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Fine Chemicals

A welcome reception

Flavour chemists are adopting development techniques from the drugs industry to speed up discovery of new flavour compounds. Dr Cynthia Challener reports

The taste process involves numerous taste receptors located in the taste buds, as well as up to 400 odour receptors, and thus is highly complex. Up to six basic flavours have been identified, including sweet, salty, bitter, savoury (also known as umami), sour and possibly also fat.

Because little was understood how these different tastes were experienced, developing new flavour compounds traditionally involved synthesising target compounds and evaluating them through actual taste tests; only about 1,000 compounds/year could be investigated. Today, much more is known about how some of the basic taste modalities are encoded.

In the case of the sweet, bitter and umami flavours, G-protein-coupled receptors (GPCRs) on the surface of a taste receptor cell have been shown to bind flavour compounds specific for each modality. This binding activates a G-protein which, in turn, activates a cell signaling pathway that ultimately produces a nerve impulse that is sensed as taste.

Each functional receptor for sweet and umami is a heterodimer incorporating a common receptor sub-unit (T1R3/T1R2 and T1R3/T1R1 respectively). Humans appear to detect bitter flavours via at least 25 different receptors.

No specific receptors have been identified yet for salt and sour tastes. These flavours are believed to be sensed via the activation of the taste cells expressing ion channels specific for sodium (salt) or hydrogen ions (sour). It is also believed that spicy and cool flavours are detected through ligand-gated ion channels called TRP channels.

GPCRs are a very large group of trans-membrane receptors that sense molecules outside the cell. When a specific molecule is detected through ligand binding, signal transduction pathways inside the cell are activated, leading to specific cellular responses. GPCRs play a role in many diseases and numerous drug development efforts are targeting compounds from small molecule to peptides designed to interact with them.

It was therefore only natural for flavour developers looking for new compounds that could bind to taste receptors to turn to high throughput screening techniques that had been used to develop drug compounds targeting GPCRs. With these methods, one company active in this field, Senomyx, says that it is now able to investigate up to 10,000 compounds/week.

The new screening approach relies on cell-based assays, in which engineered cells express the receptor for a single taste modality through the introduction of a cloned receptor into a mammalian cell line, which translates the signal from the activated receptor into an increase in free intracellular calcium ion. Such increases are detected by fluorescence of calcium-sensitive dyes.

This method is similar to the methods used in the pharmaceuticals industry, but the compounds chosen for screening are very different. Desirable flavour substances are not required to enter the blood stream or to cross the blood brain barrier. In addition, they are preferably low molecular weight, non-volatile and highly water-soluble.

As with drugs, though, it is necessary for the compounds to be selective and only active on the desired taste receptor. They must also be stable under a wide range of temperatures and in hydrolytic conditions. Some flavours companies possess combinatorial libraries of both natural and synthetic candidates, developed in-house or from outside sources.

Senomyx and Redpoint Bio are two companies focused on using drug discovery techniques for identification of new flavour compounds. Givaudan and IFF, two of the largest global suppliers of flavours and fragrances, have also invested in high throughput technologies. Mark Dewis, vice president of flavours R&D at IFF, notes that this approach is one of many ways to identify new compounds but it is particularly effective in identifying disruptive technologies.

Taste science at Senomyx

 

Sweet enhancements

Identifiying flavour compounds that can enhance the sweet sensation without adding calories has been a main focus of R&D efforts at most flavours companies. Both governments and retailers have demanded that they reduce the quantity of sugar in many prepared foods to combat the growing waves of obesity and diabetes.

Companies have therefore searched for compounds that enhance the sweetness of both caloric and non-caloric sweeteners. These work because the taste receptors involved in the sweetness experience have multiple binding sites that interact with different types of substances.

Positive allosteric modulators are flavour enhancers that bind to the receptor somewhere other than the primary binding site and thus improve the binding affinity of the major tastant. As a result, less of the primary taste compound is required to evoke the same level of taste sensation. Redpoint Bio and Senomyx have both been active in this area.

For Redpoint Bio, the focus has been on the identification of natural sweeteners, as consumer interest in such products has become a growing trend, according to CEO Ray Salemme. Most recently, it has been working on RP44 or Rebaudioside C (Reb C). Reb C does not impart any sweetness itself but can help to reduce the need for sucrose, fructose and high fructose corn syrup by 25-30% when used at levels of ~150-250 ppm in products.

