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Olefin Isomers of a Triazole Bisphosphonate Synergistically Inhibit Geranylgeranyl Diphosphate Synthase S

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Olefin Isomers of a Triazole Bisphosphonate Synergistically Inhibit Geranylgeranyl Diphosphate Synthase S Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2017/01/05/mol.116.107326.DC1 1521-0111/91/3/229–236$25.00 http://dx.doi.org/10.1124/mol.116.107326 MOLECULAR PHARMACOLOGY Mol Pharmacol 91:229–236, March 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics Olefin Isomers of a Triazole Bisphosphonate Synergistically Inhibit Geranylgeranyl Diphosphate Synthase s Cheryl Allen, Sandhya Kortagere, Huaxiang Tong, Robert A. Matthiesen, Joseph I. Metzger, David F. Wiemer, and Sarah A. Holstein Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York (C.A.); Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania (S.K.); Penn State Cancer Institute, Hershey, Pennsylvania (H. T.); Department of Chemistry, University of Iowa, Iowa City, Iowa (R.A.M., J.I.M., D.F.W.); and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska (S.A.H.) Downloaded from Received November 3, 2016; accepted December 28, 2016 ABSTRACT The isoprenoid donor for protein geranylgeranylation reactions, in which cells were treated with varying concentrations of each geranylgeranyl diphosphate (GGDP), is the product of the isomer alone and in different combinations revealed that the enzyme GGDP synthase (GGDPS) that condenses farnesyl two isomers potentiate the induced-inhibition of protein ger- molpharm.aspetjournals.org diphosphate (FDP) and isopentenyl pyrophosphate. GGDPS anylgeranylation when used in a 3:1 HG:HN combination. A inhibition is of interest from a therapeutic perspective for synergistic interaction was observed between the two isomers multiple myeloma because we have shown that targeting Rab in the GGDPS enzyme assay. These results suggested that the GTPase geranylgeranylation impairs monoclonal protein traf- two isomers bind simultaneously to the enzyme but within ficking, leading to endoplasmic reticulum stress and apoptosis. different domains. Computational modeling studies revealed We reported a series of triazole bisphosphonate GGDPS inhibi- that HN is preferred at the FDP site, that HG is preferred at the tors, of which the most potent was a 3:1 mixture of homogeranyl GGDP site, and that both isomers may bind to the enzyme (HG) and homoneryl (HN) isomers. Here we determined the activity simultaneously. These studies are the first to report a set of of the individual olefin isomers. Enzymatic and cellular assays olefin isomers that synergistically inhibit GGDPS, thus estab- revealed that although HN is approximately threefold more potent lishing a new paradigm for the future development of GGDPS at ASPET Journals on September 23, 2021 than HG, HN is not more potent than the original mixture. Studies inhibitors. Introduction (GGDP). The FDP and GGDP isoprenoid moieties derived from these prenyl synthases play important roles in pro- In animals, the isoprenoid biosynthetic pathway begins tein prenylation, a post-translational modification. This mod- with the conversion of hydroxymethyl glutaryl-coenzyme A ification is necessary for proper intracellular localization and (HMG-CoA) to mevalonate by HMG-CoA reductase. Mevalo- function of proteins such as members of the Ras small GTPase nate undergoes phosphorylation and decarboxylation to form superfamily, many of which are involved in signal trans- isopentenyl pyrophosphate (IPP), which reversibly isomerizes duction pathways. There has been significant focus on the to dimethylallyl pyrophosphate. IPP and dimethylallyl pyro- development of inhibitors of the prenyl transferases for phosphate serve as substrates for farnesyl disphosphate pharmacological activity and therapeutic applications (Holstein synthase (FDPS), which generates the C15 farnesyl diphos- and Hohl, 2012; Palsuledesai and Distefano, 2015). In the phate (FDP) from these C5 precursors, whereas FDP and IPP setting of multiple myeloma, we have been focused on the serve as substrates for geranylgeranyl diphosphate synthase disruption of Rab GTPase geranylgeranylation as a novel (GGDPS), generating the C20 geranylgeranyl diphosphate therapeutic strategy, because our studies have demonstrated agents that impair Rab geranylgeranylation lead to a disrup- This project was supported by the National Institutes of Health National tion of monoclonal protein trafficking, resulting in induction of Cancer Institute [Grant R01 CA172070], the American Society of Hematology, ER stress and apoptosis (Holstein and Hohl, 2011; Dykstra and the Roy J. Carver Charitable Trust. Portions of these data were presented as abstracts at the following meeting: et al., 2015). Allen C, Metzger JI, Wills VS, Tong