of wild type larvae. Intestinal fluorescence derived from the casein reporter was minimally reduced in larvae treated with 5 of 7 compounds. Treatment with 2 compounds caused a profound reduction in the metabolism of the casein reporter. Changes in gallbladder and intestinal fluorescence detected in the primary screening assay detected could have arisen from a reduction in either intestinal and or hepatic lipid processing. We fed compound treated larvae egg yolk and after allowing time for its absorption, we performed whole mount stainings using the lipophilic dye oil red o to determine whether yolk-derived lipids accumulated in either organ. Wild type larvae fed egg yolk had strong ORO staining of the anterior intestine, as well as the blood stream, the latter arising from lipid in circulating lipoproteins. Manual dissection of the intestine showed that the ORO staining derived from small lipid droplets within the enterocyte cytoplasm. Lipid within the intestinal lumen was not detected in any wild type larvae. Each of the 7 active compounds tested reduced intestinal lipid. Lipid was detected in enterocytes of all compound treated larvae, but at far lower levels than in wild type, except in larvae treated with compound 10. Here luminal lipid was detected. No evidence of hepatic lipid accumulation was evident. Collectively, these findings are compatible with reduced intestinal lipid absorption in compound treated larvae. Compound synergy was examined in binary combinations of the 7 remaining active compounds with each other and with ezetimibe. Each compound was assayed at the highest concentration deemed inactive and the lowest dose considered active in the visual dose response experiment. These experiments identified potential synergism between compounds 2 and 10. The two most commonly prescribed lipid absorption inhibitors, orlistat and ezetimibe, are 1532533-67-7 generally considered to be selective inhibitors of triglyceride, and cholesterol and phytosterol absorption, respectively. To gain a better understanding of the mechanism of action of the novel compounds identified in our screen, we examined how these drugs affected absorption of fluorescent lipid reporters in zebrafish larvae. Both drugs were assayed in an 1187431-43-1 identical fashion as the screen compounds. Orlistat had no effect on the metabolism of any of the lipid reporters. This was predicted, however because none are processed by pancreatic lipase, which is responsible for hydrolysis of triglycerides. In contrast to orlistat, ezetimibe was predicted to inhibit absorption of NBDcholesterol because the amino acid domain of dog Npc1l1 required for high affinity binding to ezetimibe is highly conserved in both human NPC1L1 and the predicted zebrafish Npc1l1 protein. Indeed, larvae treated overnight with the
