A novel bisphosphonate inhibitor of squalene synthase combined with a statin or a nitrogenous bisphosphonate in vitro
2011; Elsevier BV; Volume: 52; Issue: 11 Linguagem: Inglês
10.1194/jlr.m016089
ISSN1539-7262
AutoresBrian M. Wasko, Jacqueline P. Smits, Larry W. Shull, David F. Wiemer, Raymond J. Hohl,
Tópico(s)Ubiquitin and proteasome pathways
ResumoStatins and nitrogenous bisphosphonates (NBP) inhibit 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR) and farnesyl diphosphate synthase (FDPS), respectively, leading to depletion of farnesyl diphosphate (FPP) and disruption of protein prenylation. Squalene synthase (SQS) utilizes FPP in the first committed step from the mevalonate pathway toward cholesterol biosynthesis. Herein, we have identified novel bisphosphonates as potent and specific inhibitors of SQS, including the tetrasodium salt of 9-biphenyl-4,8-dimethyl-nona-3,7-dienyl-1,1-bisphosphonic acid (compound 5). Compound 5 reduced cholesterol biosynthesis and lead to a substantial intracellular accumulation of FPP without reducing cell viability in HepG2 cells. At high concentrations, lovastatin and zoledronate impaired protein prenylation and decreased cell viability, which limits their potential use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced FPP depletion and impairment of protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented zoledronate-induced impairment of both protein farnesylation and geranylgeranylation. Cotreatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly prevent the reduction of cell viability caused by lovastatin or zoledronate alone. The combination of an SQS inhibitor with an HMGCR or FDPS inhibitor provides a rational approach for reducing cholesterol synthesis while preventing nonsterol isoprenoid depletion. Statins and nitrogenous bisphosphonates (NBP) inhibit 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR) and farnesyl diphosphate synthase (FDPS), respectively, leading to depletion of farnesyl diphosphate (FPP) and disruption of protein prenylation. Squalene synthase (SQS) utilizes FPP in the first committed step from the mevalonate pathway toward cholesterol biosynthesis. Herein, we have identified novel bisphosphonates as potent and specific inhibitors of SQS, including the tetrasodium salt of 9-biphenyl-4,8-dimethyl-nona-3,7-dienyl-1,1-bisphosphonic acid (compound 5). Compound 5 reduced cholesterol biosynthesis and lead to a substantial intracellular accumulation of FPP without reducing cell viability in HepG2 cells. At high concentrations, lovastatin and zoledronate impaired protein prenylation and decreased cell viability, which limits their potential use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced FPP depletion and impairment of protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented zoledronate-induced impairment of both protein farnesylation and geranylgeranylation. Cotreatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly prevent the reduction of cell viability caused by lovastatin or zoledronate alone. The combination of an SQS inhibitor with an HMGCR or FDPS inhibitor provides a rational approach for reducing cholesterol synthesis while preventing nonsterol isoprenoid depletion. The mevalonate pathway (Fig. 1) is responsible for production of the core 5-carbon isoprenoid isopentenyl diphosphate (IPP) from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) (1Goldstein J.L. Brown M.S. Regulation of the mevalonate pathway.Nature. 1990; 343: 425-430Crossref PubMed Scopus (4566) Google Scholar). HMG-CoA is first reduced to mevalonate by HMG-CoA reductase (HMGCR). Through a short series of enzymatic reactions, mevalonate is then converted into IPP. Both IPP and its isomer dimethylallyl diphosphate (DMAPP) are utilized by farnesyl diphosphate synthase (FDPS) to generate the 15-carbon farnesyl diphosphate (FPP), which resides at the major branch point of the mevalonate pathway. Addition of an isoprene unit from IPP to FPP yields geranylgeranyl diphosphate (GGPP), a process mediated by the enzyme geranylgeranyl diphosphate synthase (GGDPS). The isoprene moiety of FPP or GGPP can be posttranslationally adducted onto proteins by protein farnesyltransferase or