Amino acid derivatives as bitter taste receptor (T2R) blockers.

In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only five antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis, we elucidated the ligand-binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn-173 and Thr-174 are essential for quinine binding. Guided by a molecular model of T2R4, a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays that led to identification of γ-aminobutryic acid (GABA) and Nα,Nα-bis(carboxymethyl)-L-lysine (BCML) as competitive inhibitors of quinine-activated T2R4 with an IC50 of 3.2 ± 0.3 µM and 59 ± 18 nM, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, whereas BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala-90 and Lys-270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understanding the roles of T2Rs in extraoral tissues.

Reactive astrogliosis after spinal cord injury-beneficial and detrimental effects.

Reactive astrogliosis is a pathologic hallmark of spinal cord injury (SCI). It is characterised by profound morphological, molecular, and functional changes in astrocytes that occur within hours of SCI and evolves as time elapses after injury. Astrogliosis is a defense mechanism to minimize and repair the initial damage but eventually leads to some detrimental effects. Reactive astrocytes secrete a plethora of both growth promoting and inhibitory factors after SCI. However, the production of inhibitory components surpasses the growth stimulating factors, thus, causing inhibitory effects. In severe cases of injury, astrogliosis results in the formation of irreversible glial scarring that acts as regeneration barrier due to the expression of inhibitory components such as chondroitin sulfate proteoglycans. Scar formation was therefore recognized from a negative perspective for many years. Accumulating evidence from pharmacological and genetic studies now signifies the importance of astrogliosis and its timing for spinal cord repair. These studies have advanced our knowledge regarding signaling pathways and molecular mediators, which trigger and modulate reactive astrocytes and scar formation. In this review, we discuss the recent advances in this field. We also review therapeutic strategies that have been developed to target astrocytes reactivity and glial scaring in the environment of SCI. Astrocytes play pivotal roles in governing SCI mechanisms, and it is therefore crucial to understand how their activities can be targeted efficiently to harness their potential for repair and regeneration after SCI.