A Novel Class of Geranylgeranyl Diphosphate Synthase Inhibitors: Structure-Activity Relationships of Homoisoprenoid Triazoles in Myeloma Cells

The secretion of monoclonal protein and the dependence on the surrounding bone marrow microenvironment are key features of malignant plasma cells. We have previously demonstrated that targeting the Rab family of small GTPases via disruption of the post-translational modification geranylgeranylation not only impairs intracellular monoclonal protein trafficking, thus leading to endoplasmic reticulum stress and apoptosis, but also disrupts the interaction of the malignant plasma cells with key components of the bone marrow microenvironment. While developing agents that directly target the enzyme that geranylgeranylates the Rabs, we discovered several compounds that specifically and potently inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS). This enzyme is responsible for synthesis of the isoprenoid substrate which is used in geranylgeranylation reactions, thus inhibition of this enzyme is an alternative approach by which to target Rabs. Families of compounds were therefore designed which incorporated a hydrophobic chain, a triazole ring, and a bisphosphonic acid head group. The central triazole provides a potential zinc-binding element, and allows for divergent assembly of potential inhibitors through click chemistry. Through this strategy, prenyl, homoprenyl, geranyl, homogeranyl, farnesyl, homofarnesyl, geranylgeranyl, and homogeranylgeranyl triazole bisphosphonates have been prepared. All of these agents were subjected to in vitro enzyme assays for GGDPS as well as for the related enzyme farnesyl diphosphate synthase (FDPS), and IC₅₀’s were determined. Cellular activity in human myeloma cell lines (RPMI-8226, U266, MM.1S) was assessed via immunoblot analysis of representative prenylated proteins. These studies revealed that in all cases, the homologated-version of the allylic triazole is more potent than the corresponding isoprenoid against GGDPS, ranging from 1.1-fold improvement in the homogeranylgeranyl:geranylgeranyl pairing to almost 300-fold in the homogeranyl:geranyl pairing. Chain length was found to be an important factor which determines the inhibitory activity within the homoisoprenoid series: the order of increasing potency against GGDPS was homogeranylgeranyl < homofarnesyl < homoprenyl << homogeranyl. The latter compound was determined to be the lead compound in this series with an IC₅₀ of 58 nM against GGDPS and 480-fold selectivity for GGDPS compared with FDPS. Disruption of protein geranylgeranylation in myeloma cells with accompanying accumulation of intracellular monoclonal protein levels was observed at concentrations as low as 100 nM. This activity could be further potentiated with co-incubation with submicromolar concentrations of lovastatin, an HMG-CoA reductase inhibitor. Co-incubation studies with the isoprenoid intermediates mevalonate, farnesyl diphosphate and geranylgeranyl diphosphate (GGPP) revealed that the cellular activity of the homogeranyl triazole bisphosphonate is due to depletion of GGPP, consistent with the activity seen in the enzyme assays. Further studies demonstrated that this agent, in a concentration-dependent manner, induces elements of the unfolded protein response pathway and disrupts autophagy, as determined by immunoblot analysis of calnexin cleavage, levels of phosphorylated eIF2a, and LC3 cleavage. Treatment of human bone marrow stromal cells (HS-5 cell line) with the lead compound induced a concentration-dependent decrease in secreted IL-6 levels as measured via ELISA. In addition, myeloma cells pre-treated with the homogeranyl triazole compound displayed a decreased ability to adhere to HS-5 cells. In conclusion, these studies establish homogeranyl triazole bisphosphonate as the lead compound in a novel series of inhibitors against GGDPS and support the further development of GGDPS inhibitors as potential therapeutic agents for multiple myeloma.