geranylgeranyltransferases, respectively, in a process collectively referred to as protein prenylation. In the first committed step of de novo cholesterol synthesis, two molecules of FPP are condensed in a head-to-head orientation to first form presqualene diphosphate and subsequently squalene. This reaction is catalyzed by the enzyme squalene synthase (SQS), which is encoded for by the gene farnesyl diphosphate farnesyl transferase 1 (FDFT1). Inhibition of mevalonate pathway targets has yielded multiple drugs with clinical success. Statins (e.g., lovastatin and atorvastatin) are a class of drugs commonly prescribed to reduce cholesterol levels. Statins inhibit HMGCR (2Endo A. Kuroda M. Tanzawa K. Competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by ML-236A and ML-236B fungal metabolites, having hypocholesterolemic activity.FEBS Lett. 1976; 72: 323-326Crossref PubMed Scopus (624) Google Scholar), which is the rate-limiting step of cholesterol biosynthesis (3Siperstein M.D. Fagan V.M. Feedback control of mevalonate synthesis by dietary cholesterol.J. Biol. Chem. 1966; 241: 602-609Abstract Full Text PDF PubMed Google Scholar). This leads to upregulation of the low-density lipoprotein (LDL) receptor (LDLR) in the liver and clearance of cholesterol-containing LDL particles from the bloodstream. The use of statin drugs is prevalent because elevated total cholesterol and LDL levels are major risk factors for coronary heart disease (4Wilson P.W. D'Agostino R.B. Levy D. Belanger A.M. Silbershatz H. Kannel W.B. Prediction of coronary heart disease using risk factor categories.Circulation. 1998; 97: 1837-1847Crossref PubMed Scopus (7495) Google Scholar). Although the statins are used abundantly and effectively, there are various reasons for developing novel inhibitors of cholesterol biosynthesis. There can be side effects associated with statin use, such as myopathy and hepatotoxicity (5Armitage J. The safety of statins in clinical practice.Lancet. 2007; 370: 1781-1790Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar), which are commonly speculated to be due to the depletion of nonsterol components of the mevalonate pathway (6Bełtowski J. Wójcicka G. Jamroz-Wiśniewska A. Adverse effects of statins - mechanisms and consequences.Curr. Drug Saf. 2009; 4: 209-228Crossref PubMed Scopus (160) Google Scholar). Furthermore, statin use does not always reduce LDL to desired levels (7El Harchaoui K. Akdim F. Stroes E.S. Trip M.D. Kastelein J.J. Current and future pharmacologic options for the management of patients unable to achieve low-density lipoprotein-cholesterol goals with statins.Am. J. Cardiovasc. Drugs. 2008; 8: 233-242Crossref PubMed Scopus (24) Google Scholar), which is particularly important as lower LDL target levels are suggested for some patients (8Shepherd J. Barter P. Carmena R. Deedwania P. Fruchart J.C. Haffner S. Hsia J. Breazna A. LaRosa J. Grundy S. et al.Effect of lowering LDL cholesterol substantially below currently recommended levels in patients with coronary heart disease and diabetes: the Treating to New Targets (TNT) study.Diabetes Care. 2006; 29: 1220-1226Crossref PubMed Scopus (457) Google Scholar, 9Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in AdultsExecutive summary of the third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III).JAMA. 2001; 285: 2486-2497Crossref PubMed Scopus (24461) Google Scholar, 10Javed U. Deedwania P.C. Bhatt D.L. Cannon C.P. Dai D. Hernandez A.F. Peterson E.D. Fonarow G.C. Use of intensive lipid-lowering therapy in patients hospitalized with acute coronary syndrome: an analysis of 65,396 hospitalizations from 344 hospitals participating in Get With The Guidelines (GWTG).Am. Heart J. 2010; 160 (1136.e1–3): 1130-1136Crossref PubMed Scopus (33) Google Scholar). Inhibition of SQS has attracted much interest as a pharmacological target, and various compounds have been identified as inhibitors (11Seiki S. Frishman W.H. Pharmacologic inhibition of squalene synthase and other downstream enzymes of the cholesterol synthesis pathway: a new therapeutic approach to treatment of hypercholesterolemia.Cardiol. Rev. 2009; 17: 70-76Crossref PubMed Scopus (41) Google Scholar, 12Elsayed R.K. Evans J.D. Emerging lipid-lowering drugs: squalene synthase inhibitors.Expert Opin. Emerg. Drugs. 