Positive allosteric modulators of the human sweet taste receptor enhance sweet taste Guy Servant a,1,2 , Catherine Tachdjian a,1 , Xiao-Qing Tang a , Sara Werner a , Feng Zhang a , Xiaodong Li a , Poonit Kamdar a , Goran Petrovic a , Tanya Ditschun a , Antoniette Java a , Paul Brust a , Nicole Brune a , Grant E. DuBois b , Mark Zoller a , and Donald S. Karanewsky a a Senomyx, Inc., La Jolla, CA 92121; and b Global Research and Technology, The Coca-Cola Company, Atlanta, GA 30301 Edited* by Solomon Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved January 25, 2010 (received for review October 12, 2009) To identify molecules that could enhance sweetness perception, we undertook the screening of a compound library using a cell- based assay for the human sweet taste receptor and a panel of selected sweeteners. In one of these screens we found a hit, SE-1, which significantly enhanced the activity of sucralose in the assay. At 50 μM, SE-1 increased the sucralose potency by >20-fold. On the other hand, SE-1 exhibited little or no agonist activity on its own. SE-1 effects were strikingly selective for sucralose. Other popular sweeteners such as aspartame, cyclamate, and saccharin were not enhanced by SE-1 whereas sucrose and neotame potency were increased only by 1.3- to 2.5-fold at 50 μM. Further assay- guided chemical optimization of the initial hit SE-1 led to the dis- covery of SE-2 and SE-3, selective enhancers of sucralose and sucrose, respectively. SE-2 (50 μM) and SE-3 (200 μM) increased sucralose and sucrose potencies in the assay by 24- and 4.7-fold, respectively. In human taste tests, 100 μM of SE-1 and SE-2 allowed for a reduction of 50% to >80% in the concentration of sucralose, respectively, while maintaining the sweetness intensity, and 100 μM SE-3 allowed for a reduction of 33% in the concen- tration of sucrose while maintaining the sweetness intensity. These enhancers did not exhibit any sweetness when tasted on their own. Positive allosteric modulators of the human sweet taste receptor could help reduce the caloric content in food and bever- ages while maintaining the desired taste. enhancer | sweetness | perception | sucrose T he steady increase of the daily consumption of dietary sugar over the last decades may have contributed to the obesity crisis and the early onset of type-II diabetes observed in many developed countries (1, 2). As a result, food and beverage companies have launched a plethora of diet brands where sugar has been partly or fully replaced by noncaloric sweeteners to decrease caloric intake. Currently some of the commonly used noncaloric sweeteners include saccharin, aspartame, cyclamate, sucralose, and acesulfame K (3, 4). However, none of these substances can completely reproduce the taste of sugar. These sweeteners all suffer from one or more shortcomings including a bitter or metallic aftertaste at high concentrations, limiting their use to lower concentrations, or temporal issues such as a delayed sweet taste onset, a lingering sweet aftertaste, or a limited maximum sweetness intensity (4, 5). Another appealing approach to address the problem, in addition to looking for novel noncaloric sweeteners, would be to find molecules capable of enhancing sweetness perception. Ideally, such an enhancer molecule would not elicit sweetness on its own but it would boost the sweetness intensity of a lower amount of sweetener or sugar. Such enhancers could therefore allow for a reduction in the amount of sugar, and calories, in food and beverages while maintaining the desired taste. Similarly, a sweet taste enhancer could allow for a reduction in the amount of noncaloric sweet- eners used in “0”-calorie diet brands, decreasing the associated bitterness, lingering tastes, and other off-tastes observed at high concentrations (4, 5) and therefore potentially improving the palatability and flavor profile of several consumer products. Sweet taste is mediated by an obligate heterodimeric receptor composed of two distinct subunits (G protein-coupled receptors, GPCRs), T1R2 and T1R3, located at the surface of taste receptor cells in the taste buds (6, 7). These subunits, members of family C GPCRs, possess a large extracellular N-terminal domain, the Venus flytrap domain (VFT), linked to the seven- transmembrane C-terminal domain (TMD) by a shorter cys- teine-rich domain (6–8). Every one of these three domains contains agonist binding sites, explaining sweet receptor activa- tion by a vast repertoire of chemically distinct molecules. Sucrose and noncaloric sweeteners such as aspartame and neotame interact within the VFT of T1R2 (9, 10), other noncaloric sweeteners such as cyclamate and neohesperidin dihy- drochalcone (NHDC) interact within the TMD of T1R3 (10–12), S819, a synthetic sweet agonist, interacts with the TMD of T1R2 (10), and the sweet-tasting protein brazzein requires the cys- teine-rich domain of human T1R3 to activate the receptor (13). Family C GPCRs seem ideal targets for allosteric modulation. The savory (umami) taste receptor, a close relative to the human sweet taste receptor, is significantly enhanced by 5′-ribonucleo- tides (6, 14). Positive allosteric modulators (PAMs) have also been identified for the GABA B receptor, the calcium sensing receptor, and several subtypes of the metabotropic glutamate receptor (mGluR) family (15, 16). In all cases, the PAMs show little or no agonist activity on their own but significantly enhance the activity of the agonist on the receptor and, in functional assays, this behavior is depicted by a leftward shift of the agonist dose– response in the presence of the PAM (16–18). We report here the identification of a unique chemical class of PAMs for the human sweet taste receptor. These PAMs considerably increase the sucralose and sucrose potencies in a sweet taste receptor cell- based assay, are not sweet on their own, and significantly enhance the sweetness of sucralose or sucrose in taste tests. Results and Discussion Identification and Characterization of a PAM for the Human Sweet Taste Receptor. We have developed a very sensitive cell-based assay for the detection of human sweet taste receptor modu- lators. In this assay system, the human sweet taste receptor couples to the promiscuous G protein Gα 15 to induce PLC Author contributions: G.S., C.T., and X.L. designed research; X.-Q.T., S.W., F.Z., P.K., G.P., A.J., P.B., and N.B. performed research; G.S., C.T., X.L., T.D., G.E.D., M.Z., and D.S.K. ana- lyzed data; and G.S. wrote the paper. The authors declare no conflict of interest. Freely available online through the PNAS open access option. *This Direct Submission article had a prearranged editor. 1 G.S. and C.T. contributed equally to this work. 2 To whom correspondence should be addressed. E-mail: guy.servant@senomyx.com This article contains supporting information online at www.pnas.org/cgi/content/full/ 0911670107/DCSupplemental. 4746–4751 | PNAS | March 9, 2010 | vol. 107 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.0911670107 Downloaded by guest on June 1, 2020