Novel bisphosphonates as inhibitors of isoprenoid biosynthesis

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1 University of Iowa Iowa Research Online Theses and Dissertations 2011 Novel bisphosphonates as inhibitors of isoprenoid biosynthesis Brian M. Wasko University of Iowa Copyright 2011 Brian M Wasko This dissertation is available at Iowa Research Online: Recommended Citation Wasko, Brian M.. "Novel bisphosphonates as inhibitors of isoprenoid biosynthesis." PhD (Doctor of Philosophy) thesis, University of Iowa, Follow this and additional works at: Part of the Cell Biology Commons

2 NOVEL BISPHOSPHONATES AS INHIBITORS OF ISOPRENOID BIOSYNTHESIS by Brian M. Wasko An Abstract Of a thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Molecular and Cellular Biology in the Graduate College of The University of Iowa May 2011 Thesis Supervisor: Professor Raymond J. Hohl

3 1 Products of the isoprenoid biosynthetic pathway are involved in diverse biological functions. For example, the isoprenoid diphosphate, farnesyl diphosphate (FPP), is used for synthesis of squalene, a precursor of cholesterol. In addition, FPP and geranylgeranyl diphosphate (GGPP) are used for protein prenylation, which is a post-translational modification of certain proteins required for their proper membrane localization and function. Enzymes within the isoprenoid biosynthetic pathway have been inhibited successfully by drugs that are now used clinically, including statins and nitrogenous bisphosphonates (NBPs). Statins and NBPs are inhibitors of isoprenoid biosynthetic enzymes, due to their structural resemblance to substrates within the pathway. The bisphosphonate core resembles the diphosphate portion of isoprenoid diphosphate intermediates within the isoprenoid biosynthetic pathway. It is hypothesized that distinct isoprenoid biosynthetic enzymes can be inhibited by bisphosphonates in a manner dependent upon the overall bisphosphonate structure. Along with our collaborators, we have developed novel bisphosphonate inhibitors of multiple isoprenoid biosynthetic enzymes. Potent in vitro inhibitors of squalene synthase (SQS) were identified and evaluated in HepG2 liver cells. A lead inhibitor of squalene synthase was combined with a statin and a nitrogenous bisphosphonate, and focus was placed on these combinations as potential novel mechanisms to reduce cholesterol synthesis while minimizing impairment of non-sterol synthesis. Specifically, it was found that the lead SQS inhibitor prevents lovastatin-mediated impairment of protein farnesylation but not geranylgeranylation. Also, the lead SQS inhibitor prevented both zoledronate-induced impairment of protein farnesylation and geranylgeranylation.

4 2 Novel bisphosphonates were also identified as inhibitors of geranylgeranyl diphosphate synthase (GGDPS) and protein prenylation in K562 leukemia cells. A novel cellular consequence of GGPP depletion was also established. In PC3 cells, zoledronate and digeranyl bisphosphonate (DGBP; a lead inhibitor of GGDPS) were determined to induce autophagy as measured by accumulation of the autophagic marker LC3-II. GGPP depletion was implicated as the cause of autophagic induction in this system. Specifically, results suggest that impairment of proteins geranylgeranylated by geranylgeranyl transferase II is responsible for the induction of autophagy. Mycobacterium isoprenoid biosynthetic enzymes were also evaluated as inhibitory targets for bisphosphonates. Novel inhibitors of Mycobacteria tuberculosis omega-e,z-fpp synthase and decaprenyl diphosphate synthase were identified. A lead inhibitor of decaprenyl diphosphate synthase was also evaluated in Mycobacterium smegmatis, which was utilized as a surrogate model. The lead inhibitor was found to have no effect on M. smegmatis growth; however it enhanced growth inhibition mediated by ethambutol. This effect was prevented by addition of exogenous decaprenyl diphosphate, suggesting that the growth inhibition was due to decaprenyl diphosphate depletion. Decaprenyl diphosphate was also found to prevent the growth inhibitory effect of SQ109, a novel anti-mycobacterial drug in clinical development with an unknown mechanism of action. Abstract Approved: Thesis Supervisor Title and Department Date

5 NOVEL BISPHOSPHONATES AS INHIBITORS OF ISOPRENOID BIOSYNTHESIS by Brian M. Wasko A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Molecular and Cellular Biology in the Graduate College of The University of Iowa May 2011 Thesis Supervisor: Professor Raymond J. Hohl

6 Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL PH.D. THESIS This is to certify that the Ph.D. thesis of Brian M. Wasko has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Molecular and Cellular Biology at the May 2011 graduation. Thesis Committee: Raymond J. Hohl, Thesis Supervisor David F. Wiemer Marc S. Wold John G. Koland W. Scott Moye-Rowley

7 Science is a way of thinking much more than it is a body of knowledge. Carl Sagan ii

8 ACKNOWLEDGMENTS I am grateful to Dr. Raymond Hohl for his support and for giving me the opportunity to perform this thesis work within his laboratory. I m also thankful to the University of Iowa Interdisciplinary Program in Molecular and Cellular Biology. I would like to thank all the members of my thesis committee: David Wiemer, Scott Moye- Rowley, Marc Wold, and John Koland for providing their time and advice during my studies. I wish to thank all the past and present members of Dr. Hohl s laboratory group, of which there are too many to thank individually. In particular, I would like to thank Amel Dudakovic and Megan Moore for the insightful caffeine-aided discussions on research and beyond. I would also like to acknowledge current and former members of Dr. Wiemer s organic chemistry group for synthesizing and providing bisphosphonates. I am grateful for the unending support of my mother and father, and of all of my friends and family. Lastly, thank you Jaci, for your love and support throughout. iii

9 TABLE OF CONTENTS LIST OF TABLES... vi LIST OF FIGURES... vii LIST OF ABBREVIATIONS...x CHAPTERS I. INTRODUCTION...1 The Isoprenoid Biosynthetic Pathway...1 Protein Prenylation...3 Cholesterol Biosynthesis...4 Inhibitors of the Mevalonate Pathway...5 Non-Mevalonate Pathway of Isoprenoid Biosynthesis...8 Hypothesis...10 II. NOVEL BISPHOSPHONATE INHIBITORS OF SQUALENE SYNTHASE COMBINED WITH A STATIN AND NITROGENOUS BISPHOSPHONATE...15 Abstract...15 Introduction...16 Results...18 Discussion...22 Experimental Procedures...25 III. NOVEL BISPHOSPHONATE INHIBITORS OF GGDPS...39 Abstract...39 Introduction...39 Results...41 Discussion...44 Experimental Procedures...46 IV. BISPHOSPHONATE INHIBITORS OF FDPS AND GGDPS INDUCE AUTOPHAGY...59 Abstract...59 Introduction...60 Results...62 Discussion...66 Experimental Procedures...79 iv

10 V. INHIBITORS OF MYCOBACTERIUM ISOPRENOID BIOSYNTHESIS...81 Abstract...81 Introduction...81 Results...84 Discussion...87 Experimental Procedures...92 VI. SUMMARY Summary of Results Future Directions Conclusions REFERENCES v

11 LIST OF TABLES Table 1. GGDPS in vitro IC 50 values (µm) generated from concentration-response curves for compounds 6-12 and Table 2. Qualitative assessment for impairment of Rap1a geranylgeranylation by compounds 1 and Table 3. IC 50 values (nm) for zoledronate and compounds versus mycobacterial ω-e,z-fpp synthase Table 4. IC 50 values (µm) of zoledronate, and compounds 1, 15 and for mycobacterial decaprenyl diphosphate synthase vi

12 LIST OF FIGURES Figure 1. Overview of mevalonate pathway of isoprenoid biosynthesis...11 Figure 2. Timeline of bisphosphonate development...12 Figure 3. Chemical structures Figure 4. Non-mevalonate pathway of isoprenoid biosynthesis...14 Figure 5. SQS reaction mechanism...29 Figure 6. Coomassie Blue stained SDS-PAGE of purified recombinant GST-SQS...30 Figure 7. SQS reaction rate in response to varied FPP concentration...31 Figure 8. Structures of compounds Figure 9. In vitro SQS concentration-response curves...32 Figure 10. Protein prenylation in HepG2 cells Figure 11. FPP and GGPP levels in HepG2 cells...36 Figure 12. De novo cholesterol biosynthesis in HepG2 cells...37 Figure 13. MTT assay in HepG2 cells...38 Figure 14. Structures of aromatic bisphosphonates...48 Figure 15. Structures of compounds Figure 16. Compounds 6-12 tested for impairment of protein prenylation in K562 cells...51 Figure 17. Impairment of protein geranylgeranylation by compounds 9 and 11 was prevented by exogenous GGPP...52 Figure 18. Effect of compounds 9 and 11 on DNA synthesis Figure 19. Concentration-response of compounds 1, 14, and Figure 20. Concentration-response of DGBP and compound Figure 21. Isoprenoid diphosphate levels from K562 cells treated for 48 hours with compound 16 and DGBP at indicated concentrations Figure 22. Schematic of autophagy vii