2008; 13: 309-322Crossref PubMed Scopus (23) Google Scholar). Lapaquistat (TAK-475, Takeda) progressed to phase III clinical trials, but studies were discontinued after the US Food and Drug Administration recommended suspension of studies with high-dose (100 mg/kg) monotherapy due to hepatotoxicity manifested as elevated levels of liver transaminases (12Elsayed R.K. Evans J.D. Emerging lipid-lowering drugs: squalene synthase inhibitors.Expert Opin. Emerg. Drugs. 2008; 13: 309-322Crossref PubMed Scopus (23) Google Scholar). It is currently unknown whether this was due to an enzyme inhibitory class effect or whether it was specific to the drug. Inhibition of SQS can result in the accumulation of both FPP and FPP metabolites, such as farnesol-derived dicarboxylic acids (13Bostedor R.G. Karkas J.D. Arison B.H. Bansal V.S. Vaidya S. Germershausen J.I. Kurtz M.M. Bergstrom J.D. Farnesol-derived dicarboxylic acids in the urine of animals treated with zaragozic acid A or with farnesol.J. Biol. Chem. 1997; 272: 9197-9203Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), which could be responsible for the possible hepatotoxicity with the high-dose monotherapy of lapaquistat. Farnesol itself can be proapoptotic at high concentrations (14Joo J.H. Jetten A.M. Molecular mechanisms involved in farnesol-induced apoptosis.Cancer Lett. 2010; 287: 123-135Crossref PubMed Scopus (145) Google Scholar). Other reported results appeared promising with lapaquistat, with cholesterol levels decreasing in monotherapy treatment. Moreover, the combination therapy of lapaquistat with statins showed additional LDL reduction compared with statins alone (12Elsayed R.K. Evans J.D. Emerging lipid-lowering drugs: squalene synthase inhibitors.Expert Opin. Emerg. Drugs. 2008; 13: 309-322Crossref PubMed Scopus (23) Google Scholar). Also of interest, lapaquistat's active metabolite T-91485 was capable of preventing statin-induced myotoxicity in a human skeletal muscle cell model (15Nishimoto T. Tozawa R. Amano Y. Wada T. Imura Y. Sugiyama Y. Comparing myotoxic effects of squalene synthase inhibitor, T-91485, and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors in human myocytes.Biochem. Pharmacol. 2003; 66: 2133-2139Crossref PubMed Scopus (72) Google Scholar). Similarly, lapaquistat was able to prevent statin-induced myotoxicity in a guinea pig model (16Nishimoto T. Ishikawa E. Anayama H. Hamajyo H. Nagai H. Hirakata M. Tozawa R. Protective effects of a squalene synthase inhibitor, lapaquistat acetate (TAK-475), on statin-induced myotoxicity in guinea pigs.Toxicol. Appl. Pharmacol. 2007; 223: 39-45Crossref PubMed Scopus (31) Google Scholar). In addition to the expected cholesterol depletion, other SQS inhibitors have shown the potential for added benefits due to decreased triglyceride biosynthesis (17Hiyoshi H. Yanagimachi M. Ito M. Saeki T. Yoshida I. Okada T. Ikuta H. Shinmyo D. Tanaka K. Kurusu N. et al.Squalene synthase inhibitors reduce plasma triglyceride through a low-density lipoprotein receptor-independent mechanism.Eur. J. Pharmacol. 2001; 431: 345-352Crossref PubMed Scopus (38) Google Scholar), likely resulting from a farnesol-induced mechanism (18Hiyoshi H. Yanagimachi M. Ito M. Yasuda N. Okada T. Ikuta H. Shinmyo D. Tanaka K. Kurusu N. Yoshida I. et al.Squalene synthase inhibitors suppress triglyceride biosynthesis through the farnesol pathway in rat hepatocytes.J. Lipid Res. 2003; 44: 128-135Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Nitrogenous bisphosphonates (NBP; e.g., zoledronate and alendronate) are a second class of clinical drugs targeting the mevalonate pathway, and they are used for treatment of bone-related disorders such as osteoporosis. NBPs function by inhibition of FDPS, thus depleting cellular levels of FPP and other downstream isoprenoids (19van Beek E. Pieterman E. Cohen L. Lowik C. Papapoulos S. Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates.Biochem. Biophys. Res. Commun. 