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Chapter 24 Development of Transient Receptor Potential Melanostatin 5 Modulators for Sweetness Enhancement 1

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Downloaded by IOWA STATE UNIV on April 19, 2017 | http://pubs.acs.org Publication Date: March 4, 2008 | doi: 10.1021/bk-2008-0979.ch024

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R. W. Bryant , K. S. Atwal , I. Bakaj , M. T. Buber , S. Carlucci , R. Cerne , R. Cortés , H. R. Devantier , C. J. Hendrix , S. P. Lee , R. K. Palmer , C. Wilson , Q. Yang , and F. R. Salemme 1

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Redpoint Bio Corporation (formerly Linguagen Corporation), 2005 Eastpark Boulevard, Cranbury, NJ 08512-3515 Current address: Merck & Company, Inc., 126 East Lincoln Avenue, Rahway,NJ07065 2

The discovery o f a family o f G protein-coupled receptors (GPCRs) that can bind sweet, bitter, or umami tastants has established that these taste sensations are mediated by classical signal transduction cascades. Proteins downstream from the tastant binding GPCRs, such as the G protein gustducin, phospholipase C (PLC ) and the Transient Receptor Potential Melanostatin 5 (TRPM5) ion channel, have been identified as critical components in the transduction of these taste sensations. Using modern pharmaceutical discovery technology, we have discovered prototype compounds that specifically enhance TRPM5 activity in the presence of low levels of surrogate tastants. Enhancers operating through this novel mechanism could potentially allow for full taste sensations to be experienced from reduced concentrations of nutritive sweeteners. α

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© 2008 American Chemical Society

Weerasinghe and DuBois; Sweetness and Sweeteners ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


387 Excess sugar in food has been identified as a key contributor to important public health problems facing developed countries worldwide. These include the rising trend in obesity (1) and its associated increased incidence of diabetes and other diseases (2), as well as oral health concerns (3). A s a result, there is a growing interest, both from the general public and from the government, to develop cost-effective methods of reducing the amount of caloric sweetener that is added to processed foods and beverages while retaining their palatability and nutritional value. The food industry has made a large research and development investment that has led to the production of acceptable low-calorie sweeteners. Indeed, a large number of synthetic sweeteners have been identified and several have been successfully commercialized (4). Although many sugar substitutes are currently available, no one sweetener is thus far ideal. In particular, many have off-tastes and do not accurately replicate the temporal sensory experience of real sugar (5-8). A n alternative to artificial sweeteners is the identification of non-nutritive "sweetness enhancers" that can be used in concert with reduced quantities of nutritive sweeteners to enhance the perception of sweet taste. The ideal sweetness enhancer would have no taste of its own, but would increase the natural sweetness of sugar while preserving the temporal characteristics of the sugar taste. Although an enhancer providing a 1.5x reduction in the amount of sugar required for a specific sweetening application could be commercially useful, a larger reduction factor of 2-5x is more desirable from the public health perspective. Research is currently in progress to identify molecules that could function as sweetness enhancers. One strategy involves identification of positive allosteric modulators of the G-protein coupled receptors (GPCRs) that bind sweet tastants (9). We describe an alternative approach involving identification of positive modulators of taste signaling components acting downstream of taste G P C R receptors. We focused initially on finding positive modulators of the Transient Receptor Potential Melanostatin 5 (TRPM5) ion channel, and have identified prototype compounds which selectively and strongly enhance the response of T R P M 5 to low levels of surrogate tastants.

Signal transduction pathways involved in T R P M 5 activation during sweet taste reception The signaling cascade downstream of G P C R activation provides several targets for discovery of taste modulation, namely GPCRs, signal transduction proteins, and ion channels. The first step in the proposed pathway that ultimately leads to the perception of taste is the binding of a tastant to the extracellular domain of a G P C R of the T1R family (10, 11) (Figure 1). This


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To Brain Figure I. Potential targets for small molecule discovery in taste cell sensing.

family includes different subunits that form heteromeric receptors which mediate individual taste modalities. For example, sweet taste in mice and humans is transduced by the T1R2:T1R3 receptor (12, 13), while the umami taste is transduced by the T1R1.T1R3 receptor (13, 14). Approximately 25 cognate bitter receptors of the T2Rn class also exist (15-17). Sweet tastant binding to the T1R2:T1R3 receptor leads to the dissociation of heterotrimeric G proteins into their α and βγ subunits (reviewed in (11)), although it is not completely clear yet which specific α and βγ subunits are involved (18-23). Following a classical signal transduction cascade, the βγ subunit activates PLCp (24). This leads to inositol triphosphate (IP ) generation, which then results in C a release from intracellular stores. This C a release is presumably mediated by the type HI IP receptor (IP3R3) (25). The resultant increase in intracellular calcium concentration ([Ca ]0 causes the TRPM5 channel to open (26). TRPM5 activation depolarizes the taste cell which contributes to neurotransmitter release, thus transmitting tastant-mediated taste bud activation to the gustatory nerves. While GPCRs have been very successful targets for pharmaceutical development (27, 28), discovery programs for direct modulators of signal transduction targets such as G-proteins and phospholipases have not been as productive. Ion channels, however, have been successful targets for discovery of modulators in many areas of pharmaceutical research (29-32). 2

