Synthesis and Structure-Function Relationship Of Novel Triazole Phosphonates As Inhibitors Of Rab Geranylgeranyl Transferase

Multiple myeloma is characterized by the proliferation of malignant plasma cells which secrete large quantities of monoclonal protein (MP). As yet, no therapeutic strategies have been developed which directly target MP secretion. The Rab family of small GTPases plays key roles in intracellular vesicle trafficking. We have demonstrated that agents which impair the geranylgeranylation of Rab proteins induce apoptosis of myeloma cells by disrupting MP secretion, resulting in accumulation of MP within the cells and activation of the unfolded protein response pathway (UPR). Previously identified inhibitors of Rab geranylgeranyl transferase (Rab GGTase), the enzyme responsible for post-translational modification of Rab proteins, lack potency and have limited therapeutic potential. We hypothesized that potent and selective inhibitors of Rab GGTase could be designed based on available crystallographic data which reveals how the isoprenoid substrate interacts with the enzyme’s active site. Families of compounds were therefore designed incorporating three motifs to allow for interaction with key components of the enzyme’s active site: a non-hydrolyzable polar head group, a zinc-binding motif, and an isoprenoid or isoprenoid-like chain. The isoprenoid-based compounds were prepared using a divergent synthetic strategy based on azide-acetylene cycloadditions or “click” chemistry. We have previously demonstrated that direct use of an isoprenoid azide in a click reaction results in a triazole product that is a mixture of olefin isomers, due to an unavoidable [3,3] sigmatropic rearrangement that scrambles the stereochemistry of the first olefin prior to cycloaddition. This problem now has been circumvented by the development of a new synthetic strategy which is based on a regiospecific epoxidation of the C-2 olefin in the isoprenoid alcohol, followed by introduction of the azide and formation of the triazole. After completion of a click reaction the product can be reduced back to the parent olefin with good stereocontrol, to afford isoprenylated triazoles of the same chain length as those prepared directly from isoprenoid azides. Series of compounds were thus prepared in which chain length (including isoprenoid and isoprenoid-like), olefin stereochemistry, and polar head group (including bisphosphonic acids and carboxyphosphonic acids) were varied. All novel agents were subjected to in vitro enzyme assays for Rab GGTase as well as the related enzymes farnesyl transferase (FTase) and geranylgeranyl transferase I (GGTase I) and IC₅₀ values were obtained. The cytotoxic activities of these agents were determined via MTT assays in human myeloma cell lines. Disruption of protein prenylation in myeloma cells was assessed via immunoblot analysis of farnesylated (Ras) and geranylgeranylated (Rap1a and Rab6) proteins. The results from these enzymatic and cell-based assays enabled the determination of a detailed structure-function relationship. These studies reveal that both chain length and olefin stereochemistry have a significant impact on both inhibitor potency and selectivity with respect to the target enzyme and the related prenyltransferases. For example, when the chain length was increased by just one methylene unit (farnesyl to homofarnesyl), the potency against Rab GGTase was enhanced by approximately 4-fold while changing the stereochemistry at the second olefin site from trans to cis diminished the potency by approximately 5-fold. These studies are guiding the design and synthesis of future generations of inhibitors with the ultimate goal of developing a potent and selective Rab GGTase inhibitor which can be utilized as a therapeutic agent for myeloma.