title: Lead discovery for mammalian elongation of long chain fatty acids family 6 using a combination of high-throughput fluorescent-based assay and RapidFire mass spectrometry assay.
abstract: A high-throughput RapidFire mass spectrometry assay is described for elongation of very long-chain fatty acids family 6 (Elovl6). Elovl6 is a microsomal enzyme that regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 may be a new therapeutic target for fat metabolism disorders such as obesity, type 2 diabetes, and nonalcoholic steatohepatitis. To identify new Elovl6 inhibitors, we developed a high-throughput fluorescence screening assay in 1536-well format. However, a number of false positives caused by fluorescent interference have been identified. To pick up the real active compounds among the primary hits from the fluorescence assay, we developed a RapidFire mass spectrometry assay and a conventional radioisotope assay. These assays have the advantage of detecting the main products directly without using fluorescent-labeled substrates. As a result, 276 compounds (30%) of the primary hits (921 compounds) in a fluorescence ultra-high-throughput screening method were identified as common active compounds in these two assays. It is concluded that both methods are very effective to eliminate false positives. Compared with the radioisotope method using an expensive14C-labeled substrate, the RapidFire mass spectrometry method using unlabeled substrates is a high-accuracy, high-throughput method. In addition, some of the hit compounds selected from the screening inhibited cellular fatty acid elongation in HEK293 cells expressing Elovl6 transiently. This result suggests that these compounds may be promising lead candidates for therapeutic drugs. Ultra-high-throughput fluorescence screening followed by a RapidFire mass spectrometry assay was a suitable strategy for lead discovery against Elovl6.
authors: Centre for Drug Discovery, Graduate School of Pharmaceutical Science, University of Shizuoka, Suruga-ku, Shizuoka, Shizuoka, Japan; Discovery Technology Laboratories, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda-shi, Saitama, Japan.
authors: Mari
authors: M
authors: Takamiya
authors: Advanced Drug Research Laboratories, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda-shi, Saitama, Japan.
authors: Masaaki
authors: M
authors: Sakurai
authors: Advanced Drug Research Laboratories, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda-shi, Saitama, Japan.
authors: Fumie
authors: F
authors: Teranishi
authors: Advanced Drug Research Laboratories, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda-shi, Saitama, Japan.
authors: Tomoko
authors: T
authors: Ikeda
authors: Discovery Technology Laboratories, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda-shi, Saitama, Japan.
authors: Tsutomu
authors: T
authors: Kamiyama
authors: Centre for Drug Discovery, Graduate School of Pharmaceutical Science, University of Shizuoka, Suruga-ku, Shizuoka, Shizuoka, Japan. Electronic address: aasai@u-shizuoka-ken.ac.jp.
authors: Akira
authors: A
authors: Asai
mesh_terms: D000123:Acetyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D004353:Drug Evaluation, Preclinical; D000081031:Fatty Acid Elongases; D057166:High-Throughput Screening Assays; D013058:Mass Spectrometry; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
chemical_list: D004364:Pharmaceutical Preparations; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases
keywords: Drug discovery; Elongation of long chain fatty acids family 6 (Elovl6); High-throughput screening (HTS); RapidFire mass spectrometry (RF-MS)