Synthesis and Evaluation of Structurally Diverse C-2-Substituted Thienopyrimidine-Based Inhibitors of the Human Geranylgeranyl Pyrophosphate Synthase.

Novel analogues of C-2-substituted thienopyrimidine-based bisphosphonates (C2-ThP-BPs) are described that are potent inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS). Members of this class of compounds induce target-selective apoptosis of multiple myeloma (MM) cells and exhibit antimyeloma activity in vivo. A key structural element of these inhibitors is a linker moiety that connects their (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)bisphosphonic acid core to various side chains. The structural diversity of this linker moiety, as well as the side chains attached to it, was investigated and found to significantly impact the toxicity of these compounds in MM cells. The most potent inhibitor identified was evaluated in mouse and rat for liver toxicity and systemic exposure, respectively, providing further optimism for the potential value of such compounds as human therapeutics.

Novel bisphosphonates with antiresorptive effect in bone mineralization and osteoclastogenesis.

Bisphosphonates such as zoledronic, alendronic and risedronic acids are a class of drugs clinically used to prevent bone density loss and osteoporosis. Novel P-C-P bisphosphonates were synthesized for targeting human farnesyl pyrophosphate synthase (hFPPS) and human geranylgeranyl pyrophosphate synthase (hGGPPS), key enzymes of the mevalonate pathway, and capable of anti-proliferative action on a number of cell lines (PC3, MG63, MC3T3, RAW 264.7, J774A.1, bone marrow cells and their co-colture with PC3) involved in bone homeostasis, bone formation and death. Among sixteen compounds, [1-hydroxy-2-(pyrimidin-2-ylamino)ethane-1,1-diyl]bis(phosphonic acid) (10) was effective in reducing PC3 and RAW 264.7 cell number in crystal-violet and cell-dehydrogenase activity assays at 100 µM concentration. 10 reduced differentiated osteoclasts number similarly with zoledronic acid in osteoclastogenesis assay. At nanomolar concentrations, 10 was more effective than zoledronic acid in inducing mineralization in MC3T3 and murine bone marrow cells. Further, 10 significantly inhibited the activity of hFPPS showing an IC50 of 0.31 µM and a remarkable hydroxyapatite binding of 90%. Docking calculations were performed identifying putative interactions between some representative novel bisphosphonates and both hFPPS and hGGPPS. Then, 10 was found to behave similarly or even better than zoledronic acid as a anti-resorptive agent.

Unraveling the Prenylation-Cancer Paradox in Multiple Myeloma with Novel Geranylgeranyl Pyrophosphate Synthase (GGPPS) Inhibitors.

Post-translational prenylation of the small GTP-binding proteins (GTPases) is vital to a plethora of biological processes, including cellular proliferation. We have identified a new class of thienopyrimidine-based bisphosphonate (ThP-BP) inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS) that block protein prenylation in multiple myeloma (MM) cells leading to cellular apoptosis. These inhibitors are also effective in blocking the proliferation of other types of cancer cells. We confirmed intracellular target engagement, demonstrated the mechanism of action leading to apoptosis, and determined a direct correlation between apoptosis and intracellular inhibition of hGGPPS. Administration of a ThP-BP inhibitor to a MM mouse model confirmed in vivo downregulation of Rap1A geranylgeranylation and reduction of monoclonal immunoglobulins (M-protein, a biomarker of disease burden) in the serum. These results provide the first proof-of-principle that hGGPPS is a valuable therapeutic target in oncology and more specifically for the treatment of multiple myeloma.

Measurement of protein farnesylation and geranylgeranylation in vitro, in cultured cells and in biopsies, and the effects of prenyl transferase inhibitors.

The importance of the post-translational lipid modifications farnesylation and geranylgeranylation in protein localization and function coupled with the critical role of prenylated proteins in malignant transformation has prompted interest in their biology and the development of farnesyl transferase and geranylgeranyl transferase inhibitors (FTIs and GGTIs) as chemical probes and anticancer agents. The ability to measure protein prenylation before and after FTI and GGTI treatment is important to understanding and interpreting the effects of these agents on signal transduction pathways and cellular phenotypes, as well as to the use of prenylation as a biomarker. Here we describe protocols to measure the degree of protein prenylation by farnesyl transferase or geranylgeranyl transferase in vitro, in cultured cells and in tumors from animals and humans. The assays use [(3)H]farnesyl diphosphate and [(3)H]geranylgeranyl diphosphate, electrophoretic mobility shift, membrane association using subcellular fractionation or immunofluorescence of intact cells, [(3)H]mevalonic acid labeling, followed by immunoprecipitation and SDS-PAGE, and in vitro transcription, translation and prenylation in reticulocyte lysates. These protocols require from 1 d (enzyme assays) to up to 3 months (autoradiography of [(3)H]-labeled proteins).

Digeranyl bisphosphonate inhibits geranylgeranyl pyrophosphate synthase.

A primary cellular target of the clinical nitrogenous bisphosphonates is the isoprenoid biosynthetic pathway. Specifically these drugs inhibit the enzyme farnesyl pyrophosphate synthase and deplete cells of larger isoprenoids. Inhibition of this enzyme results in impaired processing of both farnesylated and geranylgeranylated proteins. We recently showed that isoprenoid-containing bisphosphonates such as digeranyl bisphosphonate inhibit protein geranylgeranylation and not farnesylation. Here, we show that this impairment results from potent and specific inhibition of geranylgeranyl pyrophosphate synthase, which leads to enhanced depletion of intracellular geranylgeranyl pyrophosphate relative to the nitrogenous bisphosphonate zoledronate.

The crystal structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding.

Modification of GTPases with isoprenoid molecules derived from geranylgeranyl pyrophosphate or farnesyl pyrophosphate is an essential requisite for cellular signaling pathways. The synthesis of these isoprenoids proceeds in mammals through the mevalonate pathway, and the final steps in the synthesis are catalyzed by the related enzymes farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase. Both enzymes play crucial roles in cell survival, and inhibition of farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates is an established concept in the treatment of bone disorders such as osteoporosis or certain forms of cancer in bone. Here we report the crystal structure of human geranylgeranyl pyrophosphate synthase, the first mammalian ortholog to have its x-ray structure determined. It reveals that three dimers join together to form a propeller-bladed hexameric molecule with a mass of approximately 200 kDa. Structure-based sequence alignments predict this quaternary structure to be restricted to mammalian and insect orthologs, whereas fungal, bacterial, archaeal, and plant forms exhibit the dimeric organization also observed in farnesyl pyrophosphate synthase. Geranylgeranyl pyrophosphate derived from heterologous bacterial expression is tightly bound in a cavity distinct from the chain elongation site described for farnesyl pyrophosphate synthase. The structure most likely represents an inhibitory complex, which is further corroborated by steady-state kinetics, suggesting a possible feedback mechanism for regulating enzyme activity. Structural comparisons between members of this enzyme class give deeper insights into conserved features important for catalysis.