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389 T R P M 5 is a monovalent cation channel gated by Ca (26, 33, 34), temperature (35), voltage (34), pH (36), and PIP (33). Expression patterns suggest, and knockout animal data validates, the importance of the T R P M 5 protein in taste transduction* Northern blot experiments show that T R P M 5 m R N A is selectively expressed in taste tissue compared to non-taste tissue (37), and immunohistological staining of circumvallate papilla tissue localizes the protein to taste buds (38). In addition, the TRPM5 protein co-localizes with G i3> α-gustducin, and PLCp (38), strongly implicating it as a component of the taste signaling pathway in which these three signaling molecules are involved. Consistent with these observations, TRPM5 knockout mice have greatly reduced responses to sweet, bitter, and umami tastants (39, 40). For example, there is a six-fold reduction in the chorda tympani nerve response to 400 m M sucrose and a seven-fold reduction in the glossopharyngeal nerve response to 10 m M quinine in Trpm5 null mice, compared to wild-type control animals (39). The identification of small molecule modulators of the TRPM5 ion channel could therefore result in compounds that amplify the signaling cascade downstream of the sweet GPCRs. O f course, a modulator of TRPM5 could in theory also alter bitter and umami tastes, based on the mechanism described in Figure L The effect of TRPM5 enhancers would therefore be context sensitive. We describe here the results o f our discovery program, which has led to the identification of several TRPM5 enhancers that could represent a novel approach to sweetness enhancement particularly for beverages where sugar is the predominant taste. 2


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Results A Pharmaceutical-Based Discovery Process for Identification of Taste Modulators We have implemented a process for the identification of novel taste modulators using technology originally developed for pharmaceutical discovery. The discovery process starts with data from knockout animals validating T R P M 5 as a component of the sweet signal transduction pathway, as was previously discussed. To isolate TRPM5 as a target and develop an assay for the rapid screening of small molecules that would enhance TRPM5 activity, stably transfected cell lines were developed. The full-length hTRPM5 c D N A sequence was cloned from an intestinal c D N A library and subcloned into the pcDNA 3.2/v5-DEST vector (Invitrogen). This construct was transfected into human embryonic kidney-293 (HEK293) and Chinese hamster ovary (CHO) cell lines in order to generate stable cell lines expressing hTRPMS. Stably transfected clones were selected using Geneticin (Invitrogen). Control cell lines containing the empty vector and counter-screening cell lines expressing other T R P family receptors were developed following the same methods.



390 A n engineered cell assay, using stably transfected TRPM5-expressing cell lines, was established. This assay incorporated an automated high-throughput screening (HTS) process involving 384-well format liquid handling and a 384well fluorescence imaging plate reader (FLIPR-Tetra®, Molecular Devices). Data were processed using an Excel-based data analysis package and Accessbased data management system. Hits generated from the initial library screen were validated through chemical and pharmacologic means, incorporating L C / M S structure confirmation and specific assays to rule out blockade of calcium mobilization or artifactual membrane potential responses. The specificity of validated hits were examined with a range of counterscreens using cell lines expressing other ion channels having potential pharmacological relevance or potentially acting as modulators of taste sensations. Compounds were additionally evaluated using whole-cell electrophysiological recordings of the TRPM5-expressing cells together with the engineered counter-screen cell lines. Validated hits with appropriate specificity and potency that emerged from this process define the starting point for a chemical optimization process aimed at the eventual identification of development compounds with all of the properties (e.g. potency, safety, process stability, manufacturing feasibility) required for a commercially viable product.

High-Throughput Screening Assay Development for T R P M S Modulator Identification The FLIPR screening assay took advantage of the native expression of purinergic metabotropic P2Y receptors in the parental HEK293 (41) or C H O (42) cell lines used for stable recombinant expression of T R P M 5 . In the assay, 10 μΜ adenosine 5'- triphosphate (ATP) acted as a surrogate tastant molecule to activate the GPCR-initiated signaling cascade that causes cytoplasmic [Ca ]j to rise, thus employing an endogenous mechanism to activate the TRPM5 ion channel and depolarize the cell. The TRPM5-expressing cell lines were plated in 384-well format plates one day prior to the experiment. The cells were loaded with membrane potential or calcium-sensitive dyes one hour before the assay. Changes in fluorescence intensity resulting from the addition of A T P were then recorded by the FLIPR-Tetra™ instrument (Figure 2A). A T P stimulation caused an equivalent transient increase in [Ca ]i in T R P M 5 - and vector-transfected HEK293 cells, as shown in Figure 2B, indicating that both cell lines are responsive to A T P . A T P stimulation, however, resulted in significant membrane depolarization only in cells expressing T R P M 5 . The ratio of maximum amplitude response for the TRPMS transfectant was generally four-fold greater than the vector-transfected cells (Figure 2C). This assay provided very reliable data, reporting with Z'>0.5 for over 90% of plates. 2+

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Figure 2. Basis of TRPMS HTS membrane potential dye assay. A. Activation strategy for TRPMS-exprèssing HEK293 cells utilizing endogenous GPCRs. B. [Ca *]j increase as reported by calcium dye in FLIPR assay. C. Membrane potential change as reported by membrane potential dye in FLIPR assay. 2

Following this development and optimization process, the FLIPR assay was used to screen a chemical library comprised of 83,580 diverse synthetic compounds. In this two-addition FLIPR assay, library compounds were applied at approximately 10 μΜ, followed by addition of 10 μΜ A T P three minutes later. Figure 3 shows the results of a portion of the compounds (59,238) from the initial screen as a frequency distribution of the percentage of inhibition of the TRPMS response. A response of less than ± 2 5 % inhibition indicated an inactive compound. Potential T R P M 5 blockers showed greater than 50% inhibition of T R P M S activation, while enhancers showed -50% inhibition or better; i.e. they increased the activation of TRPMS in the assay. At a compound concentration of approximately 10 μΜ, most compounds were shown to be inactive. The screen identified 601 compounds, or 0.7% of the compounds in the library, as potential TRPMS blockers (

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