13 Figure 23. Isoprenoid pathway inhibitors interfere with protein prenylation, decrease MTT activity, and decrease DNA synthesis in PC3 cells Figure 24. Isoprenoid pathway inhibitor-induced LC3-II accumulation and impairment of GGTase-II protein geranylgeranylation, but not impairment of GGTase I geranylgeranylation is prevented by GGPP addition in PC3 cells Figure 25. Bisphosphonates induce autophagic flux in PC3 cells...77 Figure 26. Bisphosphonates induce LC3-II accumulation in MDA-MB-231 but not in MDA-MB-468 and HepG2 cells...78 Figure 27. Inhibition of either FTase or GGTase I does not induce LC3-II accumulation in PC3 cells...79 Figure 28. Inhibition of autophagy enhances DGBP-induced reduction in MTT activity and DNA synthesis in PC3 cells Figure 29. Schematic of mycobacterial cell wall...94 Figure 30. Coomassie Blue stained SDS-PAGE of purified mycobacterial ω-e,z- FPP synthase Figure 31. ω-e,z-fpp reaction rate versus substrate concentration Figure 32. Structures of compounds Figure 33. Coomassie Blue stained SDS-PAGE of purified recombinant decaprenyl diphosphate synthase...99 Figure 34. Decaprenyl diphosphate synthase reaction rate versus substrate concentration Figure 35. Structures of compounds 1, 15, and Figure 36. Structures of stilbene-containing compounds Figure 37. Effect of compound 25 effect on decaprenyl diphosphate synthase kinetics Figure 38. Protein sequence alignment of decaprenyl diphosphate synthase from M. tuberculosis and homolog from M. smegmatis Figure 39. Compound 25 enhances ethambutol-mediated growth inhibition and decaprenyl diphosphate prevents this effect Figure 40. Compound 26 impairs M. smegmatis growth as measured in a CFU assay viii

14 Figure 41. Structures of ethambutol (EMB) and SQ Figure 42. SQ109-mediated growth inhibition is prevented by addition of decaprenyl mono- or diphosphate ix

15 LIST OF ABBREVIATIONS BCA DGBP DMAPP ECL FBS FDPS FOH FPP FTase FTIs GGDPS GGOH GGPP GPP GGTase I GGTase II GGTIs HMGCR bicinchoninic acid digeranyl bisphosphonate dimethylallyl diphosphate enhanced chemiluminescent fetal bovine serum farnesyl diphosphate synthase farnesol farnesyl diphosphate farnesyl transferase farnesyl transferase inhibitors geranylgeranyl diphosphate synthase geranylgeraniol geranylgeranyl diphosphate geranyl diphosphate geranylgeranyl transferases I geranylgeranyl transferases II geranylgeranyl transferase inhibitors HMG-CoA reductase IC 50 inhibitory concentration 50% IPP IPTG LDL LDLR MTT isopentenyl diphosphate isopropyl β-d-1-thiogalactopyranoside low-density lipoprotein low-density lipoprotein receptor 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide x

16 NBPs PARP PBS PVDF SDS SDS-PAGE SQS nitrogenous bisphosphonates poly(adp-ribose) polymerase phosphate buffered saline polyvinylidene fluoride sodium dodecyl sulfate SDS polyacrylamide gel electrophoresis squalene synthase xi

17 1 CHAPTER I: INTRODUCTION The Isoprenoid Biosynthetic Pathway Isoprenoids (also commonly referred to as terpenoids) are a diverse and immense class of compounds, with over 23,000 characterized (1). Isoprenoids are built from five carbon isoprene units, which are combined and derivatized in nature to form molecules of varied function. Isopentenyl diphosphate (IPP) may be considered the fundamental source of carbon for all isoprenoid biosynthesis. There are two known metabolic pathways involved in the synthesis of IPP, the mevalonate pathway and the nonmevalonate pathway. Humans, as well as other higher eukaryotes, utilize the mevalonate pathway (Figure 1) for production of IPP (2). Acetate is converted to acetyl-coa, which can then be converted to acetoacetyl-coa by acetoacetyl-coa thiolase. Acetoacetyl-CoA is the substrate for synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) by the enzyme HMG-CoA synthase (EC ). HMG-CoA is then reduced to mevalonate by HMG-CoA reductase (HMGCR, EC ) in a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reaction. Mevalonate is then ATP-dependently phosphorylated by mevalonate kinase (MVK, EC ), and again by phosphomevalonate kinase (PMVK, EC ). Deficiency of MVK can result in mevalonic aciduria and hyperimmunoglobulinemia D syndrome (3). Diphosphomevalonate decarboxylase (EC ) converts diphosphate-mevalonate into IPP, and requires ATP. IPP can be interconverted to its isomer DMAPP by isopentenyl diphosphate isomerase, and two isoforms of this gene exist in humans (4) (IDI, EC ).

18 2 The prenyl group of DMAPP can be attached to adenosine residues of specific trnas by trna isopentenyltransferase (TRIT1) (5). The trna encoding for the 21 st amino acid, selenocysteine, requires isopentylation of adenosine 37 (6). In humans, there are approximately 25 selenocysteine-containing proteins identified, which typically have antioxidant related functions (e.g., glutathione peroxidase) (7). Two equivalents of IPP and one equivalent of DMAPP are used to generate the 15 carbon farnesyl diphosphate (FPP) via farnesyl diphosphate synthase (FDPS, EC ). FPP is the major branch point of the mevalonate pathway. FPP can have an additional isoprene unit from IPP added to form geranylgeranyl diphosphate (GGPP), catalyzed by the enzyme GGPP synthase (GGDPS, EC ). FPP and GGPP can both be utilized for protein prenylation, as discussed below. FPP also can have multiple isoprene units from IPP added by a cis-prenyltransferase, dehydrodolichyl diphosphate synthase (DHDDS). This results in the formation of the long chain dehydrodolichyl diphosphate, which undergoes further processing to yield α-saturated dolichol. Dolichols are glycosyl carrier molecules involved in the cellular processes of N-linked glycosylation, which is important for protein folding. Dolichol is also involved in the synthesis of glycosylphosphatidylinositol (GPI) anchors, which are reversible posttranslational membrane anchors (8). Mutations in DHDDS can result in retinitis pigmentosa, potentially due to defects in rhodopsin glycosylation (9,10). The farnesyl group of FPP can also be used for synthesis of heme a, a porphyrin ring system containing an iron atom. Cytochrome c oxidase (complex IV) utilizes heme a in oxidative phosphorylation (11). GGPP and IPP are substrates for the prenyl diphosphate synthase (PDSS1 and PDSS2) complex, which forms the isoprenoid side-chain of

19 3 ubiquinone (coenzyme Q-10) (12). Ubiquinone is another important molecule involved in oxidative phosphorylation, as it functions as an electron carrier from complex I or II to complex III. GGPP can also be utilized for the synthesis of menaquinone-4 (vitamin K2), a cofactor required for posttranslational carboxylation of some proteins, such as the osteoblastic marker osteocalcin (13). FPP and GGPP can also be reversibly converted to their respective alcohol forms, FOH and GGOH (14), by an integral membrane phosphatase, PDP1/PPAPDC2, (15) and as yet unidentified kinases (16). Protein Prenylation The isoprene portion of either FPP or GGPP can be post-translationally linked onto the carboxy-terminus of select proteins, in a process collectively referred to as protein prenylation (17). Protein farnesylation utilizes FPP and is catalyzed by protein farnesyl transferase (FTase, EC ). Geranylgeranylation utilizes GGPP and is catalyzed by either protein geranylgeranyltransferase I (GGTase I, EC ) or II (GGTase II or Rab GGTase, EC ). FTase and GGTase I recognize CaaX (C = Cysteine, a = aliphatic amino acid, X = any amino acid) box motifs, and the prenyl group is added onto the cysteine via a thioester linkage (18). FTase displays preference for proteins containing X as methionine, serine, alanine, or glutamate, while GGTase I preferentially acts on CaaL sequences (17). Following prenylation, the three C-terminal residues (aax) are cleaved and the terminal cysteine is methyl esterified at the carboxyl group (18). GGTase II requires additional factors for protein recognition and does not utilize a CAAX motif, but instead the consensus sequences CC, CXC, or CXXX (19,20). The CXC geranylgeranylated proteins are methyl esterified but CC geranylgeranylated proteins are not. GGTase II can add two geranylgeranyl groups onto a single protein, and

20 4 the majority of Rab proteins contain two cysteine residues, and two geranylgeranyl groups are added during prenylation (20). It is estimated that 2% of proteins in the proteome of mammals are prenylated (21), including many small GTP-hydrolyzing enzymes, such as the Ras protein. Protein prenylation is thought to facilitate interaction with membranes and modulate proteinprotein interactions (22). Cholesterol Biosynthesis Cholesterol can be synthesized within cells and can be obtained from the diet. Two units of FPP can form squalene in a two-step reaction catalyzed by squalene synthase (SQS, EC ). SQS is encoded by the gene farnesyl diphosphate farnesyltransferase 1 (FDFT1). In the first step of the reaction, a cyclopropylcarbinyl diphosphate intermediate is formed, known as pre-squalene diphosphate (PSDP) (23). In the second NADPH-dependent step, PSDP is converted to squalene. Interestingly, PSDP has been identified within cells and can mediate the cellular immune response by inhibiting superoxide anion production in neutrophils (24). Squalene is oxidized to 2,3- oxidosqualene by squalene epoxidase. Lanosterol synthase then uses 2,3-oxidosqualene for the synthesis of lanosterol, which is followed by approximately 20 additional enzymatic steps to form cholesterol (25). Cholesterol is a vital component of cell membranes and is also necessary for the synthesis of steroid hormones, bile acids, and vitamin D. In addition to endogenous biosynthesis, cholesterol can also be obtained extracellularly from low density lipoprotein (LDL) particles by the LDL receptor (LDLR). Many genes controlling cholesterol biosynthesis and uptake are

