Class a gpcr-binding compound modifier
A compound and binding technology, applied in the direction of DNA / RNA fragments, antibodies, sugar derivatives, etc., can solve problems that are not suitable for G protein coupling or agonist binding
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Embodiment 1
[0165] 1.1 Expression of BLT1 variants for the purpose of BLT1 characterization
[0166] For evaluation by the consistency method ( Figure 8 A) Thermostability of designed guinea pig BLT1 variants, using QuikChange kit (Agilent) to thermally stabilized BLT1 variant cDNA previously constructed in pcDNA3 vector (removal of residues 1-14, with H83G / K88G / S309A mutations and N-terminal with FLAG tag) introduced point mutation (Hori, T. et al., Protein Expr. Purif. 72 (2010) 66-74, Hori, T. et al., Biochem. Biophys. Rep. 4 (2015) 243-249). COS-7 cells were transfected with expression vectors encoding BLT1 variants using Lipofectamine2000. To analyze the BIIL260-binding residue of BLT1, the cDNA introduction point of wild-type BLT1 with a FLAG tag at the N-terminus in the pcDNA3 vector was mutated using In-Fusion enzyme (Clontech). Expression vectors were transfected into HEK293 cells using polyethyleneimine. For the benzamidine competition assay, HEK293 cells were transfected w...
Embodiment 2
[0240] 2.1 Functional analysis of compounds having a structure in which class A GPCR-binding compounds bind to benzamidine groups
[0241] 2.1.1 Synthesis of compounds
[0242] A compound (compound 1) having a structure in which a benzamidine group is bound to carbazole (adrenoceptor ligand), and a structure in which a benzamidine group is bound to adenosine (A 2A Adenosine receptor ligand) compound (compound 2), and histamine (compound 3, 4) compounds having a structure in which a benzamidine group is bound to histamine (histamine receptor ligand). Proceed as follows. It should be noted that the following compounds 43-28 correspond to compound 4, compounds 42-28 correspond to compound 3, compounds 7-28 correspond to compound 2, and compounds 15-28 correspond to compound 1.
[0243] 2.1.1.1 Synthesis of compounds 43-28, 42-28
[0244]
[0245] Nitrobenzenesulfonylation of histamine dihydrochloride and Mitsunobu reaction followed by click reaction synthesis with an additi...
Embodiment 3
[0310] 3.1 Functional analysis of benzamidine derivatives
[0311] 3.1.1 Compounds
[0312] Compound 0 (benzamidine hydrochloride hydrate) and compounds 1, 3-5, 7, 11-13 (benzamidine derivatives) listed in Table 4 were purchased. Compound 14 (benzamidine derivative) used the above S1. The structure of the protonation state of each compound is shown in Figure 19 .
[0313] 【Table 4】
[0314] serial number Compound name seller Catalog number 0 Benzamidine Hydrochloride Hydrate sigma-aldrich corp. B6506-5G 1 1H-pyrazole-1-carboxamidine hydrochloride sigma-aldrich corp. 402516-10G 3 Pyrrolidine-1-carboxamidine hydroiodide sigma-aldrich corp. CDS014943-100MG 4 benzamide oxime sigma-aldrich corp. CDS001188-50MG 5 Piperidine-1-carboxamide hydrobromic acid sigma-aldrich corp. CDS014887-100MG 7 Cyclopropanecarboxamide hydrochloride Tokyo chemical C2344 11 1-(o-tolyl)biguanide Tokyo chemical T0314 ...
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