ω-Hydroxy isoprenoid bisphosphonates as linkable GGDPS inhibitors

Abstract

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential therapeutic target for multiple myeloma. Malignant plasma cells produce and secrete large amounts of monoclonal protein, and inhibition of GGDPS results in disruption of protein geranylgeranylation which in turn impairs intracellular protein trafficking. Our previous work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective inhibitors of GGDPS. To explore the possibility of selective delivery of such compounds to plasma cells, new analogues with an ω-hydroxy group have been synthesized and examined for their enzymatic and cellular activity. These studies demonstrate that incorporation of the ω-hydroxy group minimally impairs GGDPS inhibitory activity. Furthermore conjugation of one of the novel ω-hydroxy GGDPS inhibitors to hyaluronic acid resulted in enhanced cellular activity. These results will allow future studies to focus on the in vivo biodistribution of HA-conjugated GGDPS inhibitors.

Keywords: Bisphosphonate; GGDP synthase; Inhibition; Isoprenoid biosynthesis; Triazole.

Figure 1.

Geranylgeranyl diphosphate and known inhibitors of geranylgeranyl diphosphate synthase.

Figure 2.. Comparison of the effects of the novel ω-hydroxyl triazole bisphosphonates to the parent analogues on protein geranylgeranylation.

RPMI-8226 cells were incubated for 48 hours in the presence or absence of lovastatin (Lov, 10 μM) or varying concentrations of the test compounds. A) Immunoblot analysis of uRap1a (antibody detects only unmodified protein) and β-tubulin (as a loading control). B) Intracellular lambda light chain concentrations were determined via ELISA. Data are expressed as a percentage of control (mean ± SD, n=3). The * denotes p < 0.05 per unpaired two-tailed t-test.

Figure 3.. Comparison of the effects of the ω-hydroxyl triazole bisphosphonate 15 and the HA-conjugate 27 on protein geranylgeranylation.

RPMI-8226 (R) or MM.1S (M) cells were incubated for 48 hours in the presence or absence of lovastatin (Lov, 10 μM) or varying concentrations of the test compounds (for the HA-conjugate 27, concentration is based on the percent weight of the inhibitor in the HA polymer conjugate). Immunoblot analysis of uRap1a (antibody detects only unmodified protein) and β-tubulin (as a loading control). These blots are representative of three independent experiments.

Scheme 1.

Synthesis of compound 15, the ω-hydroxy analogue of compound 3.

Scheme 2.

Synthesis of compound 25, the ω-hydroxy analogue of compound 6.

Scheme 3.

Generation of HA-GGDPS inhibitor polymer conjugate 27.