21 5 transcriptionally regulated due to sterol response elements (SREs) within their promoters (26). SRE binding proteins (SREBPs) sense cholesterol levels via an interaction with the sterol-sensing cleavage-activating protein (SCAP) in the endoplasmic reticulum. When cholesterol levels are adequate, cholesterol is bound by a sterol sensing domain within SCAP, and SCAP and insulin-regulated protein (Insig) maintain an interaction with SREBP. When sterol levels are not sufficient, SCAP no longer interacts with Insig, allowing the SREBP-SCAP complex to migrate from the ER to the golgi. SREBP is cleaved within the golgi by site-1 and site-2 proteases, releasing active SREBP. Active SREBP migrates to the nucleus where it plays a role in the transcriptional activation of genes containing SREs (26). HMGCR, FDPS, SQS, and LDLR are SREBP-responsive due to SREs located within their respective gene promoter regions. Inhibitors of the Mevalonate Pathway Statins Akira Endo isolated the first statin (mevastatin) from Penicillium citrinum in the 1970s (27). Lovastatin was the first commercial statin approved by the FDA in Statins structurally resemble HMG-CoA and act as competitive inhibitors of HMGCR (28). Statins are used to decrease cholesterol biosynthesis, as HMGCR is responsible for the rate limiting step of cholesterol biosynthesis (29). By inhibiting upstream in the isoprenoid biosynthetic pathway, the statins decrease mevalonate and thus downstream intermediates such as FPP, GGPP, and cholesterol. Statin-mediated cholesterol reduction results in increased levels of LDLR and other SRE-responsive proteins (30). An increase in LDL receptors in the liver results in clearance of cholesterol-containing LDL particles

22 6 from the bloodstream (30). The use of statin drugs is prevalent because elevated total cholesterol and LDL levels and are major risk factors for coronary heart disease (31). While statins are used primarily for lowering serum cholesterol levels, they have other effects due to their inhibition upstream within the isoprenoid biosynthetic pathway. By depleting FPP and GGPP statins can decrease protein prenylation (32). Due to this reduction of prenylation, statins are being evaluated as potential anti-cancer agents in a manner similar to FTIs and GGTIs (discussed below) (33). Interestingly, there are some clinical studies suggesting decreased incidence of certain cancer types with long-term statin use (34). However, strong evidence is lacking, except in the case of association of statin use with reduced risk of aggressive prostate cancer (35). Bisphosphonates The first geminal bisphosphonates were synthesized by Hoffman and von Beyer in 1897 (Figure 2) (36). The early applications of bisphosphonates included use as corrosion preventatives and as water softeners (37). Herbert Fleisch et al. found that inorganic pyrophosphate (diphosphate) inhibited crystallization of calcium phosphate, and subsequently it was determined that the diphosphate-like non-nitrogenous bisphosphonate etidronate could inhibit bone resorption (36). Bone resorption involves the recycling of bone by osteoclasts, which is normally balanced by osteoblast-mediated bone synthesis. In 1977 the FDA approved etidronate for Paget s disease of the bone, which is a disorder with a phenotype of weakened bone (38). In the 1990s the FDA approved two nitrogen containing bisphosphonates (NBPs): pamidronate for hypercalcemia of malignancy and aledronate for Paget s disease of the bone and osteoporosis. Subsequently, the mechanism of non-nitrogenous bisphosphonate

23 7 therapeutic action was elucidated, when they were found to incorporate into nonhydrolyzable ATP analogs (39). It was later determined that NBPs function by inhibition of FDPS, thus depleting FPP and other downstream isoprenoids (40). Another NBP, zoledronate (Figure 3B), has more recently been approved for multiple myeloma and cancers metastasized to bone (41). Much recent interest in NBPs has been in regard to anti-cancer use. 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) (Figure 3A). This modification allows for chemical functionalization at the bisphosphonate core as well as increased metabolic stability when compared to diphosphates. The P-C-P linkage when combined with the nitrogenous and α-hydroxy groups facilitates considerable bone targeting (42). FTIs and GGTIs Aside from the nitrogenous bisphosphonates and statins, there are multiple other inhibitors of isoprenoid biosynthetic enzymes under development. FTase and GGTase I are heterodimers with a shared alpha subunit and distinct beta subunits. Inhibitors of protein farnesyl transferase (FTIs) have been evaluated in part due to the requirement of farnesylation of Ras proteins for their proper membrane localization and function. Ras proteins are small GTPase protein aberrantly activated in ~30% of all human cancers (43), hence the interest in disruption of these specific proteins. FTIs have not fared well in clinical trials (44), perhaps in part due to alternate geranylgeranylation of N-Ras and K-Ras when FTase is inhibited (45). There has been some resurgent interest in the well tolerated FTIs as treatment for Hutchinson-Gilford Progeria Syndrome, a premature

24 8 aging disease caused by the constitutive farnesylation of lamin A (46). However, the occurance of cross-prenylation is also an issue with lamin A (47). Inhibitors of GGTase I have been developed with potential anti-cancer applications in mind, in part due to the alternative prenylation of Ras when FTIs are employed (45). Inhibitors of GGTase II are in early development and under investigation as possible treatments for cancer metastasized to bone (48). SQS inhibitors Squalene synthase is another prospective therapeutic target within the isoprenoid biosynthetic pathway. SQS is a potential target for agents to lower cholesterol levels, because it catalyzes the first committed step toward cholesterol biosynthesis from the mevalonate pathway. Multiple inhibitors of SQS have been identified, including the zaragozic acids, quinuclidines, 2-biphenylmorpholines, and 4,1-benzoxazepines (49,50). The furthest progressed of the SQS inhibitors has been lapaquistat (TAK-475, Takeda); however, clinical trials involving this drug were halted after elevated markers of liver damage were noted in patients receiving the highest dose mono-therapy (50). Non-Mevalonate Pathway of Isoprenoid Biosynthesis The non-mevalonate pathway (Figure 4), also know as the deoxy-xylulose phosphate (DOXP) or methylerythritol phosphate (MEP) pathway, is an alternate route for the production of IPP utilized in some eukaryotic parasites and some bacteria including those in the Mycobacterium genus. Pyruvate and glyceraldehyde 3-phosphate are converted to DOXP by DOXP synthase (DXS). DOXP is then NADPH-dependently reduced by DOXP reductase to MEP. MEP undergoes four further enzyme-catalyzed

25 9 transformations to yield (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) that can then be reduced to DMAPP and IPP, the core isoprenoid building blocks. Isoprenoid biosynthesis is an attractive target for developing anti-pathogenics due to the vital functions of isoprenoid pathway products in various pathogenic organisms (51). Fosmidomycin is an experimental drug used for malaria, which inhibits DOXP reductase in the malaria-causing Plasmodium falciparum (52). In many bacteria, cispolyprenyl diphosphates generated from FPP and IPP are important in synthesis of the cell wall, and this area of isoprenoid biosynthesis is also under investigation for enzymatic targets and novel inhibitors. For example, inhibitors of E.coli undecaprenyl diphosphate synthase (UPPS) are being developed (53). Other distinct pathogen isoprenoid biosynthetic enzymes are also being targeted. For example, Staphylococcus aureus dehydrosqualene synthase (CrtM) has been targeted in vitro by bisphosphonate and phosphonosulfonate inhibitors (54,55). The product of this enzyme, dehydrosqualene, is a precursor of staphyloxanthin, which is the pigment responsible for the aureus (golden in Latin) portion of the name of S. aureus. Aside from color, this pigment provides S. aureus an antioxidant defense against host macrophages. A phosphonosulfonate inhibitor of CrtM that also inhibited human SQS (BPH-652) caused S. aureus colonies to grow white in color instead of golden and enhanced clearance of the bacterium in mice (55). This finding was novel and exciting in that it provided a proof of principle for a virulence factor-based therapy. Instead of directly inhibiting growth of a microorganism, it was rendered susceptible to the host immune defense. Numerous other intriguing potential targets exist within pathogen isoprenoid biosynthetic pathways, and there are many possibilities yet to be explored.

26 10 Hypothesis The isoprenoid biosynthetic pathway is of vast importance in relation to various diseases, and inhibition of isoprenoid biosynthetic enzymes has yielded successful drugs, including the statins and nitrogenous bisphosphonates. Due to the structural similarly between the bisphosphonate core and that of diphosphates within the isoprenoid biosynthetic pathway, it is hypothesized that dependent upon chemical modification of the side chains, bisphosphonates can be employed to inhibit distinct isoprenoid biosynthetic enzymes. This will result in disruption of cellular functions dependent upon the specific isoprenoid diphosphate that has been depleted. Herein is described the identification and development of novel bisphosphonates as inhibitors of various isoprenoid biosynthetic enzymes and the cellular consequences that follow. Specifically, novel inhibitors of human SQS are identified. SQS inhibitors are assessed in combination with a statin and NBP and evaluated with an emphasis on novel mechanisms to specifically deplete cholesterol while maintaining non-sterol synthesis. Novel inhibitors of GGDPS are also identified, and a NBP and GGDPS inhibitor are determined to induce autophagy via GGPP depletion. Inhibitors of mycobacterial isoprenoid biosynthetic enzymes are also identified and evaluated in vivo using Mycobacterium smegmatis.

27 Figure 1. Overview of mevalonate pathway of isoprenoid biosynthesis. Enzymes illustrated include HMGCR, FDPS, SQS, GGDPS, FTase and GGTase I and II. Statins and nitrogenous bisphosphonates inhibit HGMCR and FDPS, respectively. 11

28 Figure 2. Timeline of bisphosphonate development. 12

29 13 A B Figure 3. Chemical structures. (A) Generalized core structure of bisphosphonate and pyrophosphate (or diphosphate). (B) Structures of lovastatin, zoledronate and DGBP.

30 14 Figure 4. Non-mevalonate pathway of isoprenoid biosynthesis. Adapted from Barry et al. (56). DXS utilizes pyruvate and glyceraldehyde 3-phosphate to form DOXP, with release of CO 2. DOXP is the substrate for DOXP reductase, yielding MEP. Four further transformations occur via the indicated enzymes, forming HMB-PP, which can then be reduced to IPP and DMAPP.

