Here were identify three additional compounds recovered in this screen and provide a detailed account of prioritize compounds for testing in mammalian models. Larvae tolerated overnight incubation in the majority of the 3,840 compounds analyzed in the primary screen, however 67 compounds caused Afatinib larval death or severely compromised LY2109761 cardiac circulation and were therefore deemed toxic. 50 compounds caused either complete or partial inhibition gallbladder fluorescence. When re-tested in a qualitative visual assay of PED-6 metabolism, 15 of these compounds were considered active in a dose responsive fashion. 12 of the 15 compounds identified in the primary screen were tested in adult fish; 5 compounds were deemed active based on reduced gallbladder fluorescence derived from PED-6 while 7 were either inactive and or toxic in adult fish and not studied further. Together with the 3 compounds that were not available in sufficient quantity to be tested in adult fish, this left 8 compounds for testing in secondary assays. The visual dose response assays conducted in larvae arrayed in the 96 well plates showed that 2 of the 8 compounds first inhibited PED-6 processing at 6.25 uM, whereas the remaining compounds were first active at 25 uM. In separate experiments, combined gallbladder and intestinal fluorescence of individual compound treated larvae was quantified using fluorescence microscopy. This showed that the active compounds reduced PED-6 metabolism between 51%�C67%. Of the 8 active compounds, only 1 has been used in humans; clofazimine, a rhiminophenazine dye with antimicrobial and anti-inflammatory activity used to treat leprosy and other types of mycobacterial infections. Although intestinal toxicity has been reported with long term use of high doses of this drug, no prior reports of altered lipid absorption have been reported. We devised a series of secondary assays that allowed us to further characterize the active compounds�� mechanism of action and prioritize them for testing in mammals. We assayed the effect of the active compounds on the ingestion of fluorescent microspheres to control for the possibility that they prevented swallowing of PED-6 from the larvae��s aqueous media. This assay confirmed normal swallowing in 7 of 8 active compounds. Interestingly, the 1 compound that inhibited swallowing had no obvious effect on larval motility or cardiac function. We assayed the effect of the active compounds on the metabolism of fluorescent cholesterol and fatty acid analogues because these dietary lipids are differentially absorbed and or processed by enterocytes compared with the phospholipid used for the primary screen, PED6. Recent studies have shown that the intestinal absorption of dietary cholesterol is dependent on the Neiman Pick Type C 1Like 1 protein. Although the function of NPC1L1 is still debated, it is generally agreed upon that it as a cholesterol transporter embedded within the apical enterocyte membrane. NPC1L1 has not been implicated in phospholipid absorption, thus it was not predicted that the screen compounds, which were identified by their inhibition of phospholipid absorption, would interfere with absorption of a fluorescent cholesterol analog, NBD-cholesterol. Surprisingly, each of the 7 active compounds inhibited metabolism of NBDcholesterol, as determined by levels of biliary and intestinal fluorescence. We next measured the effect of the active compounds on the absorption of fluorescent short chain fatty acid and long chain fatty acid analogs.