Nitrogen-containing bisphophonates (N-BPs) are a blockbuster class of drugs for the treatment of common metabolic bone diseases.  Recently, N-BPs have been shown to inhibit FPP synthase and/or isopentenyl diphosphate (IPP) isomerase (both enzymes in the mevalonate pathway), thereby preventing the synthesis of the isoprenoid lipids farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), which are vital substrates for protein prenylation.  By preventing the synthesis of FPP and GGPP, N-BPs prevent the prenylation of small GTPases and inhibit osteoclast function. This study demonstrates conclusively that the major pharmacological target of N-BPs is FPP synthase.  Furthermore, minor structural modifications to the N-BPs that govern <i>n vivo </i>potency have a marked effect on potency for inhibition of FPP synthase <i>in vitro.  </i>Non-N-BPs, such as clodronate and etidronate, did not inhibit FPP synthase, consistent with other studies suggesting that the non-N-BPs and N-BPs act by different molecular mechanisms. Clinically, N-BPs have been shown to exhibit marked differences in efficacy between patients and may lie resident in the skeleton for many years.  J774 macrophage-like cells resistant to the effects of N-BP (J774-RES) were generated to study the possible cellular mechanisms underlying clinical resistance to treatment.  The J774-RES cells accumulated N-BP to a lesser extent than parental cells and also exhibited increased expression of the MCSF receptor, although further studies are required to clarify the exact mechanism of the resistance of J774-RES cells. Finally, N-BPs have been shown to induce the proliferation of the major subset (V<span style='font-family:Symbol'>g9V<span style='font-family:Symbol'>d2) of <span style='font-family:Symbol'>g,<span style='font-family:Symbol'>d-T cells in humans, attributed to an agnostic effect on the <span style='font-family:Symbol'>g,<span style='font-family:Symbol'>d-T cell receptor (TCR).  The findings of this study indicate that the N-BPs act indirectly, by inhibiting FPP synthase and causing the accumulation of IPP, a known agonist of the <span style='font-family:Symbol'>g,<span style='font-family:Symbol'>d-TCR.  Furthermore, this proliferative effect of N-BPs could be abrogated by statins, possibly indicating a means of preventing the acute-phase response, the major side effect to intravenously-administrated N-BPs.

Farnesyl pyrophosphate synthase (FPPS) is a key branch point enzyme in the mevalonate pathway and the main molecular target of nitrogen-containing bisphosphonates (N-BPs), potent inhibitors of osteoclastic activity and the leading drug of choice for conditions characterized by excessive bone resorption. The main aim of this thesis is to investigate the interaction of N-BPs with FPPS in order to gain further insights into the mechanism of drug inhibition. Kinetic and crystallographic studies following site-directed mutagenesis of FPPS reveal key residues involved in stabilization of carbocation intermediate, substrate binding and formation of a tight enzyme-inhibitor complex. The aromatic ring of Tyr204 is involved in N-BP binding but not in the catalytic mechanism, where the hydroxyl moiety plays an important role. Lys200 is implicated in regulation of substrate binding, product specificity and enzyme isomerization which leads to a tight binding inhibition. Phe239 is considered important for the FPPS C-terminal switch which stabilizes substrate binding and promotes the inhibitor induced isomerized state. The highly conserved Arg112, Asp103 and Asp107 are pivotal for catalysis. Successful purification of the full length of Rab geranylgeranyl transferase (RGGT) complex downstream of the FPPS in the mevalonate pathway was achieved and may lead to co-crystallization with BP analogues and identification of the putative site of drug binding. Investigation of the in vitro effect of N-BPs on osteoclastogenesis suggest a correlation with FPPS inhibition kinetics for the most potent N-BPs but indicate an alternative mechanism of the disruption of bone resorption by alendronate. Together these results highlight the importance of the multiple interactions of N-BPs with side-chain residues of FPPS which dictate their strength of binding and advance the understanding of their pharmacophore effect.