31 15 CHAPTER II: NOVEL BISPHOSPHONATE INHIBITORS OF SQUALENE SYNTHASE COMBINED WITH A STATIN AND NITROGENOUS BISPHOSPHONATE Abstract Statins and nitrogenous bisphosphonates (NBPs) are clinically used inhibitors of the mevalonate pathway, which is the pathway responsible for cholesterol production as well as other isoprenoid-derived molecules. Through the inhibition of HMGCR and FDPS, respectively, these drugs deplete cells of FPP and can disrupt downstream cellular processes such as protein prenylation. At the major branch point of the mevalonate pathway, SQS utilizes FPP in the first committed step toward cholesterol biosynthesis. In these studies, novel bisphosphonates are identified as potent and specific inhibitors of SQS, including 9-biphenyl-4,8-dimethyl-nona-3,7-dienyl-1,1- bisphosphonic acid, tetrasodium salt (compound 5; Figure 8). Compound 5 reduced cholesterol biosynthesis, did not effect cell viability, and lead to a substantial intracellular accumulation of FPP in HepG2 cells. At high concentrations, lovastatin and zoledronate impair protein prenylation and are cytotoxic, limiting their use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced depletion of FPP levels and protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented both zoledronate-induced impairment of protein farnesylation and geranylgeranylation. Co-treatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly ameliorate the reduction of cell

32 16 viability activity caused by treatment with lovastatin or zoledronate alone. The combination of an SQS inhibitor with HMGCR or FDPS inhibitors provides a rational approach for reducing cholesterol synthesis, while maintaining non-sterol isoprenoid biosynthesis closer to basal levels than treatment with single inhibitors. Introduction Squalene synthase (SQS) is responsible for the first committed step from the isoprenoid biosynthetic pathway toward cholesterol biosynthesis. SQS is a membraneassociated enzyme that localizes to the endoplasmic reticulum (57). SQS catalyzes the reductive dimerization of two units of FPP in a head-to-head orientation in a two step reaction to form squalene (23). In the first step of the reaction, pre-squalene diphosphate is formed, which is then converted to squalene in an NADPH-dependent second step (Figure 5). The structure of SQS is folded as a single domain and is entirely α-helical with two active sites located in a central channel that is lined by two DDXXD motifs containing conserved aspartate residues (58). These residues associate with multiple Mg 2+ ions that stabilize binding to the diphosphate portion of FPP, while the hydrophobic portion of FPP extends into a hydrophobic channel (58). Inhibitors containing bulky hydrophobic groups were characterized in a complex with SQS, and the hydrophobic groups were in proximity of the side chains of Phe 54 and Tyr 73 within an inhibitor-binding cavity (58). Statins inhibit HGMCR and impair cholesterol biosynthesis, which causes an upregulation of the LDL receptor in the liver resulting in clearance of cholesterolcontaining LDL particles from the bloodstream. Statins are widely used because elevated LDL levels and total cholesterol are major risk factors for coronary heart disease (31).

33 17 While the statins are used abundantly and effectively, there are various reasons for developing novel inhibitors of cholesterol biosynthesis. For example, there are sideeffects associated with statin use such as myopathy and hepatotoxicity (59), which are commonly speculated to be due to the depletion of non-sterol components of the mevalonate pathway (60). Furthermore, statin use does not always reduce LDL to desired levels (61), which is particularly important as lower LDL target levels are suggested for some patients (62,63). Inhibition of SQS has drawn much interest as a pharmacological target, and various molecules have been identified as inhibitors (49,50). Lapaquistat (TAK-475, Takeda) progressed to phase III clinical trials, but these trials were discontinued after the U.S. Food and Drug Administration (FDA) recommended suspension of studies with the high dose (100 mg/kg) mono-therapy due to cases of elevated blood levels of liver transaminases, a commonly used measure of hepatotoxicity (50). It is currently unknown whether this was due to inhibition of SQS per se, or if it was specific to the drug. Inhibition of SQS can result in the accumulation of FPP metabolites, such as farnesolderived dicarboxylic acids (64), which could be responsible for the possible hepatotoxicity with the high-dose monotherapy. Farnesol itself can also be pro-apoptotic at high concentrations (65). Other reported results appeared promising with lapaquistat, with cholesterol levels decreased in monotherapy treatment. Moreover, the combination therapy with statins showed additional LDL reduction compared to statins alone (50). Also of interest, T (the active metabolite of lapaquistat) was capable of preventing statin-induced myotoxicity in a human skeletal muscle cell model (66). Similarly, lapaquistat prevented statin-induced myotoxicity in a guinea pig model (67). In addition

34 18 to the expected cholesterol depletion, other SQS inhibitors have shown the potential for added benefits due to decreased triglyceride biosynthesis (68), likely resulting from a farnesol-induced mechanism (69). Nitrogenous bisphosphonates (e.g., zoledronate, alendronate) are another class of clinical drugs targeting the mevalonate pathway. These compounds inhibit FDPS and deplete FPP and other downstream isoprenoids (40). Although these compounds have a high affinity for bone, there are also reports of decreased cholesterol levels with patients treated with nitrogenous bisphosphonates (70). The combination of FDPS inhibitors with SQS inhibitors has not been evaluated. While various SQS inhibitors exist, relatively few are based on a bisphosphonate structure (71), and specificity for SQS relative to the prenylation arm of the mevalonate pathway has not been reported. Herein is described the identification of novel bisphosphonates as potent inhibitors of SQS. A structure activity relationship (SAR) was evaluated in the context of potency and specificity for these novel compounds. Emphasis was placed on the evaluation of a lead compound (5) in combination with lovastatin or zoledronate in HepG2 cells. These combinations of inhibitors are hypothesized to decrease cholesterol biosynthesis while preventing the depletion of non-sterol isoprenoid levels, resulting in less adverse cellular effects compared to inhibition of HMGCR or FDPS alone. Results In vitro inhibition of recombinant SQS. Squalene synthase was purified utilizing an N-terminal GST-tag (Figure 6). In vitro assays were established, and the K m and V max were determined to be 2.6 µm and 160 pmol/min/mg, respectively (Figure 7). A screen

35 19 of in-house compounds was performed to identify in vitro inhibitors of SQS. 9-Biphenyl- 4,8-dimethyl-nona-3,7-dienyl-1,1-bisphosphonic acid, tetrasodium salt (compound 5) was identified as one of the most potent compounds. This became the lead inhibitor and additional compounds were synthesized based upon this structure. A small panel of compounds (1, 2, 3, 4 and 5) was selected for SAR studies (Figure 8). Concentrationresponse curves (Figure 9) were used to determine the concentration required to elicit 50% of maximal inhibitory activity (IC 50 value). Geranyl bisphosphonate (1) had an IC 50 value of 230 nm in this assay. Addition of a phenyl ring at the C-9 position of the geranyl chain (compound 2) enhanced potency to yield an IC 50 of 8.1 nm. The addition of a biphenyl group in an ortho (3), meta (4), or para (5) substituted pattern resulted in IC 50 values of 1.9, 5.2, and 4.2 nm, respectively. Effects of compounds 1-5 on protein prenylation in HepG2 cells. Substrate-like inhibitors targeted against SQS have potential for off-target effects due to inhibition of other FPP-utilizing enzymes. Geranyl bisphosphonate (72) has previously been identified as an inhibitor of GGPPS (73). With this in mind, the compounds active against SQS were evaluated for impairment of protein farnesylation or geranylgeranylation, outputs that could identify possible inhibition of FDPS, GGPPS, or prenyltransferases. Ras is used as an indicator of protein farnesylation, and its impairment is noted by the appearance of an upper unmodified band on the western blot. Rap1a is used as an indicator for protein geranylgeranylation, and its impairment is noted by the appearance of a band on the western blot (the antibody used only detects the unmodified form of Rap1a). Lovastatin (25 µm) was used as a positive control. Lovastatin depletes mevalonate and the downstream products (e.g., FPP and GGPP) and

36 20 thus impairs both farnesylation and geranylgeranylation. HepG2 cells were treated with 50 µm concentration of compounds 1-5. As previously reported (73), mono-geranyl bisphosphonate (1) inhibits protein geranylgeranylation (Figure 10A). Compound 2 also inhibits geranylgeranylation, while compound 3 displays slight inhibition. No detectable inhibition was noted with compounds 4 or 5. Although slightly less potent in enzyme assays than compound 3, compound 5 was utilized as the lead inhibitor due to its specificity. Effect of compound 5 combined with lovastatin and zoledronate-induced impairment of protein prenylation. Treatment of HepG2 cells with 25 µm lovastatin for 24 h results in impairment of both farnesylation of Ras and geranylgeranylation of Rap1a. Lovastatin-induced impairment of Ras farnesylation is preventable by co-treatment with 25 µm exogenous FPP, while lovastatin-induced impairment of Rap1a geranylgeranylation is prevented by co-treatment with 25 µm GGPP (Figure 10B). Cotreatment of 25 µm lovastatin with 25 µm compound 5 prevents lovastatin-induced inhibition of Ras farnesylation, but does not completely restore Rap1a geranylgeranylation. Treatment of HepG2 cells for 24 h with 10 µm zoledronate causes impaired Ras farnesylation and Rap1a geranylgeranylation (Figure 10C), and co-treatment with exogenous FPP or GGPP prevents the impairment of farnesylation or geranylgeranylation, respectively. Co-treatment of 10 µm zoledronate with 2.5 µm compound 5 partially prevents zoledronate-induced impairment of Ras farnesylation and Rap1a geranylgeranylation. Co-treatment of 10 µm zoledronate with 25 µm compound

