Abstract
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 shallower. F-actin staining revealed that DMSO-treated OCs displayed clear actin rings, and F-actin forms a peripheral sealing zone. By contrast, IN04-treated OCs showed disarranged F-actin in the cytoplasm, and F-actin failed to form a sealing zone on bone slices. IN04 treatment had no effects on OC-derived coupling factor production nor on osteoblast nodule formation. Our data indicate IN04 is a potent inhibitor of LRRK1, suppressing OC function with no effect on OC formation.
Highlights
Osteoporosis is an aging-related major health problem in the United States
Homology-based protein modeling of the hLRRK1 KD indicated that the Leucine rich repeat kinase 1 (LRRK1) KD contains an extra loop in the activation site compared with the hLRRK2 KD [18], and it has a narrower active pocket for ligand binding (Figure 1A)
To test if IN04 docking to the active pocket inhibits ATP binding to the LRRK1 KD, we first purified recombinant protein from E. coli and used the protein for an in vitro pulldown assay
Summary
There are two major known causes of osteoporosis: low peak bone mineral density (BMD), typically achieved around the age of 30, and a high rate of bone loss that occurs after menopause and during the natural process of aging. Effective inhibitors of bone resorption such as bisphosphonates have widely been used in the clinic to treat high-turnover bone diseases. Treatment with bisphosphonates results in suppression of both bone resorption and bone formation and blunts the anabolic actions of PTH [1]. Because bisphosphonates are incorporated within the bone matrix with high affinity, long term treatment with these drugs may impair fracture healing, cause jaw osteonecrosis, and increase the risk for atypical fractures of the femur [2,3,4,5,6,7]. Novel anti-resorptive www.aging-us.com molecules that avoid anti-anabolic actions are needed as therapeutics to increase bone mass and reduce fracture risk
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