1999; 264: 108-111Crossref PubMed Scopus (470) Google Scholar). Bisphosphonates may be regarded as analogs of diphosphates, in which the central bridging oxygen atom (P-O-P) has been replaced with a carbon (P-C-P). This results in increased metabolic stability and allows chemical functionalization of the bisphosphonate core. Furthermore, the P-C-P linkage combined with an α-hydroxy group facilitates bone targeting (20Nancollas G.H. Tang R. Phipps R.J. Henneman Z. Gulde S. Wu W. Mangood A. Russell R.G. Ebetino F.H. Novel insights into actions of bisphosphonates on bone: differences in interactions with hydroxyapatite.Bone. 2006; 38: 617-627Crossref PubMed Scopus (709) Google Scholar). Although these compounds have a high affinity for bone, there are also reports of decreased cholesterol levels with patients treated with nitrogenous bisphosphonates (21Guney E. Kisakol G. Ozgen A.G. Yilmaz C. Kabalak T. Effects of bisphosphonates on lipid metabolism.Neuroendocrinol. Lett. 2008; 29: 252-255PubMed Google Scholar). To our knowledge, the combination of an FDPS inhibitor with an SQS inhibitor has not been evaluated. While various SQS inhibitors exist, relatively few are based on a bisphosphonate structure (22Ciosek Jr., C.P. Magnin D.R. Harrity T.W. Logan J.V. Dickson Jr., J.K. Gordon E.M. Hamilton K.A. Jolibois K.G. Kunselman L.K. Lawrence R.M. Lipophilic 1,1-bisphosphonates are potent squalene synthase inhibitors and orally active cholesterol lowering agents in vivo.J. Biol. Chem. 1993; 268: 24832-24837Abstract Full Text PDF PubMed Google Scholar), and their specificity for SQS relative to the prenylation arm of the mevalonate pathway has not been reported. Herein, we describe the synthesis and identification of novel bisphosphonates as potent inhibitors of SQS. A structure-activity relationship is evaluated in the context of potency and specificity for these novel compounds. Emphasis is placed on the evaluation of a lead compound 5 (Fig. 2) in combination with lovastatin or zoledronate in HepG2 cells. We hypothesized that these combinations of inhibitors would decrease cholesterol biosynthesis while preventing the depletion of nonsterol isoprenoid levels, resulting in reduced adverse cellular effects compared with inhibition of HMGCR or FDPS alone. Preparation of compounds 1 (23Holstein S.A. Cermak D.M. Wiemer D.F. Lewis K. Hohl R.J. Phosphonate and bisphosphonate analogues of farnesyl pyrophosphate as potential inhibitors of farnesyl protein transferase.Bioorg. Med. Chem. 1998; 6: 687-694Crossref PubMed Scopus (97) Google Scholar) and 2 (24Shull L.W. Design and Synthesis of Bisphosphonate Analogues of Farnesyl Pyrophosphate. MS Thesis.in: University of Iowa, Iowa City, IA2003Google Scholar, 25Shull L.W. Wiemer D.F. Copper-mediated displacements of allylic THP ethers on a bisphosphonate template.J. Organomet. Chem. 2005; 690: 2521-2530Crossref Scopus (9) Google Scholar) has been described, while compounds 3-5 were prepared as follows. In short, geranyl acetate was oxidized with SeO2 under reported literature procedures (26Umbreit M.A. Sharpless K.B. Allylic oxidation of olefins by catalytic and stoichiometric selenium dioxide with tert-butyl hydroperoxide.J. Am. Chem. Soc. 1977; 99: 5526-5528Crossref Scopus (537) Google Scholar) (Fig. 3). The newly formed hydroxyl group was protected by reaction with 3,4-dihydro-2H-pyran, and the acetate group was removed (27Marshall J.A. Andrews R.C. Coupling of allylic alcohol epoxides with sulfur-stabilized allylic anions.J. Org. Chem. 1985; 50: 1602-1606Crossref Scopus (49) Google Scholar). The resulting product then was split and carried forward in three divergent directions. The 2-tetrahydropyranyl (THP) protecting group of alcohol 10 was subjected to copper-mediated Grignard displacement (24Shull L.W. Design and Synthesis of Bisphosphonate Analogues of Farnesyl Pyrophosphate. MS Thesis.in: University of Iowa, Iowa City, IA2003Google Scholar, 25Shull L.W. Wiemer D.F. Copper-mediated displacements of allylic THP ethers on a bisphosphonate template.J. Organomet. Chem. 2005; 690: 2521-2530Crossref Scopus (9) Google Scholar, 28Mechelke M.F. Wiemer D.F. Synthesis of farnesol analogues through Cu(I)-mediated displacements of allylic thp ethers by Grignard reagents.J. Org. Chem. 1999; 64: 4821-4829Crossref PubMed Scopus (39) Google Scholar) with the corresponding biphenyl Grignard reagents to provide independently the ortho, meta, and para analogs 11, 12, and 13, respectively. Standard conversion of the free hydroxyl group to the allylic bromide was performed using PBr3. After workup, the bromides were used without further purification in the subsequent alkylation of tetraethyl methylenebisphosphonate (29Valentijn A.R.P.M. van den Berg O. van der Marel G.A. Cohen L.H. van Boom J.H. Synthesis of pyrophosphonic acid analogues of farnesyl pyrophosphate.Tetrahedron. 