37 21 5 completely prevents both zoledronate-induced impairment of Ras farnesylation and Rap1a geranylgeranylation. Effect of compound 5 and lovastatin on FPP and GGPP levels. We next determined the FPP and GGPP levels from HepG2 cells in response to treatment with compound 5 and lovastatin, alone or in combination for 24 h. In HepG2 cells treated with 2.5 µm compound 5, FPP levels were increased approximately 4-fold and GGPP levels were increased approximately 2-fold compared to control (Figure 11). At 25 µm, compound 5 further increased FPP levels to 22-fold of control, while GGPP appeared to be already maximally increased. Lovastatin (25 µm) reduced FPP and GGPP levels to 34 and 8 percent of control, respectively. The combination of 25 µm lovastatin with 25 µm compound 5 resulted in increased FPP levels compared to lovastatin treated and control cells; however, GGPP levels remained diminished. These data correlate with the results showing prevention of lovastatin-induced impairment of Ras farnesylation, but not Rap1a geranylgeranylation, by co-treatment with compound 5. Effect of compound 5 with lovastatin or zoledronate on cholesterol biosynthesis. Lovastatin at 50 nm or compound 5 at 50 µm inhibited de novo cholesterol biosynthesis in HepG2 cells by ~60% compared to untreated HepG2 cells (Figure 12). The combination of these concentrations of lovastatin and compound 5 significantly enhanced inhibition of cholesterol synthesis compared to treatments with the individual agents. Zoledronate at 10 µm also reduced cholesterol biosynthesis compared to control; however, the combination of 25 µm compound 5 with 10 µm zoledronate did not significantly enhance cholesterol depletion when compared to compound 5 alone.

38 22 Effect of compound 5 on lovastatin- and zoledronate-induced impairment of cell viability. The MTT assay measures the activity of enzymes that reduce the MTT substrate within metabolically active cells and is commonly used as a measure of cell viability. Treatment of HepG2 cells for 48 h with 25 µm lovastatin significantly reduced MTT activity compared to control (Figure 11A). Co-treatment with 25 µm FPP, GGPP, or compound 5 significantly reduced lovastatin-mediated reduction of MTT activity. Compound 5 (25 µm) had no significant effect on MTT levels as a single treatment. Zoledronate (10 µm) decreased MTT activity to ~50% of control cells, and this was not reduced by FPP co-treatment (Figure 11B). Co-treatment of 10 µm zoledronate with 25 µm GGPP or 25 µm compound 5 significantly ameliorated zoledronate-induced reduction of MTT activity. Discussion This work has identified several novel inhibitors of SQS. A lead inhibitor of SQS (compound 5) was identified that did not impair protein prenylation in HepG2 cells, which suggests specificity for SQS over the prenylation branch of the mevalonate pathway. Other bisphosphonates have been shown to inhibit SQS (71); however, their effects on protein prenylation were not reported. Structure-activity relationship analysis determined that the addition of a phenyl group at the C-9 position (compound 2) of geranyl bisphosphonate greatly increased potency for SQS in vitro relative to monogeranyl bisphosphonate (1). The addition of a second phenyl ring in an ortho (3), meta (4), or para (5) substituted pattern, further enhanced IC 50 potency. This suggests that the hydrophobic portion of the molecule is important for potency. It would be expected that the bisphosphonate portion of compound 5 likely interacts with magnesium atoms bound

39 23 within a DDXXD motif. Other bisphosphonates have been shown to interact in this manner with other DDXXD-containing proteins (74). The hydrophobic portion of the molecule may extend into a hydrophobic flap consisting of residues that has been found to display conformational differences with different inhibitors, including an inhibitor containing a biphenyl group (58). Compounds 1, 2 and the ortho-substituted compound 3 display at least some impairment of protein geranylgeranylation, while no impairment is detectable with the meta (4) or para (5) compounds under the conditions used. Emphasis was placed on characterization of compound 5 in combination with HMGCR inhibition (lovastatin) and FDPS inhibition (zoledronate) in HepG2 cells. Simultaneous inhibition of SQS and HMGCR or FDPS provides potential mechanisms for decreasing cholesterol synthesis with less disruption of non-sterol isoprenoid levels compared to single enzyme inhibition, potentially alleviating toxicity due to excessive isoprenoid accumulation or depletion. Treatment of HepG2 cells with compound 5 results in an inhibition of cholesterol biosynthesis and a substantial accumulation of FPP, which could result in the formation of potentially toxic farnesol (65) or farnesol-derived dicarboxylic acids (64). Bisphosphonates are notorious for poor cellular entry due to their high charge to mass ratio, likely explaining the difference in potency between the enzyme assay with SQS and cellular cholesterol synthesis assay. Future studies could evaluate prodrug approaches that could confer liver-targeting and mask the bisphosphonate negative charge, which might significantly enhance cellular uptake (75-77). With statin treatment, there is depletion of mevalonate and all downstream components of the mevalonate pathway (28). The combination of lovastatin with

40 24 compound 5 is able to further deplete cholesterol synthesis while restoring FPP levels and protein farnesylation, but not GGPP levels or protein geranylgeranylation. Many of the pleiotropic effects of statins are thought to be mediated by GGPP depletion and impairment of protein geranylgeranylation (78). For example, statins can upregulate enos and have antioxidant effects (79), which are prevented by addition of GGPP but not FPP (80)). Due to the low GGPP levels and impairment of protein geranylgeranylation with the combination of compound 5 and lovastatin, some statinmediated pleiotropic effects may be retained. Nonetheless, this combination can partially prevent statin-mediated reduction in cell viability. These results may explain why other SQS inhibitors can prevent statin-induced myopathy in cell and animal models (66,67). While adverse effects of the SQS inhibitor lapaquistat as a monotherapy were discouraging, these results suggest that the dual administration of SQS inhibitors with statins may alleviate potential problems due to treatment with either agent alone. The FPP accumulation due to SQS inhibition and FPP depletion due to HMGCR inhibition may offset each other, resulting in FPP available for other necessary cellular processes, but not in such extreme excess to produce adverse effects. With FDPS inhibition by nitrogenous bisphosphonates, there is decreased product formation and accumulation of the substrates DMAPP and IPP. Notably, nitrogenous bisphosphonate-induced IPP accumulation can facilitate the formation of the possible pro-apoptotic isoprene ATP analog known as ApppI (81). With the combination of FDPS and SQS inhibition, flux through the mevalonate pathway will be first inhibited at FDPS resulting in accumulation of IPP. The flux able to bypass the FDPS inhibition will yield FPP and encounter a second inhibition at SQS, preventing the formation of squalene

41 25 and resulting in some relative accumulation of FPP. This SQS-induced FPP accumulation when combined with FDPS-induced IPP accumulation yields the necessary substrates for GGPP synthesis. This presence of FPP and GPPP could then be utilized in other non-sterol branches of the mevalonate pathway, such as protein farnesylation and geranylgeranylation. Indeed, in HepG2 cells, the combination of compound 5 and zoledronate prevents zoledronate-induced impairment of farnesylation and geranylgeranylation and ameliorates zoledronate-induced reduction in MTT activity. In conclusion, these results suggest that dual inhibition of HMGCR and SQS or FDPS and SQS could yield a means of cholesterol reduction with the potential for minimal off target effects due to decreased depletion of non-sterol isoprenoids compared to HMGCR or FDPS inhibition alone. Treatment using multiple drugs (SQS inhibitors combined with statins being the most feasible approach in the near term) or the development of novel compounds capable of dual inhibition could be feasible. Specifically, design and synthesis of a single agent capable of potent dual FDPS and SQS inhibition is a potentially achievable goal and future studies could evaluate this approach. Experimental Procedures Protein purification. A plasmid containing N-terminal glutathione-s-transferase tagged human squalene synthase (EX-C0605-B03) was obtained from Genecopoeia (Rockville, MD). The plasmid was transformed into BL21 E.coli and expressed using 0.1 mm IPTG overnight at room temperature. Bacteria were lysed using lysozyme, and tagged protein was purified using glutathione-agarose beads (Sigma; St. Louis, MO) according to the manufacturer protocol.

42 26 SQS enzyme assays. Enzyme assays were performed in 20 µl reactions containing 50 mm phosphate buffer at ph 7.4 containing 5 mm MgCl 2, 4 mm CHAPS, 10 mm DTT, 720 ng recombinant enzyme, 0.5 µm 3 H-FPP (American Radiolabeled Chemicals, St. Louis, MO) and 2 mm NADPH. Inhibitors were added prior to substrate addition and pre-incubated for 10 min at 37 C. Substrate was then added and reactions were allowed to proceed for an additional 10 min at 37 C. Reactions were stopped by the 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 scintillation vials containing econo-safe LSC fluid (Research Products International) and radioactivity was quantitated using a liquid scintillation counter. Cell culture. HepG2 cells were obtained from American Type Culture Collection (ATCC) and grown at 37 C with 5% CO 2 in Dulbecco Minimum Essential Media (DMEM; Sigma) containing pen-strep, amphotericin B (Thermo Scientific; Walthman, MA), 2 mm Glutamax (Invitrogen; Carlsbad, CA), 1 mm sodium pyruvate (Sigma), and 10% fetal bovine serum. Western blot analysis. Protein concentrations were determined by the bicinchoninic acid (BCA) assay. Proteins were resolved on 15% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes via electrophoresis. Blocking was performed in 5% non-fat dry milk for 45 minutes, after which primary and secondary antibodies were added sequentially for one hour each at 37 o C. Proteins were visualized using an enhanced chemiluminescence detection kit. 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). Horseradish peroxidase

43 27 (HRP)-conjugated anti-mouse and anti-rabbit IgG were acquired from GE Healthcare (Buckinghamshire, UK). Cholesterol biosynthesis assay. Cells were plated in 12-well plates and grown to near confluency. Compounds were added for one hour followed by the addition of 1 µci of 1-14 C-acetate (Sigma) for 4 h. Cells were harvested using trypsin and lipids were extracted using Bligh and Dyer method (82). Chloroform extracts were dried down, resuspended in a small volume of cholorform 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 quantitated using a liquid scintillation counter. Measurement of FPP and GGPP levels. Both FPP and GGPP levels were determined as reported (83). 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. MTT Assay. 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 incubated for an additional 3 h. MTT stop solution (HCl, Triton X-100, and isopropyl alcohol) was then added, plates were gently agitated at 37 C overnight, and then aliquots were transferred

44 28 to 96-well plates. Absorbance was measured at 540 nm with a reference wavelength at 650 nm. Statistical Analysis. Unpaired two-tailed t-test was used to determine statistical significance which was set to p<0.05.

