title: Amino acid derivatives as bitter taste receptor (T2R) blockers.
abstract: In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only five antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis, we elucidated the ligand-binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn-173 and Thr-174 are essential for quinine binding. Guided by a molecular model of T2R4, a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays that led to identification of γ-aminobutryic acid (GABA) and Nα,Nα-bis(carboxymethyl)-L-lysine (BCML) as competitive inhibitors of quinine-activated T2R4 with an IC50 of 3.2 ± 0.3 μM and 59 ± 18 nM, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, whereas BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala-90 and Lys-270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understanding the roles of T2Rs in extraoral tissues.
authors: From the Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba R3E 0W4, the Manitoba Institute of Child Health, Winnipeg, Manitoba R3E 0W4, and.
authors: Sai P
authors: SP
authors: Pydi
authors: From the Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba R3E 0W4.
authors: Tyler
authors: T
authors: Sobotkiewicz
authors: From the Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba R3E 0W4, the Manitoba Institute of Child Health, Winnipeg, Manitoba R3E 0W4, and.
authors: Rohini
authors: R
authors: Billakanti
authors: From the Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba R3E 0W4.
authors: Rajinder P
authors: RP
authors: Bhullar
authors: the National Research Council of Canada, Saskatoon, Saskatchewan S7N 0W9, Canada.
authors: Michele C
authors: MC
authors: Loewen
authors: From the Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba R3E 0W4, the Manitoba Institute of Child Health, Winnipeg, Manitoba R3E 0W4, and Prashen.Chelikani@umanitoba.ca.
authors: Prashen
authors: P
authors: Chelikani
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D018682:GABA Agents / Q000737:chemistry / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011803:Quinine / Q000737:chemistry / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000737:chemistry; D013649:Taste; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000737:chemistry / Q000494:pharmacology; D000098706:Taste Receptors, Type 2
chemical_list: D000596:Amino Acids; D018682:GABA Agents; D043562:Receptors, G-Protein-Coupled; D000098706:Taste Receptors, Type 2; D013912:Threonine; D005680:gamma-Aminobutyric Acid; D001216:Asparagine; D011803:Quinine; D008239:Lysine; D000409:Alanine
keywords: Bitter Blockers; Bitter Taste Receptors; Calcium Imaging; G Protein-coupled Receptor (GPCR); Molecular Modeling; Receptor Structure-Function; Site-directed Mutagenesis