1995; 51: 2099-2108Crossref Scopus (22) Google Scholar). The resulting phosphonate esters 17, 18, and 19 were hydrolyzed to their corresponding salts (3, 4, and 5, respectively) under standard McKenna procedures (30McKenna C.E. Higa M.T. Cheung N.H. McKenna M. The facile dealkylation of phosphonic acid dialkyl esters by bromotrimethylsilane.Tetrahedron Lett. 1977; 18: 155-158Crossref Scopus (510) Google Scholar). All bisphosphonate salts were dissolved in water prior to use for biological studies. Detailed experimental and characterization data is provided in the supplementary data. A plasmid containing glutathione-S-transferase tagged human squalene synthase (EX-C0605-B03) was obtained from Genecopoeia (Rockville, MD). The plasmid was transformed into BL21 (DE3) Escherichia coli and expressed using 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) overnight at room temperature. Following lysis using lysozyme, 1.5% sarkosyl was added to increase protein solubility. Tagged protein was purified using glutathione agarose beads (Sigma; St. Louis, MO) according to the manufacturer's protocol. Enzyme assays were performed in 20 µl reactions containing 50 mM phosphate buffer (pH 7.4, 5 mM MgCl2, 4 mM CHAPS, 10 mM DTT), 400 ng recombinant enzyme, 0.25 µM [1-3H]FPP (20 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO) and 2 mM NADPH. Inhibitors were added with enzyme and incubated for 10 min at 37°C. Substrate was then added and reactions were incubated for 10 min at 37°C. Reactions were stopped by addition of 300 µl 1 mM EDTA, and then 1 ml ice-cold petroleum ether was added. After freezing the lower aqueous phase, the upper phase containing the products was transferred to a scintillation vial containing liquid scintillation fluid, and radioactivity was quantitated using a Beckman liquid scintillation counter. Data was analyzed using Prism Graphpad software. HepG2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and grown at 37°C with 5% CO2 in DMEM (Sigma) containing pen-strep (Gibco), amphotericin B (Thermo Scientific; Walthman, MA), 2 mM Glutamax (Invitrogen; Carlsbad, CA), 1 mM sodium pyruvate (Sigma), and 10% fetal bovine serum. Protein concentrations were determined by the bicinchoninic acid (BCA) method. Proteins were resolved on 12 or 15% gels and transferred to polyvinylidene difluoride membranes via electrophoresis. Blocking was performed in 5% nonfat dry milk for 45 min, after which primary and secondary antibodies were added sequentially for 1 h each at 37°C. Proteins were visualized using enhanced chemiluminescence detection. Rap1a and α-tubulin antibodies were acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-pan-Ras was acquired from InterBiotechnology (Tokyo, Japan). Cells were plated in 12-well plates and grown to near confluency. Compounds were added for 1 h followed by the addition of 1 µCi of 1-14C-acetate (Sigma) for 4 h. Cells were harvested using trypsin, and lipids were extracted using the Bligh and Dyer method (31Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43117) Google Scholar). Chloroform extracts were dried, resuspended in a 30 µl of chloroform, and loaded on S-60 silica TLC plates. TLC was performed using an eluting solvent system of toluene and isopropyl ether (1:1) as the mobile phase. Plates were stained with iodide to determine the location of a cholesterol standard. Regions corresponding to cholesterol were excised from the plate, and radioactivity was quantified using a liquid scintillation counter. Both FPP and GGPP levels were determined as reported (32Tong H. Holstein S.A. Hohl R.J. Simultaneous determination of farnesyl and geranylgeranyl pyrophosphate levels in cultured cells.Anal. Biochem. 