Receptor activator of nuclear factor-κB ligand (RANKL), a trimeric tumour necrosis factor (TNF) superfamily member, is the central mediator of osteoclast formation and bone resorption. Functional mutations in RANKL lead to human autosomal recessive osteopetrosis, whereas RANKL overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Following a forward genetics approach, we have recently shown that a novel loss-of-function allele of Rankl with a glycine-to-arginine susbstitution at codon 278, causes severe recessive osteopetrosis in mice due to inhibition of RANKL trimerisation. Notably, SPD304, a small molecule inhibitor of TNF trimerisation, also binds and inhibits RANKL, suggesting similar inhibitory mechanisms. However, SPD304 displays high cell toxicity. Based on the trimeric structure of RANKL and its interaction with SPD304, novel small molecules were designed to abrogate RANKL trimer formation and biological function while also displaying lower toxicity. Of the 72 SPD304-like derivatives synthesised and tested, 8 displayed complete inhibition of human RANKL-induced osteoclastogenesis at 5 μM without affecting the differentiation of the preosteoblastic MC3T3-E1 cells. Notably, these compounds were significantly less cytotoxic compared with SPD304 as shown by MTT. Moreover, the most effective small molecule inhibitors dissociated human RANKL trimers as shown by cross linking and western blot and suppressed the activation of NFATc1, the master regulator of osteoclast formation. Our research identified potent small molecule inhibitors of human RANKL designed to target and block its trimerisation. The more effective inhibitors will be further evaluated in vivo using our unique human RANKL-expressing transgenic mouse models of osteoporosis.
Disclosure: The authors declared no competing interests. This work was funded by project TheRAlead (09ΣϒN-21-784) which is Co-financed by the European Union (European Regional Development Fund) and Greece, Operational Program Competitiveness & Entrepreneurship, NSRF 2007-2013 in the context of GSRT-National action Cooperation.