Discovery and Development of S6821 and S7958 as Potent TAS2R8 Antagonists.

In humans, bitter taste is mediated by 25 TAS2Rs. Many compounds, including certain active pharmaceutical ingredients, excipients, and nutraceuticals, impart their bitter taste (or in part) through TAS2R8 activation. However, effective TAS2R8 blockers that can either suppress or reduce the bitterness of these compounds have not been described. We are hereby reporting a series of novel 3-(pyrazol-4-yl) imidazolidine-2,4-diones as potent and selective TAS2R8 antagonists. In human sensory tests, S6821 and S7958, two of the most potent analogues from the series, demonstrated efficacy in blocking TAS2R8-mediated bitterness and were selected for development. Following data evaluation by expert panels of a number of national and multinational regulatory bodies, including the US, the EU, and Japan, S6821 and S7958 were approved as safe under conditions of intended use as bitter taste blockers.

Toxicological evaluation of the flavour ingredient N-(1-((4-amino-2,2-dioxido-1H-benzo[c][1,2,6]thiadiazin-5-yl)oxy)-2-methylpropan-2-yl)-2,6-dimethylisonicotinamide (S2218).

A toxicological evaluation of N-(1-((4-amino-2,2-dioxido-1H-benzo[c][1,2,6]thiadiazin-5-yl)oxy)-2-methylpropan-2-yl)-2,6-dimethylisonicotinamide (S2218; CAS 1622458-34-7), a flavour with modifying properties, was completed for the purpose of assessing its safety for use in food and beverage applications. S2218 exhibited minimal oxidative metabolism in vitro, and in rat pharmacokinetic studies, the compound was poorly orally bioavailable and rapidly eliminated. S2218 was not found to be mutagenic in an in vitro bacterial reverse mutation assay, and was found to be neither clastogenic nor aneugenic in an in vitro mammalian cell micronucleus assay. In subchronic oral toxicity studies in male and female rats, the NOAEL was 140 mg/kg bw/day (highest dose tested) for S2218 sulfate salt (S8069) when administered as a food ad-mix for 13 consecutive weeks. Furthermore, S2218 sulfate salt demonstrated a lack of maternal toxicity, as well as adverse effects on fetal morphology at the highest dose tested, providing a NOAEL of 1000 mg/kg bw/day for both maternal toxicity and embryo/fetal development when administered orally during gestation to pregnant rats.

Deficits in synaptic transmission and learning in amyloid precursor protein (APP) transgenic mice require C-terminal cleavage of APP.

Synaptic dysfunction has been shown to be one of the earliest correlates of disease progression in animal models of Alzheimer's disease. Amyloid-beta protein (Abeta) is thought to play an important role in disease-related synaptic dysfunction, but the mechanism by which Abeta leads to synaptic dysfunction is not understood. Here we describe evidence that cleavage of APP in the C terminus may be necessary for the deficits present in APP transgenic mice. In APP transgenic mice with a mutated cleavage site at amino acid 664, normal synaptic transmission, synaptic plasticity, and learning were maintained despite the presence of elevated levels of APP, Abeta42, and even plaque accumulation. These results indicate that cleavage of APP may play a critical role in the development of synaptic and behavioral dysfunction in APP transgenic mice.

B-ephrin reverse signaling is required for NMDA-independent long-term potentiation of mossy fibers in the hippocampus.

The mossy fiber to CA3 pyramidal neuron synapse in the hippocampus displays an atypical form of NMDA receptor-independent long-term potentiation (LTP). Plasticity at this synapse is expressed in the presynaptic terminal as an elevated probability of neurotransmitter release. However, evidence indicates that postsynaptic mechanisms and trans-synaptic signaling through an association between postsynaptic EphB receptors and presynaptic B-ephrins are necessary for the induction of LTP. Here we show that ephrin-B3 protein is highly expressed in mossy fiber axons and terminals. There are specific deficits in mossy fiber LTP in mice in which the cytoplasmic C-terminal signaling domain of the ephrin-B3 protein is replaced with beta-galactosidase. These deficits are not observed in ephrin-B3 null mutant mice because of functional redundancy by virtue of other B-ephrins. These results indicate that B-ephrin reverse signaling into the presynaptic mossy fiber bouton is required for the induction of NMDA receptor-independent LTP at this synapse.

DPP10 modulates Kv4-mediated A-type potassium channels.

A new member of a family of proteins characterized by structural similarity to dipeptidyl peptidase (DPP) IV known as DPP10 was recently identified and linked to asthma susceptibility; however, the cellular functions of DPP10 are thus far unknown. DPP10 is highly homologous to subfamily member DPPX, which we previously reported as a modulator of Kv4-mediated A-type potassium channels (Nadal, M. S., Ozaita, A., Amarillo, Y., Vega-Saenz de Miera, E., Ma, Y., Mo, W., Goldberg, E. M., Misumi, Y., Ikehara, Y., Neubert, T. A., and Rudy, B. (2003) Neuron. 37, 449-461). We studied the ability of DPP10 protein to modulate the properties of Kv4.2 channels in heterologous expression systems. We found DPP10 activity to be nearly identical to DPPX activity and significantly different from DPPIV activity. DPPX and DPP10 facilitated Kv4.2 protein trafficking to the cell membrane, increased A-type current magnitude, and modified the voltage dependence and kinetic properties of the current such that they resembled the properties of A-type currents recorded in neurons in the central nervous system. Using in situ hybridization, we found DPP10 to be prominently expressed in brain neuronal populations that also express Kv4 subunits. Furthermore, DPP10 was detected in immunoprecipitated Kv4.2 channel complexes from rat brain membranes, confirming the association of DPP10 proteins with native Kv4.2 channels. These experiments suggest that DPP10 contributes to the molecular composition of A-type currents in the central nervous system. To dissect the structural determinants of these integral accessory proteins, we constructed chimeras of DPPX, DPP10, and DPPIV lacking the extracellular domain. Chimeras of DPPX and DPP10, but not DPPIV, were able to modulate the properties of Kv4.2 channels, highlighting the importance of the intracellular and transmembrane domains in this activity.