Reb C and Reb A are both steviol glycosides isolated from the native South American stevia plant and have received GRAS status from the FDA. This triggered a milestone payment from IFF  to Redpoint as part of a five-year agreement the two signed in June 2010, giving IFF exclusive rights to develop, manufacture and commercialise RP44 in virtually all food and beverage product categories.

Interestingly, RP44 was not identified via the high throughput assay technology. "Natural products, which are often based on plant extracts, are typically complex mixtures of substances and thus do not readily lend themselves to evaluation using typical cell-based methods," Salemme explains. Redpoint thus turned to an older approach, also developed in the pharmaceuticals industry, based on animal behavioural models.

After numerous tests to show that rats could reliably differentiate between sugar, salt and umami flavours and that known natural high-intensity sweeteners like Reb A tasted sweet to them, Redpoint used chemoinformatic methods to develop a library of natural compounds which were subsequently tested in dose-ranging experiments, in combination with nutritive sweeteners, to evaluate enhancement potential. RP44 was identified as an effective sweetness enhancer.

According to Senomyx, the use of high throughput screening to discover sweet enhancers is most effective when the screening system is based on the human sweet taste receptor. The company has patented composition claims covering these receptors, as well as claims directed to T1R1, T1R2 and T1R3 nucleic acid sequences that encode the sweet and umami receptors and the use of these receptors in screening assays designed to identify new flavour ingredients that induce or modulate sweet or umami taste.

Senomyx is therefore emphasising new natural flavours and is predominantly looking at plant extracts for this effort, according to CSO Don Karanewsky. The company has also found that a more efficient screening method is necessary with natural extracts containing multiple components because a higher false positive 'hit' rate is often observed when using the high throughput cell-based assays that are effective for screening synthetic compounds.

"To address this issue, we have developed additional secondary cell-based assays that can differentiate compounds that signal through the taste receptor and the other GPCRs present in the parental cell line," Karanewsky says. Of specific interest are natural compounds that enhance the sweetness of carbohydrates.

Molecular structure of Reb C

This effort also includes different screening paradigms, including different assays and new ways of evaluating how flavour compounds interact with the ligand binding domain of receptor sites other than in cell-based systems. The goal is to find a more direct method of measuring a substance's binding activity with the receptor rather than relying on downstream signaling pathways. Some of these new systems are based on technologies and methods used in drug discovery for identifying targets for GCPRs.

Meanwhile, Senomyx is moving forward with two synthetic sweetener enhancers for which it has received GRAS status. With S2383, the sucralose content of foods can be reduced by up to 75%, while S6973 makes it possible to reduce by 50% the sucrose present in product prototypes while maintaining the sweet taste of natural sugar. In tests, 9 ppm of S6973 in a 6% sucrose solution has a sweetness intensity equivalent to that of a 12% sucrose solution (p<0.05).

Firmenich is commercialising these two products and is expected to launch flavour systems containing S6973 to customers in 2011. The two companies are also collaborating to develop synthetic and natural flavour ingredients intended to enhance the taste of sucrose, fructose and Reb A. Senomyx has also identified some initial fructose enhancers that create a statistically significant amplification of the sweet taste of fructose, according to Karanewsky.

Other recent deals include a four-year agreement with Pepsi to develop and commercialise sweetener enhancers and natural sweeteners for non-alcoholic beverages. Both Pepsi and Firmenich will make royalty payments based on sales of products that include Senomyx's ingredients. The latter has already made some upfront payments to Senomyx.

 

Leaving bitterness behind

The need to reduce sugar in the diet has led to increased use of artificial sweeteners. Many of these, however, have a noticeably bitter aftertaste, while there is a drive to reduce the amount of these sweeteners used because of the demand for more natural ingredients. One approach has been to find bitter blockers that eliminate the bitter taste.

Via a collaboration with the German Institute of Human Nutrition, Givaudan identified two bitter receptors in the hTAS2R family of GPCRs. In June 2010, the company announced that by using a high throughput screening approach based on one of these - the more robust hTAS2R31 - it had found a new antagonist called GIV3727 that inhibits activation of the bitter receptor by the two common artificial sweeteners saccharin and acesulfame K.

GIV3727 also inhibits five additional hTAS2R receptors. Human sensory trials confirmed that it is active in vivo, according to Jay Slack, a principal investigator in Givaudan's Molecular Biotechnology group.