2005; 336: 51-59Crossref PubMed Scopus (69) Google Scholar). Briefly, FPP and GGPP were extracted from cells and incorporated into fluorescently-labeled CAAX peptides by FTase and GGTase, which were then quantified by fluorescent detection on an HPLC. Levels were normalized to total protein as measured by BCA assay. The MTT assay measures the activity of enzymes that reduce the MTT substrate within metabolically active cells. It is commonly used as a measure of cell viability. Cells were allowed to adhere in 24-well plates and grown until approximately 50% confluent. Cells were treated with indicated compounds and incubated for 45 h, followed by addition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; EMD Chemicals; La Jolla, CA) and incubation for an additional 3 h. MTT stop solution (HCl, triton X-100, and isopropyl alcohol) was then added, and plates were gently agitated at 37°C overnight. Absorbance was measured at 540 nm with a reference wavelength at 650 nm. HepG2 cells were allowed to adhere in 6-well plates and grown until approximately 50% confluent. Cells were then washed with PBS, and the cells were equilibrated in media containing 10% lipoprotein deficient serum (LPDS) for 24 h. Cells were then treated for 24 h in media containing 10% LPDS with indicated compounds. Total RNA was isolated using Qiashredders and RNase easy mini kit (Qiagen), with inclusion of a DNase step as recommended. cDNA was made from 1 µg of RNA by reverse-transcription using the iScript DNA Synthesis Kit (Bio-Rad). Primers were from Integrated DNA Technologies (Coralville, IA). The primers used were: LDLR forward, TCAACACACAACAGCAGATGGCAC; LDLR reverse, AAGGCTAACCTGGCTGTCTAGCAA; GAPDH forward, TCGACAGTCAGCCGCATCTTCTTT; GAPDH reverse, ACCAAATCCGTTGACTCCGACCTT. Real-time PCR was performed using Sybr Green Master Mix (Applied Biosystems) using an Applied Biosystems Model 7000 real-time thermalcycler. The protocol for real-time was: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 95°C for 15s and 60°C for 1 min. A screen of in-house compounds was performed to identify in vitro inhibitors of SQS. A small panel of compounds (1, 2, 3, 4, and 5) was selected for further study of their inhibitory activity (Fig. 2). These compounds were synthesized as described in "Experimental Procedures." Dose-response curves were used to determine the concentration of compound required to inhibit 50% of SQS activity (IC50 value, Table 1). Geranyl bisphosphonate (compound 1) had an IC50 value of 1,361 nM in this assay. Addition of a phenyl ring at the C-9 position of the geranyl chain (compound 2) enhanced potency to an IC50 of 26.5 nM. The addition of a biphenyl group in an ortho- (compound 3), meta- (compound 4), or para- (compound 5) substituted pattern resulted in IC50 values of 5.7, 13.4, and 7.1 nM, respectively. One-way ANOVA was used to test for statistical differences in IC50 values, and the means were significantly different across the samples (P < 0.05). Tukey post hoc analysis indicated that compounds 2-5 were each statistically different from compound 1, but they were not statistically different from each other.TABLE 1IC50 values of compounds 1–5 for inhibition of SQSCompoundIC50 (nM)11361 ± 460226.5 ± 8.935.7 ± 1.7413.4 ± 1.857.1 ± 1.3Three independent dose-response curves of compounds 1–5 were used to generate IC50 values. Values are expressed as the mean ± SE, n = 3. Open table in a new tab Three independent dose-response curves of compounds 1–5 were used to generate IC50 values. Values are expressed as the mean ± SE, n = 3. Substrate-like inhibitors targeted against SQS have potential for off-target effects due to inhibition of other FPP utilizing enzymes. Our laboratories previously identified geranyl bisphosphonate (33Shull L.W. Wiemer A.J. Hohl R.J. Wiemer D.F. Synthesis and biological activity of isoprenoid bisphosphonates.Bioorg. Med. Chem. 2006; 14: 4130-4136Crossref PubMed Scopus (64) Google Scholar) as an inhibitor of GGDPS (34Wiemer A.J. Yu J.S. Lamb K.M. Hohl R.J. Wiemer D.F. Mono- and dialkyl isoprenoid bisphosphonates as geranylgeranyl diphosphate synthase inhibitors.Bioorg. Med. Chem. 2008; 16: 390-399Crossref PubMed Scopus (38) Google Scholar). With this in mind, we set out to determine whether the compounds active against SQS impaired protein farnesylation or geranylgeranylation, outputs