Synthesis and Structure-Activity Relationship Analysis of 5-HT7 Receptor Antagonists: Piperazin-1-yl Substituted Unfused Heterobiaryls.

A series of piperazin-1-yl substituted unfused heterobiaryls was synthesized as ligands of the 5-HT7 receptors. The goal of this project was to elucidate the structural features that affect the 5-HT7 binding affinity of this class of compounds represented by the model ligand 4-(3-furyl)-2-(4-methylpiperazin-1-yl)pyrimidine (2). The SAR studies included systematical structural changes of the pyrimidine core moiety in 2 to quinazoline, pyridine and benzene, changes of the 3-furyl group to other heteroaryl substituents, the presence of various analogs of the 4-methylpiperazin-1-yl group, as well as additional substitutions at positions 5 and 6 of the pyrimidine. Substitution of position 6 of the pyrimidine in the model ligand with an alkyl group results in a substantial increase of the binding affinity (note a change in position numbers due to the nomenclature rules). It was also demonstrated that 4-(3-furyl) moiety is crucial for the 5-HT7 binding affinity of the substituted pyrimidines, although, the pyrimidine core can be replaced with a pyridine ring without a dramatic loss of the binding affinity. The selected ethylpyrimidine (12) and butylpyrimidine (13) analogs of high 5-HT7 binding affinity showed antagonistic properties in cAMP functional test and varied selectivity profile-compound 12 can be regarded as a dual 5-HT7/5-HT2AR ligand, and 13 as a multi-receptor (5-HT7, 5-HT2A, 5-HT6 and D2) agent.

In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters.

Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae.

Automated, quantitative screening assay for antiangiogenic compounds using transgenic zebrafish.

Pathologic angiogenesis has emerged as an important therapeutic target in several major diseases. Zebrafish offer the potential for high-throughput drug discovery in a whole vertebrate system. We developed the first quantitative, automated assay for antiangiogenic compound identification using zebrafish embryos. This assay uses transgenic zebrafish with fluorescent blood vessels to facilitate image analysis. We developed methods for automated drugging and imaging of zebrafish in 384-well plates and developed a custom algorithm to quantify the number of angiogenic blood vessels in zebrafish. The assay was used to screen the LOPAC1280 compound library for antiangiogenic compounds. Two known antiangiogenic compounds, SU4312 and AG1478, were identified as hits. Additionally, one compound with no previously known antiangiogenic activity, indirubin-3'-monoxime (IRO), was identified. We showed that each of the hit compounds had dose-dependent antiangiogenic activity in zebrafish. The IC(50) of SU4312, AG1478, and IRO in the zebrafish angiogenesis assay was 1.8, 8.5, and 0.31 micromol/L, respectively. IRO had the highest potency of the hit compounds. Moreover, IRO inhibited human umbilical vein endothelial cell tube formation and proliferation (IC(50) of 6.5 and 0.36 micromol/L, respectively). It is therefore the first antiangiogenic compound discovered initially in a zebrafish assay that also has demonstrable activity in human endothelial cell-based angiogenesis assays.

Neuroprotection of MPTP-induced toxicity in zebrafish dopaminergic neurons.

Parkinson's disease is characterized by a severe loss of dopaminergic neurons resulting in a range of motor deficits. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to cause a similar loss of dopaminergic neurons in the human midbrain with corresponding Parkinsonian symptoms. Several animal species have also shown sensitivity to MPTP, including primates, mice, goldfish, and, most recently, zebrafish. This study demonstrates that the effect of MPTP on dopaminergic neurons in zebrafish larvae is mediated by the same pathways that have been demonstrated in mammalian species. MPTP-induced neurodegeneration was prevented by co-incubation with either the monoamine oxidase-B (MAO-B) inhibitor l-deprenyl or the dopamine transporter (DAT) inhibitor nomifensine. Furthermore, targeted inactivation of the DAT gene by antisense morpholinos also protected neurons from MPTP damage. Thus, the mechanism for MPTP-induced dopaminergic neuron toxicity in mammals is conserved in zebrafish larvae. Effects on swimming behavior and touch response that result from MPTP damage are partially ameliorated by both l-deprenyl and DAT knockdown.