Heterologous expression of many G‐protein coupled receptors (GPCRs) is a major bottleneck in drug discovery efforts for therapeutic development of the receptor. The goal of this study was to utilize domains from a well‐trafficked GPCR to aid in improving the trafficking of a related receptor. The adenosine A2a receptor (A2aR) shows exceptional expression and trafficking to the plasma membrane in yeast; however, this is not the case for other adenosine receptors. A2aR has a longer C‐terminus than the other adenosine receptors, which may contribute to its exceptional trafficking to the plasma membrane. To test the possibility to improve trafficking of the adenosine A1 receptor (A1R), chimeric receptors containing the seven transmembrane domains of A1R and the full‐length or truncated A2aR C‐terminus were constructed. The chimeric receptor showed improved localization to the plasma membrane and was capable of binding radioligand with native A1R affinity. Functionally active A1R receptor variants were produced at a theoretical yield of 95 pmol/mg total membrane protein, estimated using radioligand binding data, which are greater than three‐fold higher than previously reported yields from other heterologous expression systems, and should facilitate biophysical characterization and drug discovery efforts. © 2018 American Institute of Chemical Engineers AIChE J, 64: 4297–4307, 2018

The Schiff base and its metal complexes are extensively studied for their adaptable metal chelating properties. Their ability to alter the design and modify it for various organic and biological applications makes them versatile compounds. The chiral Schiff base and their metal complexes possess excellent stability in very high temperatures, making them a suitable candidate for organocatalysts in reactions involving harsh conditions. Besides, these chiral complexes of Schiff base can transform their chirality in the final product formed in different chemical reactions. This stereo-induction by the chiral metal complexes of Schiff base is considerably valuable for synthesizing high optically active compounds in the field of medicinal and natural product synthesis. Thus, this review emphasizes the latest and relevant literature concerning the asymmetric catalysis of chiral Schiff base ligand-metal complexes in various synthetic organic transformation reactions. The synthesis and the catalytic mechanisms of various reactions are discussed, including their enantioselectivities, diastereoselectivities, regioselectivities, and stereoselectivities of different complexes to organic transformation.

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Recent efforts to determine the high-resolution crystal structures for the adenosine receptors (A1R and A2AR) have utilized modifications to the native receptors in order to facilitate receptor crystallization and structure determination. One common modification is a truncation of the unstructured C-terminus, which has been utilized for all the adenosine receptor crystal structures obtained to date. Ligand binding for this truncated receptor has been shown to be similar to full-length receptor for A2AR. However, the C-terminus has been identified as a location for protein-protein interactions that may be critical for the physiological function of these important drug targets. We show that variants with A2AR C-terminal truncations lacked cAMP-linked signaling compared to the full-length receptor constructs transfected into mammalian cells (HEK-293). In addition, we show that in a humanized yeast system, the absence of the full-length C-terminus affected downstream signaling using a yeast MAPK response-based fluorescence assay, though full-length receptors showed native-like G-protein coupling. To further study the G protein coupling, we used this humanized yeast platform to explore coupling to human-yeast G-protein chimeras in a cellular context. Although the C-terminus was essential for Gα protein-associated signaling, chimeras of A1R with a C-terminus of A2AR coupled to the A1R-specific Gα (i.e., Gαi1 versus Gαs). This surprising result suggests that the C-terminus is important in the signaling strength, but not specificity, of the Gα protein interaction. This result has further implications in drug discovery, both in enabling the experimental use of chimeras for ligand design, and in the cautious interpretation of structure-based drug design using truncated receptors.

The adenosine A3 receptor (A3R) is the only adenosine receptor subtype to be overexpressed in inflammatory and cancer cells and therefore is considered a novel and promising therapeutic target for inflammatory diseases and cancer. Heterologous expression of A3R at levels to allow biophysical characterization is a major bottleneck in structure-guided drug discovery efforts. Here, we apply protein engineering using chimeric receptors to improve expression and activity in yeast. Previously we had reported improved expression and trafficking of the chimeric A1R variant using a similar approach. In this report, we constructed chimeric A3/A2AR comprising the N-terminus and transmembrane domains from A3R (residues 1–284) and the cytoplasmic C-terminus of the A2AR (residues 291–412). The chimeric receptor showed approximately 2-fold improved expression with a 2-fold decreased unfolded protein response when compared to wild type A3R. Moreover, by varying culture conditions such as initial cell density and induction temperature a further 1.7-fold increase in total receptor yields was obtained. We observed native-like coupling of the chimeric receptor to Gai-Gpa1 in engineered yeast strains, activating the downstream, modified MAPK pathway. This strategy of utilizing chimeric receptor variants in yeast thus provides an exciting opportunity to improve expression and activity of “difficult-to-express” receptors, expanding the opportunity for utilizing yeast in drug discovery.