linking of DNA and ultimately cancer cell apoptosis. The therapeutic function of cisplatin on various types of cancers has been widely reported but the side effects have been discovered together and nephrotoxicity has been regarded as major side effect of cisplatin. To select candidates for new sensitive nephrotoxicity biomarker, we performed proteomic analysis using 2-DE/MALDI-TOF-MS followed by cisplatin treatment in human kidney cell line, HK-2 cells, and compared the results to the gene profi le from microarray composed of genes changed in expression by cisplatin from formerly reported article. Annexin A5 has been selected to be the most potential candidate and it has been identifi ed using Western blot, RT-PCR and cell viability assay whether annexin A5 is available to be a sensitive nephrotoxic biomarker. Treatment with cisplatin on HK-2 cells caused the increase of annexin A5 expression in protein and mRNA levels. Overexpression of annexin A5 blocked HK-2 cell proliferation, indicating correlation between annexin A5 and renal cell toxicity. Taken together, these results suggest the possibility of annexin A5 as a new biomarker for cisplatin-mediated nephrotoxicity.