Healthy bones are strong, but forming healthy bones requires a delicate balance between bone-building osteoblasts and bone-degrading osteoclasts. If the action of osteoclasts prevails and bone resorption exceeds bone deposition, bone-diminishing diseases such as osteoporosis occur.
The answer seems simple: target osteoclasts as the enemy. But drugs currently available to rein in excessive osteoclast activity can be a sledgehammer in a scenario where a scalpel would be more appropriate.
Nitrogen-containing bisphosphonates, for example, elicit osteoclast apoptosis. A humanized monoclonal antibody to RANKL inhibits osteoclast differentiation. Each is commonly employed as an antiosteolytic therapeutic agent. But osteoclasts play multiple roles: In addition to their bone resorption activity, they also secrete factors that stimulate the recruitment and action of osteoblasts. It’s a fascinating system—and one that makes pharmacological intervention tricky, because simply killing off osteoclasts can result in side effects such as atypical subtrochanteric femur fractures, osteonecrosis of the jaw, or reduced responsiveness to the bone anabolic factor parathyroid hormone.
To mediate bone resorption, osteoclasts must establish a podosome-based structure called the sealing zone. In 2011, researchers led by Virginie Vives and Anne Blangy at the CNRS in Montpellier, France, showed that the Rac-specific guanine nucleotide exchange factor (GEF), Dock5, is necessary for sealing zone formation and thus for osteoclast function.
Recently, the team advanced their work much further. In an article published in Nature Communications entitled “Pharmacological inhibition of Dock5 prevents osteolysis by affecting osteoclast podosome organization while preserving bone formation,” the group validated a small molecule Dock5 inhibitor in disease models for major osteolytic diseases. In addition to the potential impact of this work on debilitating osteolytic diseases, it is the first time that a Rac GEF has been targeted for a disease other than cancer.
In their previous work, the researchers identified C21, a small molecule that blocks Rac activation by Dock5 in cultured cells, and showed that it inhibits bone degradation by osteoclasts in vitro. Shifting to in vivo approaches in the recent study, the authors used three mouse models for the most common osteolytic diseases—post-menopause osteoporosis, chronic inflammation (rheumatoid arthritis), and bone metastasis—providing evidence that systemic administration of C21 efficiently prevents pathological bone loss while preserving bone formation and reducing undesirable side effects.
Importantly, the authors used a fluorescence assay to analyze whether C21 was a direct inhibitor of mouse Dock5 catalytic activity. Using highly purified, E. coli-expressed Dock5 catalytic and dimerization domain, the authors assayed nucleotide exchange by fluorescence kinetics. After testing the GTPases Rac1, Rac2, RhoA, and Cdc42, the authors concluded that Dock5 is strictly specific for the Rac subfamily and that C21 inhibits Dock5-DHR2-stimulated nucleotide exchange on Rac1. They also demonstrated that treating mouse bone marrow-derived osteoclasts with 100 µM C21 disrupted podosome organization.
Using bilateral ovariectomy to recapitulate post-menopause osteoporosis, the researchers found that systemic administration of C21 in mice prevented bone loss. C21 also prevented bone loss associated with rheumatoid arthritis, which was modeled by immunization of susceptible DBA/ 1 mouse strain with type II collagen, the major protein component of articular cartilage. C21 did not prevent inflammation, but did protect mice from bone erosion in the arthritic context. Finally, the authors assessed the effect of C21 on tumor-associated bone loss. Once again, C21 provided demonstrable benefit, breaking the vicious cycle between osteoclasts and metastatic cells in the bone. And systemic administration of C21 did not result in toxicity or alter osteoblast numbers or activity.
New therapeutic approaches and drug candidates are badly needed for osteolytic diseases, and advancement of C21 or a related molecule into clinical trials would be an exciting development. But perhaps the most important aspects of the study was the validation of a GEF inhibitor in a non-cancer disease. As activators of Ras family GTPases, GEFs have been intensively investigated as anti-cancer targets. However, development of GEF inhibitors has been slow, and there are few cases of pharmacological validation, even for cancer. Hopefully this work will spur investigation of the role of GEFs in diverse diseases and accelerate identification of inhibitors for pharmacological validation.
For more information on the biological significance of Rho GEFs and their value as emerging targets, we suggest “A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay,” published by researchers at Bellbrook Labs.
Learn more about the Transcreener GDP Assay, the only mix-and-read fluorescent GTPase Assay.