A small molecular inhibitor of LRRK1 identified by homology modeling and virtual screening suppresses osteoclast function, but not osteoclast differentiation, in vitro

Si, Ming-Jue; Zeng, Canjun; Goodluck, Helen; Shen, Sandi; Mohan, Subburaman; Xing, Weirong

doi:10.18632/aging.101977

Cited by 5 publications

(4 citation statements)

References 36 publications

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“…All inhibitors ablated LRRK2-mediated phosphorylation of Rab10 at Thr73 and LRRK2 at Ser935 in parallel experiments. An earlier study reported that a compound identified form a virtual homology screen termed iN04 inhibited LRRK1, but no experimental data was presented to demonstrate this [ 57 ]. We found that iN04, when used at 5 µM had no impact on LRRK1-mediated Rab7A phosphorylation ( Supplementary Figure S7D ).…”

Section: Resultsmentioning

confidence: 99%

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

Malik

Karapetsas

Nirujogi

et al. 2021

Biochemical Journal

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Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homologue of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson’s mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we were unable to confirm previous data suggesting that Rab7A phosphorylation is regulated by the PINK1/TBK1 axis. Altogether, these finding reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

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Section: Resultsmentioning

confidence: 99%

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

Malik

Karapetsas

Nirujogi

et al. 2021

Biochemical Journal

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“…(7) LRRK1 has hence been contemplated as a potential target for osteoanabolic therapy. (41) However, only few cases of LRRK1 mutations in patients are described to date and only little is known about osteoclast pathology in the LRRK1-mutated setting. A deeper understanding of the effect LRRK1 mutations in humans have on osteoclasts is inevitable in the process of considering LRRK1 as a feasible drug target.…”

Section: Discussionmentioning

confidence: 99%

Adult Osteosclerotic Metaphyseal Dysplasia With Progressive Osteonecrosis of the Jaws and Abnormal Bone Resorption Pattern Due to a LRRK1 Splice Site Mutation

Howaldt

Hennig

Rolvien

et al. 2020

Journal of Bone and Mineral Research

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Osteosclerotic metaphyseal dysplasia (OSMD) is a rare autosomal recessive sclerosing skeletal dysplasia. We report on a 34-year-old patient with sandwich vertebrae, platyspondyly, osteosclerosis of the tubular bones, pathologic fractures, and anemia. In the third decade, he developed osteonecrosis of the jaws, which was progressive in spite of repeated surgical treatment over a period of 11 years. An iliac crest bone biopsy revealed the presence of hypermineralized cartilage remnants, large multinucleated osteoclasts with abnormal morphology, and inadequate bone resorption typical for osteoclast-rich osteopetrosis. After exclusion of mutations in TCIRG1 and CLCN7 we performed trio-based exome sequencing. The novel homozygous splice-site mutation c.261G>A in the gene LRRK1 was found and co-segregated with the phenotype in the family. cDNA sequencing showed nearly complete skipping of exon 3 leading to a frameshift (p.Ala34Profs*33). Osteoclasts differentiated from the patient's peripheral blood monocytes were extremely large. Instead of resorption pits these cells were only capable of superficial erosion. Phosphorylation of L-plastin at position Ser5 was strongly reduced in patient-derived osteoclasts showing a loss of function of the mutated LRRK1 kinase protein. Our analysis indicates a strong overlap of LRRK1-related OSMD with other forms of intermediate osteopetrosis, but an exceptional abnormality of osteoclast resorption. Like in other osteoclast pathologies an increased risk for progressive osteonecrosis of the jaws should be considered in OSMD, an intermediate form of osteopetrosis.

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“…All inhibitors ablated LRRK2-mediated phosphorylation of Rab10 at Thr73 and LRRK2 at Ser935 in parallel experiments. An earlier study reported that a compound identified form a virtual homology screen termed iN04 inhibited LRRK1, but no experimental data was presented to demonstrate this [57]. We found that iN04, when used at 5 μM had no impact on LRRK1-mediated Rab7A phosphorylation (SFig 7D).…”

Section: Resultsmentioning

confidence: 64%

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

Malik

Karapetsas

Nirujogi

et al. 2020

Preprint

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Much attention has focused on LRRK2, as autosomal dominant missense mutations that enhance its kinase activity cause inherited Parkinson’s disease. LRRK2 regulates biology by phosphorylating a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. In this study we explore whether LRRK1, a less studied homologue of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that the endogenous Rab7A protein, phosphorylated at Ser72 was most impacted by LRRK1 knock-out. This residue is not phosphorylated by LRRK2 but lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson’s mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous Type-2 tyrosine kinase inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. Finally, we show that interaction of Rab7A with its effector RILP is not affected by high stoichiometry LRRK1 phosphorylation. Altogether, these finding reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology.

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A small molecular inhibitor of LRRK1 identified by homology modeling and virtual screening suppresses osteoclast function, but not osteoclast differentiation, in vitro

Cited by 5 publications

References 36 publications

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

Adult Osteosclerotic Metaphyseal Dysplasia With Progressive Osteonecrosis of the Jaws and Abnormal Bone Resorption Pattern Due to a LRRK1 Splice Site Mutation

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

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