A Dissertation Entitled Are C. elegans receptors useful targets for drug discovery: Identification of genes encoding seven potential biogenic amine receptors in the parasitic nematode Brugia malayi and pharmacological comparison of tyramine receptor homologues from Caenorhabditis elegans (TYRA-2) and B. malayi (Bm4) By Katherine Ann Smith Submitted as partial fulfilment of the requirements for The Doctor of Philosophy in Biology ___________________________ Advisor: Dr Richard Komuniecki ___________________________ College of Graduate Studies The University of Toledo May 2007 An Abstract of Are C. elegans receptors useful targets for drug discovery: Identification of genes encoding seven potential biogenic amine receptors in the parasitic nematode Brugia malayi and pharmacological comparison of tyramine receptor homologues from Caenorhabditis elegans (TYRA-2) and B. malayi (Bm4) by Katherine Ann Smith Submitted as partial fulfilment of the requirements for The Doctor of Philosophy in Biology The University of Toledo May 2007 Filarial nematodes, such as Brugia malayi, cause major health problems worldwide. At present, no vaccine against B. malayi is available and current chemotherapy is ineffective against adult parasites. Biogenic amines (BAs) regulate a number of key processes in nematodes, suggesting that nematode BA receptors may be useful targets for drug discovery. Therefore, we have used a bioinformatics approach to identify genes encoding putative B. malayi BA receptors, using BA receptors recently identified in the free-living nematode, Caenorhabditis elegans. Surprisingly, the B. malayi genome appears to contain less than half of the genes predicted to encode BA receptors in the genomes of C. elegans or C. briggsae; however, all of the predicted B. malayi receptors have clear orthologues in C. elegans. ii The B. malayi genes encode each of the major BA receptor subclasses, including three serotonin, two dopamine and two tyramine (TA) receptors. There is little overall synteny between the B. malayi and C. elegans genomes surrounding the genes encoding the predicted BA receptors; however, most of the intron/exon borders of orthologous BA receptor genes are conserved among the three species. Multiple BA receptor alignment and phylogenetic analysis suggests that potential ligand specificity and G-protein coupling of the individual receptors can be predicted via this bioinformatics approach. Further study of the seven B. malayi receptors identified a putative TA receptor (Bm4) and compared its pharmacology to its putative C. elegans orthologue, TYRA-2, under identical expression and assay conditions. In the present study, membranes from HEK-293 cells stably expressing Bm4 exhibited specific, 3 3 saturable, high-affinity [ H]LSD and [ H]TA binding with Kds of 18 ± 0.9 nM and 15 ± 0.2 nM, respectively. More importantly, both TYRA-2 and Bm4 receptors exhibited similar rank orders of potencies for a number of potential tyraminergic ligands. However, some significant differences were noted. For example, chloropromazine exhibited an order of magnitude higher affinity for Bm4 than TYRA-2 (pKis of 7.6 ± 0.2 and 6.49 ± 0.1, respectively). In contrast, TYRA-2 had significantly higher affinity for phentolamine than Bm4. These results highlight the utility of the nearly completed B. malayi genome and the importance of using receptors from individual parasitic nematodes for drug discovery. iii DEDICATION For my family: Your love, support, encouragement and belief in me through this entire journey, Thank you. This thesis is dedicated to them. iv ACKNOWLEDGEMENTS Many people at the University of Toledo have had a big impact upon my life and career and I would like to acknowledge them and say thank you. My advisor, Dr Richard Komuniecki inspired me to take this challenging path and has guided me throughout my graduate career and helped me grow not just as a scientist but as a person. Thank you for never crossing the line, despite moving it so often! Dr. Patricia Komunecki was responsible for introducing me to the Komuniecki lab, and under the initial guidance of Dr Emilio Duran, my enthusiasm for research was ignited. Both Rick and Patsy have also helped me through a tough event here in Toledo, and I can never repay the kindness you both bestowed upon me, thank you both. I would also like to acknowledge my committee members, Dr. J. Gray, Dr. W. Messer, Dr. J. Plenefish and Dr. M. Funk. In particular, I would like to express gratitude to Dr. John Gray for his insight into bioinformatics. I would also like to thank Dr. Scott Leisner and Dr. Debera Vestal for the use of their computers. Finally I would like to thank the Komuniecki lab members both past and present who provided a pleasant working environment. Dr. Emilio Duran and Dr. Sally Harmych for their initial guidance and helping me become settled in the Komuniecki lab. Dr. Hong Xiao for her advice and lastly Dr Elizabeth Rex, for her advice, training of culture assays, but mostly for her invaluable friendship. v TABLE OF CONTENTS ABSTRACT…………………………………………………………………………...ii DEDICATION...............................................................................................................iv ACKNOWLEDGEMENTS...........................................................................................v TABLE OF CONTENTS……………………………………………………...