that can identify inhibitors of FDPS, GGDPS, or prenyltransferases (35Barney R.J. Wasko B.M. Dudakovic A. Hohl R.J. Wiemer D.F. Synthesis and biological evaluation of a series of aromatic bisphosphonates.Bioorg. Med. Chem. 2010; 18: 7212-7220Crossref PubMed Scopus (24) Google Scholar, 36Wasko B.M. Dudakovic A. Hohl R.J. Bisphosphonates induce autophagy by depleting geranylgeranyl diphosphate.J. Pharmacol. Exp. Ther. 2011; 337: 540-546Crossref PubMed Scopus (53) Google Scholar). The prenylation of select individual proteins was assessed by Western blot for use as markers of the cellular status of protein farnesylation and geranylgeranylation. The Ras antibody utilized in these experiments recognizes both the modified (farnesylated) and unmodified (nonfarnesylated) forms of the protein. Impairment of farnesylation on the Ras Western blot panel was noted by the presence of a more slowly migrating upper unmodified band. The impairment of Rap1a geranylgeranylation was noted by the appearance of a band on the Western blot (the antibody used only detects the unmodified form of Rap1a). HepG2 cells were treated with 25 µM lovastatin for 24 h as a positive control, as this concentration was required to impair both farnesylation and geranylgeranylation (Fig. 4A). Lovastatin depletes mevalonate and the downstream products (e.g., FPP and GGPP) and thus impairs both protein farnesylation and geranylgeranylation. HepG2 cells were treated with 50 µM of compounds 1-5 for 24 h. Monogeranyl bisphosphonate (compound 1) impaired protein geranylgeranylation (Fig. 4A). Compound 2 also impaired geranylgeranylation, whereas compound 3 displayed a slight impairment. No detectable impairment was noted with compounds 4 or 5. Compound 5 was utilized in subsequent studies as the lead inhibitor due to its potency and specificity for SQS. Treatment of HepG2 cells with 25 µM lovastatin for 24 h resulted in impairment of both farnesylation of Ras and geranylgeranylation of Rap1a. Lovastatin-induced impairment of Ras farnesylation was prevented by cotreatment with 25 µM exogenous FPP, whereas lovastatin-induced impairment of Rap1a geranylgeranylation was prevented by cotreatment with 25 µM GGPP (Fig. 4B). Cotreatment of 25 µM lovastatin with 25 µM compound 5 prevented lovastatin-induced impairment of Ras farnesylation, but it did not completely restore Rap1a geranylgeranylation. Treatment of HepG2 cells for 24 h with 10 µM zoledronate caused impairment of Ras farnesylation and Rap1a geranylgeranylation (Fig. 4C), and cotreatment with exogenous FPP or GGPP prevented the impairment of farnesylation or geranylgeranylation, respectively. Cotreatment of HepG2 cells with 10 µM zoledronate with 25 µM compound 5 completely prevented both zoledronate-induced impairment of farnesylation and geranylgeranylation. We next measured the FPP and GGPP levels from HepG2 cells in response to treatment with either compound 5 or lovastatin alone and in combination for 24 h. In HepG2 cells treated with 25 µM compound 5, FPP levels were increased approximately 16-fold and GGPP levels were approximately 1.6-fold compared with control (Fig. 5). As expected, lovastatin (25 µM) reduced both FPP and GGPP levels compared with control. The combination of 25 µM lovastatin with 25 µM compound 5 resulted in increased FPP levels compared with lovastatin-treated cells; however, GGPP levels remained diminished. This data correlates with the results showing prevention of lovastatin-induced impairment of farnesylation, but not geranylgeranylation, by cotreatment with compound 5. Lovastatin at 50 nM or compound 5 at 50 µM significantly inhibited de novo cholesterol biosynthesis in HepG2 cells compared with untreated HepG2 cells (Fig. 6). The combination of these concentrations of lovastatin and compound 5 showed a trend toward enhanced inhibition of cholesterol synthesis compared with single treatments, but it was not statistically significant. Zoledronate at 10 µM also reduced cholesterol biosynthesis compared with control, and the combination of 25 µM compound 5 with 10 µM zoledronate did not significantly enhance cholesterol depletion compared with the respective single tre
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