By Dean A. Haycock

Special to BioWorld Today

Presumably soon after humans began cooking, they began experimenting with food. Long experience around an open fire leads to the similar experimentation in kitchens and then in the labs of food and beverage companies.

"The food and flavor industry matured in terms of its basic research tools about 1,000 years ago," said Rick Lufkin, president of Linguagen Corp., a Nutley, N.J., biotech start-up incorporated in 1995. Lufkin is referring to the refined and talented human tongues and the taste preferences of animals that have long led the search for better flavor.

Along the way, scientists have learned to break down the modalities of taste into four or five major categories. Sweet, sour, bitter and salt are the most familiar. Today, some would add taste for glutamate and fat among them.

"Molecular biology was applied to this area only in the 1990s," Ding Ming, research manager at Novartis Consumer Health in Summit, N.J., told BioWorld Today. "Obviously that is very late compared to other areas. This is a slow area but I think it is emerging."

Ming is the first author of "Blocking taste receptor activation of gustducin inhibits gustatory responses to bitter compounds" in the Aug. 17, 1999, issue of the Proceedings of the National Academy of Sciences (PNAS). In it, Ming, Yuzo Ninomiya and Robert Margolskee describe the first in vitro assay that correlates a flavor with a biochemically measurable response.

"People have known about the taste modalities for a long time," Ming said. "We know there must be a bioprocess to recognize the ligands and transmit the signals through the tongue epithelium back into your brain. We know there must be a receptor mechanism there but so far no one has demonstrated a single molecule that functions as a receptor for the taste agent."

Ming and his co-authors came up with the next best thing. Margolskee, associate investigator at the Howard Hughes Medical Institute and professor of physiology and biophysics, and of pharmacology at the Mount Sinai School of Medicine in New York, explained, "We are using the next step down from the receptor, a G protein called gustducin which is a gustatory or taste cell-expressed G protein. We are using it as an assay for receptor activity."

G proteins act as "second messengers," or transducers, of receptor-initiated responses. They help convey messages from a receptor located in the outer membrane of a cell to its interior components such as enzymes. Since they have an affinity for guanine nucleotides, they have been dubbed G proteins.

The novel assay can be used to identify compounds and define structures that could be used, for example, to block the bitter taste of artificial sweeteners or cough medicines.

"This paper," Lufkin said, "documents the first tentative, probably not commercializable, proof of principal that blockade is possible. The commercial applications of bitter blockade are really the foundation of Linguagen's business concept. We believe there are many, many applications of blocking bitter that have commercial applications; for example, liquid oral dosage form of pharmaceuticals such as cough syrup."

Margolskee, who serves as a consultant to Linguagen Corp., also is seeking the bitter taste receptor itself. "The taste receptor for bitter has not been biochemically characterized nor has it been cloned. We have used the same kind of assay in an attempt to physically characterize the receptor and isolate it and purify it and clone it. Those experiments are still going on and looking pretty promising. But until such point as it is cloned, we have to use this indirect means, this G-protein dependent assay, to monitor the receptor's own activity," he said.

The researchers monitored the activation of a G protein called gustducin, which is present in taste receptor cells on the tongue. Compounds that taste bitter to humans activate gustducin and another G protein, transducin, in bovine taste membranes. The new in vitro assay, using the G proteins as indicators, can indirectly identify compounds that inhibit the bitter taste receptor when they are activated by bitter compounds such as quinine, strychnine and atropine. The assay identified AMP as one such compound that blocks G-protein activation induced by bitter tasting substances. AMP was also shown to inhibit behavioral and electrophysiological responses in living mice to bitter substances. It could not block the tastes of salt, sour or sweet, however. GMP, a compound chemically very similar AMP, was not capable of blocking bitter-induced G-protein activation. The authors conclude that AMP and some other select compounds may be promising taste modifiers.

The literature suggests that G proteins are involved in sweet responses as well as bitter responses. Salt and sour tastes, however, seem to be mediated by ion channels. The same approach will not work for those modalities. Glutamate taste, however, may be amenable to this same type of approach with a receptor-G-protein pathway, according to Margolskee.

"Once you have applied the tools of molecular biology and the concept of high-throughput screening to the world of taste and flavor," Lufkin said, "you can therefore screen for taste active compounds at a rate several orders of magnitude faster than heretofore has been possible in the flavor industry. We are seeking new flavor-active, taste-active compounds that may well be there. They are most likely compounds buried in natural products."

Margolskee listed some potential applications of the assay. "There are a number of cases where bitterness is a problem," he said. "A lot of oral pharmaceuticals, cough medicines, cold medicines, even certain prescription compounds that either have a restricted solubility or they have to be administered as an oral medicine. Many of those will have a bitter taste including certain AIDS medicines. Something like AMP, or something that might be more potent than AMP, could be useful when mixed with cough medicines or protease inhibitors to get rid of that bitter taste and improve palatability and improve patient compliance."

Both Ming and Margolskee, whose research was supported by grants from the National Institutes of Health, noted there are not many groups taking molecular biology approaches to understanding taste transduction in either academic or biotechnology settings.