……...vi LIST OF TABLES ………………………………………………………………….....x LIST OF FIGURES …………………………………………………………………..xi ABBREVIATIONS………………………………………………………………......xii CHAPTER I OVERVIEW/SIGNIFICANCE.......…………………..………………………2 Brugia malayi………………………………………………………………….2 Caenorhabditis elegans as a model for parasitic nematodes………………….4 Biogenic amines as potential targets for anti-nematodal drug discovery……..5 G-protein coupled receptors………………………………………………….11 C. elegans BA GPCRs……………………………………………………….17 Bioinformatics………………………………………………………………..17 The Caenorhabditis and B. malayi genomes………………………………...20 HYPOTHESIS……………………………………………………………………….23 Hypothesis and Experimental Approach……………………………………..23 vi CHAPTER II Genes encoding putative biogenic amine receptors in the parasitic nematode Brugia malayi..……………………………………………….………24 ABSTRACT………………………………………………………………………….25 INTRODUCTION…………………………………………………………………...26 MATERIALS AND METHODS Mining The Institute for Genomic Research (TIGR) B. malayi database for GPCR homologous genes…………………………………….............31 Mining the C. briggsae and C. elegans databases……………………………32 Screening of 5’ and 3’ RACE libraries from B. malayi………………………32 Gene Maps……………………………………………………………………34 Protein alignments……………………………………………………………34 Phylogenetic tree……………………………………………………………...35 RESULTS Identification of genes encoding potential G-protein coupled biogenic amine receptors in C. elegans, C. briggsae and B. malayi ………………….37 Identification of serotonin receptor homologs in B. malayi………………….38 Identification of tyramine receptor homologs in B. malayi……......................42 Identification of dopamine receptor homologs in B. malayi………………….44 Phylogenetic relationships of biogenic amine receptors predict G-protein coupling and ligand binding………………………………………….46 Identification of highly conserved residues amongst diverse GPCR receptors………………………………………………………………51 vii Determining amino acids responsible for divergence in the phylogenetic tree……………………………………………………………………52 DISCUSSION………………………………………………………………………..58 CHAPTER III Are C. elegans receptors useful targets for drug discovery: Pharmacological comparison of tyramine receptor homologues from Caenorhabditis elegans (TYRA-2) and Brugia malayi (Bm4) ………………………………………...66 ABSTRACT…………………………………………………………………………..67 INTRODUCTION……………………………………………………………………68 METHODS AND MATERIALS Materials……………………………………………………………………...71 Methods………………………………………………………………………71 Mining the TIGR B. malayi database for C. elegans TYRA-2 homologues…71 Screening of 5’ and 3’ RACE libraries from B. malayi………………………72 Gene Maps……………………………………………………………………73 Protein alignments……………………………………………………………73 Phylogenetic tree.……………………………………………………………..74 Cloning and sequencing Bm4 cDNA…………………………………………74 Expression of Bm4 in COS-7 and HEK-293 cell lines.………………………75 Immunoflorescence of cells stably expressing Bm4……………………….....76 Membrane preparation………………………………………………………..76 Radioligand binding assay……………………………………………………77 viii RESULTS Cloning and sequence analysis of Bm4………………………………………78 Identification of Bm4, a putative C. elegans TYRA-2 homologue in B. malayi...............................................................................................78 Characterization of Bm4 after heterologous expression in mammalian cells…………………………………………………………………...81 Comparison of the pharmacological profiles of Bm4 and TYRA-2 ………...87 DISCUSSION………………………………………………………………………..91 REFERENCES……………………………………………………………………….96 ix LIST OF TABLES Table 1: Comparison of C. elegans and putative B. malayi biogenic amine receptor genes…………………………………………………............……...41 Table 2: Location of amino acid residues with greater than 50% conservation amongst 98 BA GPCRs………………………………………………57 Table 3: Pharmacological profile of Bm4 and TYRA-2…………………………...…90 Table 4: Comparison of pharmacological profiles of Bm4 and TYRA-2……………90 x LIST OF FIGURES Figure 1: Biosynthesis pathway of biogenic amines in C. elegans……………………6 Figure 2: Receptor activation states…………………………………………………..14 Figure 3: Gene maps and alignments of serotonin receptors…………………………39 Figure 4: Gene maps and alignments of tyramine and octopamine receptors………..43 Figure 5: Gene maps and alignments of dopamine receptors………………………...45