H, Kortagere S, Wiemer DF, and Holstein An alternative strategy to the direct inhibition of prenyl SA (2016) Synergistic inhibition of geranylgeranyl diphosphate synthase by a transferase activity is to inhibit the prenyl synthases involved mixture of olefin stereoisomers. American Society of Hematology Annual Meeting; 2016 Dec 3-6; San Diego, CA. American Society of Hematology, in the generation of FDP and GGDP. The nitrogenous Washington. bisphosphonates such as zoledronate (Fig. 1) have been widely dx.doi.org/10.1124/mol.116.107326. s This article has supplemental material available at molpharm. used in the management of bone disorders, including osteo- aspetjournals.org. porosis, metastatic bone disease, and myeloma bone disease. ABBREVIATIONS: FDP, farnesyl diphosphate; FDPS, farnesyl diphosphate synthase; GGDP, geranylgeranyl diphosphate; GGDPS, geranylgeranyl diphosphate synthase; HG, homogeranyl; HMG-CoA, hydroxymethyl glutaryl-coenzyme A; HN, homoneryl; IPP, isopentenyl pyrophosphate. 229 230 Allen et al. isomers (Matthiesen et al., 2016). Here we present our findings for the biologic activities of the homogeranyl and homoneryl triazole bisphosphonates and demonstrate that these compounds interact in a synergistic manner to inhibit GGDPS, thus establishing a new paradigm for the develop- ment of GGDPS inhibitors. Materials and Methods Reagents. Lovastatin (M2147), geranyl pyrophosphate (G6772), and farnesyl pyrophosphate (F6892) were obtained from Sigma (St. Louis, MO). [14C]-IPP was purchased from American Radiolabeled Chemicals (St. Louis, MO). The pure homogeranyl and homoneryl triazole bisphosphonates (7 and 8) and the 3:1 (HG:HN) mixture (6) were prepared as previously reported (Wills et al., 2015; Matthiesen et al., 2016). Stock solutions (50 mM) of the triazole bisphosphonate sodium salts were prepared in sterile water and stored at 220°C. Downloaded from Working dilutions (0.5–50 mM) of the triazole bisphosphonate sodium salts were also prepared in sterile water and stored at 220°C. Anti- Rap1a (sc-1482), Rab6 (sc-310), and calnexin (sc-23954) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti- b-tubulin (T5201) antibody was purchased from Sigma. Secondary horseradish peroxidase-linked antibodies were obtained from Amer- sham [GE Healthcare Life Sciences, Pittsburgh, PA; (anti-mouse (NA- molpharm.aspetjournals.org 931) and anti-rabbit (NA934)] or Santa Cruz Biotechnology (anti-goat sc-2020). Cell Culture. Human myeloma cell lines (RPMI-8226, MM.1S) were purchased from American Type Culture Collection (ATCC) Fig. 1. Inhibitors of FDPS and GGDPS. Chemical structures of FDPS and (Manassas, VA) and grown in media (per ATCC specifications) that GGDPS inhibitors. IC50 values are presented for previously published was supplemented with heat-inactivated fetal bovine serum, gluta- GGDPS inhibitors. mine, and penicillin-streptomycin at 37°C and 5% CO2. Monoclonal Protein Quantitation. Cells were incubated in the presence or absence of drugs for specified periods of time. The cells Notably, these agents are specific inhibitors of FDPS were lysed in RIPA buffer [0.15 M NaCl, 1% sodium deoxycholate, at ASPET Journals on September 23, 2021 (Bergstrom et al., 2000; Dunford et al., 2001) and their 0.1% SDS, 1% Triton (v/v) X-100, 0.05 M Tris HCl, pH 7.4] containing antiresorptive activity is primarily attributed to disruption of protease and phosphatase inhibitors. Protein content was determined protein geranylgeranylation within osteoclasts (Luckman et al., using the bicinchoninic acid method. A human lambda light chain kit 1998; Coxon et al., 2000). More recently there has also been (E80-116, Bethyl Laboratories, Montgomery, TX) was used to quantify intracellular monoclonal protein levels. interest in the therapeutic potential of GGDPS inhibitors as a Immunoblotting. After incubation with drugs, cells were col- more direct way of depleting cellular GGDP levels and thereby lected, washed with PBS, and lysed in RIPA buffer as described disrupting protein geranylgeranylation (Wiemer et al., 2011; above. Protein content was determined using the bicinchoninic acid Reilly et al., 2016). method. Equivalent amounts of cell lysate were resolved by SDS- Initial efforts in the development of GGDPS inhibitors PAGE, transferred to polyvinylidene difluoride membrane, probed yielded digeranyl bisphosphonate (Fig. 1), which was found with the appropriate primary antibodies, and detected using to have an IC50 of 260 nM against the enzyme (Shull et al., horseradish peroxidase-linked secondary antibodies and Amersham 2006; Wiemer et al., 2007). Crystallography studies revealed Pharmacia Biotech ECL Western blotting reagents per manufac- that the V-shaped compound occupied the FDP substrate turer’s protocols. For Rab6, cells were lysed with Triton X-114
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