45 29 Figure 5. SQS reaction mechanism. In the first reaction step, two units of FPP are utilized by SQS to form a pre-squalene diphosphate intermediate. In the second SQS reaction step, NADPH is oxidized to NADP + in the process of reducing PSDP to squalene. An inorganic pyrophosphate is released during each step. This figure is adapted from Pandit et al. (58).

46 30 Figure 6. Coomassie Blue stained SDS-PAGE of purified recombinant GST-SQS. Protein standards are labeled in kda.

47 V (pmol/min/mg) FPP (µm) Figure 7. SQS reaction rate in response to varied FPP concentration. K m = 2.6 µm, V max = 160 pmol/min/mg. Error bars represent standard deviation, n=2.

48 ortho 4 meta 5 para Figure 8. Structures of compounds 1-5.

49 33 Figure 9. In vitro SQS concentration-response curves. Compounds 1-5 were assayed at various concentrations for inhibition of recombinantly purified SQS in vitro. IC 50 values are given in text. Error bars represent standard deviation, n=2.

50 34 Figure 10. Protein prenylation in HepG2 cells. (A) HepG2 cells were treated with 25 µm lovastatin or 50 µm of compounds 1-5 for 24 h. (B) Co-treatment of 25 µm lovastatin and 25 µm FPP, 25 µm GGPP, or 25 µm compound 5 for 24 h in HepG2 cells. (C) Co-treatment of 10 µm zoledronate with 25 µm FPP, 25 µm GGPP, 2.5 or 25 µm compound 5 for 24 h in HepG2 cells. Impairment of Ras farnesylation is detected by the appearance of an upper unmodified band (arrow) when western blotting for Ras, while the inhibition of Rap1a geranylgeranylation is indicated by the appearance of a band when western blotting for Rap1a. α-tubulin is used as a loading control.

51 35

52 36 Figure 11. FPP and GGPP levels in HepG2 cells. FPP and GGPP levels were measured from HepG2 cells after 24 h incubation with the indicated compounds. Control FPP and GGPP levels are 0.38 and 0.56 pmol/mg protein, respectively. Error bars represent standard deviation, n = 2.

53 37 Figure 12. De novo cholesterol biosynthesis in HepG2 cells. HepG2 cells were treated with indicated compounds for one hour followed by addition of 14 C-acetate for four hours and radiolabeled cholesterol was measured as described in methods. Error bars represent standard error, n=3. * = p < 0.05, ns = not significant.

54 38 Figure 13. MTT assay in HepG2 cells. (A) MTT assay of HepG2 cells treated for 48 h with 25 µm lovastatin combined with 25 µm FPP, 25 µm GPPP, or 25 µm compound 5. (B) MTT assay of HepG2 cells treated for 48 h with 10 µm zoledronate combined with 25 µm FPP, 25 µm GPPP, or 25 µm compound 5. a = p < 0.05 compared to control, b = p < 0.05 compared to lovastatin alone treatment, c = p <0.05 compared to zoledronate alone treatment. Error bars represent standard error of the mean, n = 3.

55 39 CHAPTER III: NOVEL BISPHOSPHONATE INHIBITORS OF GGDPS Abstract This chapter is adapted in part from our published work (84). Nitrogenous bisphosphonates have been utilized clinically to reduce bone resorption in diseases such as osteoporosis and metastatic breast and prostate cancers. Interestingly, there is evidence that NBPs have direct antitumor activity as well, thought to be due to GGPP depletion and the subsequent impairment of protein geranylgeranylation. Due to the role of GGPP depletion in statin and bisphosphonate anti-proliferative mechanisms, the direct inhibition of GGPP synthase is an ongoing objective. Various novel bisphosphonates have been synthesized and evaluated for impairment of protein geranylgeranylation within cells, and structure function relationships have been evaluated. Several of these new compounds are potent inhibitors of GGDPS. Introduction The statins target HMGCR and the NBPs target FDPS. Many of the anti-cancer aspects of statins and NBPs are attributed specifically to the depletion of GGPP and subsequent impairment of geranylgeranylated proteins (85-87). Both of these drugs have potential limitations in regards to anti-cancer effects. Statins target the liver and can cause side-effects at higher doses (59), while NBPs predominantly distribute to the bone (88). Inhibition of GGDPS is a more direct method for depletion of GGPP. Many proteins are geranylgeranylated by GGTase I including various small GTPases involved in human cancers. For one example, the Rho family member RhoA is geranylgeranylated. A downstream effecter of RhoA is Rho kinase, and Rho kinase is

56 40 involved in multiple cellular functions including cancer cell migration (89). Statins and GGTIs have been shown to inhibit migration by impairing geranylgeranylation of proteins including Rho proteins (90). Recent work has also shown that inhibiting GGDPS impairs breast cancer cell migration (91). There are greater than 60 identified Rab (ras genes from rat brain) proteins, and these are geranylgeranylated by GGTase II (20). Rab proteins are important in regulation of vesicle transport and biogenesis of organelles (20). Numerous Rab proteins have been implicated in cancer. For example, Rab27B promotes invasive tumor growth in xenograft models (92). Also, constitutive Rab8 is necessary for trafficking of MT1-MMP that then activates MMP2, which facilitates cell invasion (93). GGDPS is the enzyme that catalyzes the formation of GGPP from FPP and IPP. Targeting GGDPS provides a more direct mechanism than statins or NBPs for depletion of GGPP and impairing protein geranylgeranylation. In contrast to specific GGTase I or II inhibitors, both subsets of geranylgeranylated proteins will be impaired. In addition, GGTase I inhibitors also tend to display some promiscuity for FTase and thus may impair protein farnesylation, while inhibition of GGDPS is specific for impairing protein geranylgeranylation but not farnesylation. Initial studies have identified various mono- and dialkylated bisphosphonate inhibitors of GGDPS that impair protein geranylgeranylation (73). Compared to NBPs, bisphosphonates containing large hydrophobic side-chain(s) are hypothesized to have increased cellular uptake, and reduced affinity for bone since lipophilicity is inversely correlated with bone affinity (94). DGBP is a current lead compound as a specific inhibitor of GGDPS (95). DGBP has been found to deplete cells of GGPP and increase

57 41 levels of the substrate FPP (95). DGBP also has been shown to impair protein geranylgeranylation, induce apoptosis in K562 leukemia cells (96), and inhibit MDA- MB-231 breast cancer cell migration (91). There are multiple reasons to include an aromatic moiety within the structure of potential inhibitors of GGDPS. Some of the potent inhibitors of FDPS are NBPs containing an aromatic substructure, including zoledronate. However, it is unknown if introduction of an aromatic moiety within an isoprenoid-containing bisphosphonate would enhance or diminish specificity for GGDPS inhibition. The aromatic compounds described herein have increased steric demand, and it is unknown if inhibition of GGDPS will be preserved. At physiological ph, the high charge to mass ratio in salts of bisphosphonic acids can cause difficulty crossing cellular membranes (77). Aromatic bisphosphonates are more lipophilic than the lead compound DGBP, which may result in enhanced delivery into the cell. Bisphosphonates containing isoprenoid olefins have biological activity, but the alkene can potentially undergo isomerization or transposition in vivo, which would not be an issue if replaced with an aromatic substructure. Lastly, the aromatic ring may lead to more favorable stacking interactions within the active site of GGDPS (e.g., with Y205 or F175) (97). Novel non-aromatic compounds consisting of variations of mono- or digeranyl bisphosphonate are also evaluated for impairment of protein geranylgeranylation. Results Aromatic bisphosphonates. Aromatic compounds (6-12; Figure 14) were screened at 10 µm in vitro and IC 50 values were determined for compounds which had IC 50 values under 10 µm (Table 1). DGBP was used as a positive control because it has

58 42 previously been shown to inhibit GGDPS (95). The dialkyl compounds 7, 9, 11, and 12 all displayed various degrees of GGDPS inhibition while the mono-alkyl compounds 6, 8, and 10, displayed little or no activity at 10 µm. Compounds 7 and 12 were moderately inhibitory with IC 50 values of ~5 and ~10 µm, respectively. Compounds 9 and 11 were more potent inhibitors with IC 50 values extrapolated to be 250 nm and 800 nm, respectively. For comparison, the published IC 50 value of DGBP is 200 nm (95). The same panel of compounds then was screened at 50 µm for 48 h against the K562 human myelogenous leukemia cell line and impairment of protein prenylation was assessed. Western blots were performed and the prenylation status of Ras and Rap1a was determined (Figure 16). Ras is farnesylated by FTase and impairment of Ras farnesylation is detected by the appearance of a more slowly migrating, unmodified band on the gel. In contrast, Rap1a is geranylgeranylated by GGTase I and the antibody used only detects the unmodified form of the protein such that the appearance of a detectable band represents impairment of protein geranylgeranylation. Lovastatin at 10 µm was used as a positive control, as it inhibits HMGCR and depletes mevalonate resulting in an impairment of protein farnesylation and geranylgeranylation. At 50µM, compounds 9 and 11 impair geranylgeranylation of Rap1a, while there is no detectable impairment of Ras farnesylation at this level (Figure 16A). Concentration response assays were performed in K562 cells with compounds 9 and 11 (Figure 16B). Impairment of Rap1a geranylgeranylation is detectable at 12.5 µm with bisphosphonate 9, while both compounds appeared to maximally impair Rap1a geranylgeranylation at 50 µm. Addition of mevalonate prevents lovastatin-induced impairment of protein farnesylation and protein geranylgeranylation (Figure 17). Additionally, FPP co-

