Although the advantages of sp3‐rich, sterically complicated molecules in drug development have been pointed out, modern screening libraries are filled with planar, sp2‐rich components. Compounds that are sp3‐rich are difficult to synthesize, and thus we aimed to invent an efficient method to construct sp3‐rich libraries. By modifying sp3‐rich 7‐azanorbornane scaffolds through click chemistry, we efficiently prepared a small set of compounds. These compounds were not only sp3‐rich, but also had sufficient “lead‐like” properties in view of molecular weights and hydrophobicity. Screening assays of this library provided weak κ opioid receptor agonists and growth hormone secretagogue receptor agonists with high hit rates. These results indicate that the 7‐azanorbornane scaffold may be a “privileged structure” for lead identification in drug discovery.
Ghrelin is a pleiotropic feeding hormone which also has a pivotal role in the central nervous system. Upon the activation of its receptor, growth hormone secretagogue receptor (GHSR), the Gαq/11‐mediated and the β‐arrestin‐mediated signaling pathways are activated. As the β‐arrestin pathway is a potential drug target, there is a strong need for β‐arrestin‐biased GHSR modulators. Activation of the β‐arrestin pathway should inhibit the Gαq/11‐mediated calcium flux through internalization of the receptor. Hence, we used the antagonistic activity in the calcium assay as the first screening for the β‐arrestin activation. By conducting the second screening assay for the β‐arrestin activation based on extracellular signal regulated kinase (ERK) 1/2 phosphorylation, we discovered a putative β‐arrestin‐biased superagonist. The activity of the compound was not completely blocked with the competitive antagonist, which implies that the effect is mediated, at least partly, by allosteric binding of the compound.
One of the most prominent features of microinjection is that foreign macromolecules can be introduced directly into specific intracellular compartments of plant cells. Target sites for injection have been vacuole, 1) cytoplasm, 2) and nucleus3) of plant protoplasts. In the previous paper, 4) the authors applied the technique to callus cells and established an intranuclear microinjection for transforming plant cells. Moreover, our previous works have presented the system for a cytoplasmic microinjection by which tobacco mosaic virus was introduced into single cells5) or cell-aggregates6'7) obtained from tomato callus tissues. These approaches enabled us to examine the mechanisms of virus multiplication or translocation and the expression of virus-disease resistance in the multi-cellular system of cultured plant cells. For further expanding the applicability of this technique to callus tissues, the present study describes the transformation of constituent cells of tomato callus aggregates by an intranuclear microinjection.Friable callus tissues obtained from leaf-explants of tomatos) (Lycopersicon esculentum cv. Fukuju No. 2) were used in the present experiment. Callus tissues were shake-cultured in liquid MurashigeSkoog9) (MS) medium supplemented with 0.1 ig/ml 2, 4-dichlorophenoxy acetic acid and 0.05 ig/ml kinetin (pH 6. 5), and filtered with a stainless sieve (pore size, 250 im diameter). Single and 2-to 10-cell-aggregates were involved in cell suspension which passed through a sieve. For microinjection, cellaggregates were embedded in agar medium and cultured using the plate culture method previously described.4) Transformation vector for higher plants, pBI 12110' containing such two reporter genes as NPT II gene for kanamycin resistance and GUS gene for /3-glucuronidase production was injected into the nuclei of constituent cells of the aggregate according to the conditions for microinjection previously reported. 4) After injection, cells were incubated at 26C for 2 days, subjected to 200ag/ml kanamycin and vitally stained with fluorescenn diacetate (FDA).11) Alternatively, injected cells were incubated and fixed with formaldehyde for histochemical analysis of 9-glucuronidase activity.12)In the culture method used in the present study, as described in the previous paper, 4) it was possible that injected cells of the aggregates were specified according to the grid lines scored onto the surface of a Petri dish. This method allowed us to critically distinguish between injected and non-injected cells of the aggregates and to analyze an expression of introduced genes in these cells. Figure 1 shows light and fluorescent micrographs of pBI 121-injected cells which were stained with FDA. The data clearly indicated that only injected cells of the aggregate expressed a resistance against kanamycin. Moreover, Fig. 2 shows an enzymatic activity of /3-glucuronidase in injected cell of the aggregate. In the present study, each 500 aggregates was used for an assay of kanamycin resistance or /3-glucuronidase production and ...
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