Bisphosphonate drugs such as zoledronic acid (ZOL) are used to inhibit bone destruction in patients with metastatic bone disease. These calcium-seeking compounds bind to bone with high affinity and block the resorbing activity of osteoclasts by preventing the prenylation of small GTPase proteins. Increasing evidence suggests that nanomolar concentrations of circulating bisphosphonates also target cells of the monocyte/macrophage lineage outside the skeleton. This could account for the apparent anti-tumour actions of these drugs in mice, as well as beneficial effects on patient survival observed in clinical trials. However, no effects of such low concentrations of ZOL on protein prenylation have been described for immune cells using existing approaches.
We have optimised a highly sensitive in vitro prenylation assay utilising recombinant Rab geranylgeranyltransferase to enable the detection of subtle effects of ZOL on the prenylation of Rab-family GTPases. In vitro treatment of J774 macrophages with 10nM ZOL resulted in a clear inhibition of Rab prenylation. Furthermore, quantitative mass spectrometry revealed 18 different unprenylated Rabs in J774 cells in the presence of nanomolar concentrations of ZOL, with a >7-fold increase in the unprenylated form of Rab proteins associated with the endophagosome pathway (Rab1, Rab5, Rab6, Rab7, Rab11, Rab14 and Rab21). Importantly, whilst a single subcutaneous injection of ZOL had no detectable effect, repeated ZOL administration inhibited the prenylation of Rab1A, Rab5B, Rab7A and Rab14 in mouse peritoneal macrophages in vivo, confirming that prolonged systemic bisphosphonate treatment can inhibit prenylation in myeloid cells in vivo outside the skeleton. These observations begin a new era in defining the precise pharmacological actions of bisphosphonate drugs on the prenylation of small GTPases in vivo in cells within and outside bone. In particular, these studies add weight to the possibility that the anti-tumour actions of bisphosphonates are due to effects on myeloid cells outside the skeleton.