59 43 treatment prevents lovastatin-induced impairment of farnesylation, but not geranylgeranylation. GGPP treatment prevents lovastatin-induced impairment of Rap1a geranylgeranylation but not Ras farnesylation. For compounds 9 and 11, no differences are noticed with co-treatment of mevalonate or FPP, while the co-treatment of GGPP prevents the impairment of Rap1a geranylgeranylation. At 50 µm, compound 9 reduces 3 H-thymidine incorporation to approximately 70% of control after 48 or 72 h of treatment (Figure 18). At 100 µm, compound 9 reduces 3 H-thymidine incorporation to approximately 40% of control after 48 h and 20% after 72 h. Compound 11 reduces 3 H-thymidine incorporation when treated at 100 µm to approximately 80% of control after 48 h and 50% after 72 h. Non-aromatic bisphosphonates. Compounds 1 and were screened at 100 µm for 48 h in K562 cells, and assessed for impairment of Rap1a geranylgeranylation (Table 2). Compounds 1, 14, 16, and 19 impaired Rap1a prenylation at 100 µm, and were selected for further analysis. The effects of compound 17 on Rap1a prenylation at 100 µm were modest in comparison to the other identified compounds, and this compound was not analyzed further. Compound 1 was previously shown to impair protein geranylgeranylation (72). Compound 14 was more potent than compound 1, as impairment of Rap1a at 25 µm was more prominent (Figure 19A). Compound 19 impaired Rap1a geranylgeranylation in a concentration-dependent manner, but with less potency than DGBP (Figure 19B). Compound 16 was found to potently impair Rap1a geranylgeranylation, and co-treatment with GGPP was able to prevent this impairment (Figure 20A). At lower concentrations (Figure 20B) compound 16 impaired Rap1a geranylgeranylation detectibly at 2.5 µm and more prominently at 5 µm, while DGBP-

60 44 induced impairment of Rap1a geranylgeranylation is not detectable below 5 µm. The effects of compound 16 were further compared to DGBP by measurement of FPP and GGPP levels from K562 cells (Figure 21). Compound 16, in a concentration-dependent manner increases FPP levels and decreases GGPP levels. At 2.5 µm, compound 16 results in similar FPP and GGPP levels as does 5 µm DGBP. Discussion The structure of GGDPS has been co-crystallized with DGBP, which bound to an inhibitor binding site (98) in a V-shaped conformation occupying portions of both the GGPP and FPP binding sites (97). Based on the di-substituted structure of inhibitors 9, 11, and 16, it would be anticipated that these molecules bind GGDPS in a similar conformation. The novel aromatic bisphosphonates (6-12) were evaluated in enzyme and various whole cell assays. Numerous mono- and dialkyl bisphosphonates have been identified as inhibitors of GGDPS, and many of these compounds have been shown to impair protein geranylgeranylation in cell lines (72,73). It was hypothesized that these synthesized novel aromatic bisphosphonates would also inhibit GGDPS. Indeed, aromatic bisphosphonate 9 was found to be a relatively potent inhibitor. The in vitro data correlated with the cellular data, as the two potent aromatic bisphosphonate in vitro GGDPS inhibitors (9 and 11) were the only compounds to show significantly diminished protein geranylgeranylation in K562 cells. To define further the specificity of these compounds for GGDPS in cells, experiments were performed with simultaneous treatment of isoprenoid biosynthetic pathway intermediates and the lead aromatic compounds (9 and 11). The GGPP co-treatment restored protein geranylgeranylation,

61 45 which supports the conclusion that only GGDPS is inhibited within cells and not the protein prenyltransferases. Cell viability in response to compounds 9 and 11 was determined by measurement of 3 H-thymidine incorporation into newly synthesized DNA (Figure 18). Bisphosphonates 9 and 11 inhibited DNA synthesis in a time and concentration dependent manner with the meta isomer 9 being more potent. This data suggests that the degree of geranylgeranylation impairment correlates with impairment of cell proliferation. Non-aromatic compounds were also assessed for impairment of prenylation in K562 cells. Of the compounds containing a geranyl chain with the second R group containing a three carbon linker followed by a primary amine (aledronate like, 17), a primary alcohol (18), or an azide (19), impairment of Rap1a geranylgeranylation is only detected when cells were treated with the azide containing compound 19. Data with compounds 17 and 12 suggests that replacing the α-hydroxy group of NBPs with a geranyl chain is not an effective strategy for inhibiting GGDPS. Compared to monogeranyl bisphosphonate (compound 1), the addition of an α-amine linking a single geranyl chain to the bisphosphonate (compound 14) appears to increase potency for impairment of protein geranylgeranylation in intact cells. However, the addition of a primary amine at the 9-position (compound 15) diminishes activity. Insertion of an oxygen at the 1 position before a single geranyl chain (compound 13) does not impair protein geranylgeranylation in cells at 100 µm, while monogeranyl bisphosphonate (1) does. Compound 16, which contains the side chain of 13 in addition to a geranyl chain, appears more potent than the previous lead compound DGBP. Although there has been

62 46 some pursuit of multi-enzyme inhibitors (i.e., FDPS and GGDPS) within the mevalonate pathway as potential anti-cancer agents, (74) specificity may be ideal for molecular intervention. Compounds with the ability to inhibit a single enzyme are useful tools to study the interrelationships of this complex system, and may have use as antiproliferative agents (99). The bisphosphonates reported herein, particularly compound 16, demonstrate selective inhibition of GGDPS, and thus expand the list of tools available for manipulation of isoprenoid biosynthesis. Compound 16 is the most potent impairer of protein geranylgeranylation identified to date. Novel analogs can now be built based on this structure to further enhance potency and to study the cellular effects of GGDPS inhibition. Experimental Procedures Cell culture. The K562 leukemia cell line was obtained from American Type Culture Collection (Manassas, VA) and was grown in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum incubated in 5% CO2 at 37 C. GGDPS in vitro assay. The GGDPS in vitro assay was performed as described previously (95). Briefly, a plasmid containing N-terminal GST-tagged human recombinant GGDPS was expressed in BL21 bacteria by induction with IPTG. Proteins were purified by batch centrifugation with glutathione agarose. The GGDPS reaction mixtures contained 20 µm FPP and 40 µm 14 C-IPP in 20 µl of 50 mm imidazole ph 7.5 buffer containing 500 µm MgCl 2 and 500 µm ZnCl 2. Compounds were pre-incubated with enzyme for 10 minutes at 37 C followed by addition of both substrates. Reactions proceeded for one hour at 37 C, and then the products were extracted using 1 ml of water-saturated butanol and the extracts were washed twice with 300 µl butanol-

63 47 saturated water. Radioactivity in the butanol extracts was quantified using a liquid scintillation counter. Western blot analysis. K562 cells were diluted to a 5x10 5 cells/ml in 5 ml in 6- well plates. Cells were incubated in the presence or absence of indicated compounds for 48 h. Cells were then centrifuged and lysed by passing cells several times through a 27G needle in 66 mm Tris containing 2% SDS. Lysates were cleared using centrifugation and protein concentration was determined using the bicinchoninic acid (BCA) assay. The remaining western blotting steps were performed as described in Chapter II Experimental Procedures. The Rab6 (sc-310) antibody was acquired from Santa Cruz biotechnology, Inc. (Santa Cruz, CA). HRP-conjugated anti-goat was obtained from Santa Cruz. Mevalonate, FPP, or GGPP was added at concentrations indicated simultaneously with isoprenoid pathway inhibitors. DNA synthesis assay. K562 cells were incubated in a 200 µl volume in 96-well plates and treated with compounds as described previously (73). Experiments with 48 h treatments were seeded with 2x10 5 cells/ml while 72 h experiments used 1x10 5 cells/ml. Four hours prior to the end of the experiments, 20 µl of [ 3 H]-thymidine (3.75 Ci/mmol in media) was added to each well. At the end time point (48 or 72 h), cells were filtered through glass microfiber paper using a cell harvester. Cellular DNA containing tritiated thymidine was quantified using a liquid scintillation counter. Measurement of FPP and GGPP levels. Measurement of FPP and GGPP levels was performed as described in Chapter II Experimental Procedures.

64 Figure 14. Structures of aromatic bisphosphonates.

65 Figure 15. Structures of compounds

66 Table 1. GGDPS in vitro IC 50 values (µm) generated from concentration-response curves for compounds 6-12 and Compound IC 50 (µm) DGBP 0.2 (95) 6 >10 7 ~5 8 > > ~

67 51 A B Figure 16. Compounds 6-12 tested for impairment of protein prenylation in K562 cells. (A) Compound 6-12 screen. (B) Concentration-response of compounds 9 and 11. K562 cells were treated with lovastatin (Lov) and compounds as indicated for 48 h. Western blots were performed as described in Experimental Procedures for Ras, Rap1a, and α- tubulin.