For its screening programme, the group engineered human embryonic kidney cells, commonly used in drug discovery for their robustness and lack of endogenous receptors. The receptor cell and a reporter G-protein were inserted into the HEK cells and tied to a calcium-signalling pathway.  The cells were then exposed to both the sweetener and the compound.

Substances identified as potential candidates were then evaluated for unwanted activity against other receptors, toxic side effects and other typical medicinal chemistry type studies. Givaudan has received GRAS approval for the new bitter blocker and its customers are now commercialising products containing it.

Senomyx also received GRAS approval in 2010 for two bitter blockers, S6821 and S7958, which reduce the bitter aftertaste associated with Reb A and the bitter notes associated with hydrolysed soy and whey proteins, menthol, caffeine and cocoa, according to Karanewsky. The GRAS designations also triggered a milestone payment from a collaborative partner.

Senomyx is working on synthetic sweeteners

 

A salty challenge

Reducing salt in foods is another important goal of consumers looking to follow a more healthy diet. As a result, companies are seeking a better understanding of this complex taste process. In addition, many countries around the world will be introducing stricter guidelines on salt content in foods in the near future.

Finding a solution is not easy, though. "There are no clear examples of natural products that provide salty flavours comparable in effect to sweet natural products like the steviosides that interact with sweet receptors," Salemme says. Redpoint is exploring the use of its behavior models for identification of natural salt enhancers as it is with the sweet modality.

"Savoury flavours have been used in the past to reduce the amount of salt in foods, but the mechanism by which this approach works remains unclear," adds Salemme. "It has been difficult to define a specific salt taste receptor, making the engineered cell-assay approach questionable. We are consequently using our rodent-based operant MOG technology."

As with its sweetener programme, the rats have been trained to distinguish salt rapidly from other essential tastes in small samples with a high level of accuracy, providing an effective means of evaluating the taste properties of complex natural product extracts. The ultimate goal is to identify active components from ingredients already used in the food industry.

Meanwhile, Givaudan has developed what it calls "a unique sensory language" called Sense It Salt in order to create a more accurate description of the complex taste effects of salt in food. This is already being developed to reduce sodium levels in soups, sauces and snacks, ready-meals and cereals.

The system's 'Salt Curve' describes the taste effects of salt through distinct time phases that strongly influence the flavour profile of the food product application being developed and for more accurate assessment of the consequences of reducing salt and the performance of flavours or ingredients that are used to restore the taste of low sodium products.

Senomyx is also working to understand how salty taste is perceived and the role that salt plays in other taste modalities, according to Karanewsky. The short-term target is to identify the protein or proteins involved in human salty taste perception, based on the work of one of the firm's scientific founders, Charles Zuker, who proved that more than one taste cell type is involved in salty taste in rodents. The receptors involved in rodent salty taste will probably have human homologues, he believes.

In addition to GPCRs, several industry programmes have used high-throughput cell-based assays to discover compounds active at the TRP channels, which are involved in sensing heat, cold and spicy compounds and are an important component of taste-signalling circuits on the tongue. One member of the TRP family, TRPM5, has also recently been found in the pancreas and gastrointestinal tract, suggesting a potential role in the regulation of metabolism and satiety.

Redpoint is consequently investigating the role of TRPM5 modulators in diabetes and obesity. TRPM5 may be involved in the secretion of important hormones like GLP1 and insulin that control sugar uptake and metabolism. Modulators could potentially find application as a new therapy for adult-onset diabetes and obesity, according to Salemme. Drugs acting as incretin and insulin secretogogues in response to the presence of sweeteners could be safer than existing oral secretoguge drugs for diabetes.

 

The genetics of taste

Clearly, the biochemistry of taste is very similar to the biochemical processes involved in disease development and treatment. The interest of flavour developers in the role of genetics in taste sensation is further confirmation of the close relationship between the two fields.

"Genetic variability in taste reception is significant," states Slack. For example, only some people are sensitive to the bitter aftertaste of artificial sweeteners. "We want to learn more about how these differences work and how and why they arise. This knowledge could be very valuable for the development of future flavour compounds and possibly to the drug industry for use in the discovery of novel therapeutics."

With the successes that these companies have had to date in adopting drug discovery technologies for flavour compound identification, it will not be surprising if advances in an understanding of taste perception will one day be used by the pharmaceutical industry for the development of novel and potentially safer therapeutics.

 

 

From Online Issue: April 2011