Are Caenorhabditis elegans receptors useful targets for drug discovery: pharmacological comparison of tyramine receptors with high identity from C. elegans (TYRA-2) and Brugia malayi (Bm4)

Abstract

The biogenic amine, tyramine (TA), modulates a number of key processes in nematodes and a number of TA-specific receptors have been identified. In the present study, we have identified a putative TA receptor (Bm4) in the recently completed Brugia malayi genome and compared its pharmacology to its putative Caenorhabditis elegans orthologue, TYRA-2, under identical expression and assay conditions. TYRA-2 and Bm4 are the most closely related C. elegans and B. malayi BA receptors and differ by only 14aa in the TM regions directly involved in ligand binding. Membranes from HEK-293 cells stably expressing Bm4 exhibited specific, saturable, high affinity, [(3)H]LSD and [(3)H]TA binding with K(d)s of 18.1+/-0.93 and 15.1+/-0.2 nM, respectively. More importantly, both TYRA-2 and Bm4 TA 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 (pK(i)s 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.

Figure 1. Gene maps and protein alignment

A, Gene maps were constructed from EST data from Wormbase for Ce tyra-2 and from 5’ and 3’ RACE data for Bm4. Exons are boxes and intronic sequence is depicted by solid lines. Gray boxes within exons depict sequence coding for TM regions. B, Annotated protein sequences were aligned by MegAlign using Clustal W and sequence analysis by BOXSHADE. White type on black background indicates identical aas; Black type on grey background indicates similar aas. Predicted TM regions are indicated by a solid line above the alignment.

Figure 2. Unrooted phylogenetic tree of Tyramine and Octopamine receptors

Protein sequences were annotated to include primarily the 7 TM regions; specifically the N-termini were deleted including 19 aa before the first conserved aa in TM I (N1.50), the third intracellular loop was deleted 10 aa after TM V and before TM VI and C-termini were removed 15 aa after TM VII. Annotated sequences were initially aligned in DNAStar with Clustal W using default parameters and fine tuned by hand (all alignments available on request). Bootstrapping was undertaken in DNAStar (1000 replicates with random seed) and trees were compiled in PAUP. Tyramine/Octopamine receptors; Anopheles gambiae (AgOA, AgTA2,), Aplysia californica (AcOA), Aplysia kurdai (AkOA), Apis mellifera (AmOA, AmTA2, AmPTA), Ascaris suum (AsTA/OA), Boophilus microplus (BmiOA), Bombyx mori (BmoOA), (Drosophila melanogaster [DmOA, DmOA2 (OCTB1_DROME), DmOA3 (OCTB3_DROME), DmTA, DmTA2], Heliothis virescens (HvOA), Locusta migratoria (LmOA), Lymnaea stagnalis (LsOA1, LsOA2), Manduca sexta (MsOA), Papilio xuthus (PxTA), Periplaneta americana (PaOA). Trace amine associated receptors; Homo sapiens (HsTAAR1) and Mus musculus (MmTAAR1).

Figure 3. Alignment of characterized Tyramine, Octopamine and Trace Amine Associated receptors

Characterized TA, OA and TAA receptor sequences were aligned by MegAlign using Clustal W and sequence analysis by BOXSHADE. White type on black background indicates identical aas; Black type on grey background indicates similar aas. Predicted TM regions are indicated by a solid line above the alignment, identical aa are marked with a * and clade-specific aa are marked with a ^. Abbreviations as follows: AcOA (Aplysia californica), AkOA (Aplysia kurdai), AmOA (Apis mellifera), BmiOA (Boophilus microplus), BmoOA (Bombyx mori), DmOA and DmTA (Drosophila melanogaster), HvOA (Heliothis virescens), PaOA (Periplaneta americana), HsTAAR1 (Homo sapiens) and MmTAAR1 (Mus musculus).

Figure 4. Saturation binding for [³H]LSD and [³H]Tyramine with membranes expressing Bm4

Membranes prepared from stably expressing HEK293 cells were incubated with [³H]LSD (A) [³H]TA or (B) at concentrations ranging from 1 nM–40 nM in the presence or absence of 1000-fold unlabelled LSD or TA, respectively. Specific binding is represented by closed squares and non-specific binding by triangles. Bm4 displays affinity for [³H]LSD with a K_d of 18.1 ± 0.93 nM and B_max of 1.79 ± 0.2 pmol/ mg and for [³H]TA a with K_d of 15.1 ± 0.2 nM and B_max of 1.15 ± 0.05 pmol/mg.

Figure 5. Pharmacological profile of Bm4

Membranes of HEK293 cells expressing Bm4 were incubated with 10 nM [³H]TA in the presence of ligands at a range of concentrations. Data are representative of at least three independent experiments performed in triplicate (see Table 1 for summary of pharmacology).

Figure 6. Pharmacological profile of TYRA-2

Membranes of HEK293 cells expressing TYRA-2 were incubated with 10 nM [³H]TA in the presence of ligands at a range of concentrations. Data are representative of at least three independent experiments performed in triplicate (see Table 1 for summary of pharmacology).