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Gene Synthesis> | Plasmid of pUC57-hLrrk1 KD encoding human LRRK1 kinase domain (hLRRK1 KD) from amino acids 1240–1530 was synthesized by GenScript (Piscataway, NJ) and sub-cloned into the pET28a plasmid with 6xHis tag at the N-terminus for recombinant protein expression in E. coli | Get A Quote |
We used TGFβ activation kinase 1 as a template to build a 3D structure of the human LRRK1 kinase domain (hLRRK1 KD) and performed small molecule docking. One of the chemicals (IN04) that docked into the pocket was chosen for evaluation of biological effects on osteoclasts (OCs) in vitro. INO4 at 16 nM completely blocked ATP binding to hLRRK1 KD in an in vitro pulldown assay. In differentiation and pit assays, while the number of OCs on bone slices were comparable for OCs treated with IN04 and DMSO, IN04 treatment of OCs significantly impaired their ability to resorb bone. The area of pits on bone slices was reduced by 43% at 5 μM and 83% at 10 μM as compared to DMSO. Individual pits appeared smaller and shal... More