68 Figure 17. Impairment of protein geranylgeranylation by compounds 9 and 11 was prevented by exogenous GGPP. K562 cells were treated with lovastatin (Lov, 10 µm) and novel bisphosphonates (9 and 11 at 50 µm) in the presence or absence of exogenous mevalonate (Mev, 500 µm), FPP (F, 20 µm), and GGPP (GG, 20 µm) for 48 h. Western blots were performed as described in Experimental Procedures for Ras, Rap1a, and αtubulin.

69 53 Compound 9 [µm] Compound 11 [µm] Figure 18. Effect of compounds 9 and 11 on DNA synthesis. Cell proliferation as a percentage of untreated control cells at 48 and 72 h was evaluated by [ 3 H]-thymidine incorporation (mean ± S.E., n = 4). (A) Compound 9. (B) Compound 11. Error bars represent standard error, n=4.

70 54 Table 2. Qualitative assessment for impairment of Rap1a geranylgeranylation by compounds 1 and K562 cells were treated with compounds at 100 µm for 48 h and western blotting was performed for Rap1a. Impairment of Rap1a Compound geranylgeranylation at 100 µm 1 Yes 13 No 14 Yes 15 No 16 Yes 17 Yes 18 No 19 Yes

71 55 A B Figure 19. Concentration-response of compounds 1, 14, and 19. K562 cells treated at indicated concentrations for 48 h with (A) compound 1, 14, and DGBP or (B) compound 19 or DGBP. Western blots were performed as described in Experimental Procedures for Rap1a and α-tubulin.

72 56 A B Figure 20. Concentration-response of DGBP and compound 16. K562 cells were treated with compounds as indicated for 48 h with (A) GGPP co-treatment and (B) at lower concentrations. Western blots were performed as described in Experimental Procedures for Rap1a, and α-tubulin.

73 57 Figure 21. Isoprenoid diphosphate levels from K562 cells treated for 48 h with compound 16 and DGBP at indicated concentrations. (A) FPP levels (pmol/mg protein) (B) GGPP levels (pmol/mg protein). Error bars represent standard deviation, n=2.

74 58 A 7 FPP (pmol / mg protein) Control 5 µm DGBP 2.5 µm 16 5 µm µm µm 16 B 2 GGPP (pmol / mg protein) Control 5 µm DGBP 2.5 µm 16 5 µm µm µm 16

75 59 CHAPTER IV: BISPHOSPHONATE INHIBITORS OF FDPS AND GGDPS INDUCE AUTOPHAGY Abstract This chapter has been adapted from our published work (100). Multiple studies have implicated the depletion of isoprenoid biosynthetic pathway intermediates with induction of autophagy. However, the precise mechanism by which inhibitors of isoprenoid biosynthesis induce autophagy has not been well established. It is hypothesized herein that inhibition of FDPS and GGDPS by bisphosphonates would induce autophagy by depleting cellular GGPP and impairing protein geranylgeranylation. An inhibitor of FDPS (zoledronate) and an inhibitor of GGDPS (digeranyl bisphosphonate, DGBP) induce autophagy in PC3 prostate cancer and MDA-MB-231 breast cancer cells as measured by accumulation of the autophagic marker LC3-II. Treatment of PC3 cells with lysosomal protease inhibitors (E-64d and pepstatin A) in combination with zoledronate or DGBP further enhances LC3-II formation, indicating that these compounds induce autophagic flux. Importantly, the addition of exogenous GGPP prevented the accumulation of LC3-II and impairment of Rab6 (a GGTase II substrate) geranylgeranylation by isoprenoid pathway inhibitors (lovastatin, zoledronate, and DGBP). However, exogenous GGPP did not restore isoprenoid pathway inhibitorinduced impairment of Rap1a (a GGTase I substrate) geranylgeranylation under identical experimental conditions. In addition, specific inhibitors of farnesyl transferase and geranylgeranyl transferase I did not induce autophagy in this system. Furthermore, the addition of bafilomycin A1 (an inhibitor of autophagy processing) enhanced the anti-

76 60 proliferative effects of DGBP. These results are the first to demonstrate that bisphosphonates induce autophagy. This study suggests that induction of autophagy in PC3 cells with these agents is likely dependent upon impairment of geranylgeranylation of GGTase II substrates. Introduction Macroautophagy (hereafter referred to as autophagy) is a cellular process which degrades damaged cytoplasmic organelles as well as long-lived, misfolded, or aggregated proteins (101). During autophagy, a target substrate is first encapsulated in a double membrane vesicle known as an autophagosome. Autophagosomes can then fuse with lysosomes to form autolysosomes, where the contents are degraded (Figure 22). This process ultimately allows for the recycling of amino acids and other degraded products and can be upregulated in response to cellular stresses such as starvation. Two major classes of clinical inhibitors target isoprenoid biosynthetic enzymes, statins and nitrogenous bisphosphonates. While statins and nitrogenous bisphosphonates are used for distinct clinical disorders, they have common effects within cells, including depletion of pathway intermediates FPP and GGPP. Inhibitors of the isoprenoid biosynthetic pathway have been linked to autophagy. Studies have shown that statins are capable of inducing autophagy in A204 human rhabdomyosarcoma cells (102). More recently, statins have been shown to induce autophagy in PC3 prostate cancer cells, and the induction of autophagy was prevented by the addition of the geranylgeraniol (GGOH), the alcohol form of GGPP (103). It remains uncertain whether this prevention is due to restoration of isoprenoid levels or protein prenylation. In addition, a novel GGTase I/II inhibitor when combined with a statin induced autophagy in the STS-26T

77 61 malignant peripheral nerve sheath tumor cell line (104), suggesting that the impairment of prenylation can induce autophagy. However, this drug combination does not allow for the distinction of whether the impairment of GGTase I or GGTase II substrate geranylgeranylation was responsible for autophagic induction. Further complicating the interrelationship of prenylation and autophagy, farnesyl transferase inhibitors have been shown to induce autophagy in Panc-1 pancreatic cancer and U2OS osteosarcoma cells (105). In addition, an inhibitor of isoprenylcysteine carboxyl methyltransferase, an enzyme required in the later steps of prenylation processing, induced autophagy in HepG2 and PC3 cells (106,107). A yeast deletion collection was treated with nitrogenous bisphosphonates, which identified ATG4, ATG11, ATG14 and ATG16 (all autophagy related genes) hemizygous strains as having increased sensitivity to nitrogenous bisphosphonates (108). Many types of cancer metastasize to bone, and more than 80% of metastatic bone disease occurs with breast and prostate cancer (109). By reducing bone resorption, NBPs decrease skeletal related events associated with bone metastasis, and NBPs may also have direct anti-cancer activity as well. Understanding the effects of NBPs on cancer cells is important and may reveal pharmacological adaptations that could enhance their anticancer activity. Many effects of NBPs are attributed specifically to the resulting GGPP depletion and the successive impairment of protein geranylgeranylation (85-87). DGBP has been shown it to be a specific inhibitor GGDPS and to impair protein geranylgeranylation via GGPP depletion (72,95). Furthermore, DGBP facilitated GGPP depletion results in the inhibition of cancer cell migration (91) and induction of apoptosis (96). It is

78 62 hypothesized that depletion of GGPP by bisphosphonate inhibitors of FDPS (i.e. zoledronate) or GGDPS (i.e. DGBP) results in the induction of autophagy. Results Isoprenoid biosynthetic pathway inhibitors interfere with protein prenylation in a concentration-dependent manner in PC3 cells. To determine the potency that isoprenoid pathway inhibitors interfere with protein prenylation, PC3 cells were treated with various concentrations of lovastatin, zoledronate, or DGBP for 24 h (Figure 23A). The Ras antibody utilized in these experiments recognizes the modified (farnesylated) and the unmodified (non-farnesylated) form of the protein. The unmodified form of Ras is the slower migrating, upper-band, on the western blot Ras panel. In contrast, the antibody used to detect Rap1a only detects the unmodified form of this protein, which is normally geranylgeranylated by GGTase I. Therefore, the appearance of a band on the western blot Rap1a panel indicates impairment of its geranylgeranylation. Detection of alpha tubulin (αtub), a house keeping gene, was used as a loading control for all western blotting experiments. Lovastatin and zoledronate interfere with both farnesylation and geranylgeranylation of proteins as indicated by the detection of the unmodified forms of Ras and Rap1a. DGBP interferes with protein geranylgeranylation without disturbing protein farnesylation. Maximal impairment of Rap1a geranylgeranylation occurs at 0.5 to 1 µm lovastatin, 50 to 100 µm zoledronate, and 10 to 25 µm DGBP. These concentrations of lovastatin and zoledronate did not maximally impair protein farnesylation. The concentration that maximally impairs protein geranylgeranylation for each inhibitor (1 µm lovastatin, 100 µm zoledronate, and 25 µm DGBP) was utilized for subsequent experiments.

79 63 Isoprenoid biosynthetic pathway inhibitors reduce MTT activity and DNA synthesis concentration-dependently in PC3 cells. In order to assess cell viability in response to the isoprenoid biosynthetic pathway inhibitors, MTT assays were performed at 48 h (Figure 23B). Additionally, cell proliferation was assessed by 3 H-thymidine incorporation assay (Figure 23C). A concentration-dependent inhibition of DNA synthesis ( 3 H-thymidine incorporation assay) and reduction of MTT activity are observed with each inhibitor tested. Lovastatin is the most potent while zoledronate is least potent at decreasing DNA synthesis and MTT activity. Bisphosphonates induce LC3-II accumulation in PC3 cells. PC3 cells were treated with lovastatin, zoledronate, and DGBP for 24 and 48 h to determine if these compounds induce LC3-II accumulation (Figure 24). Statins have been reported to induce autophagy in PC3 cells (103) and thus lovastatin is used as a positive control for LC3-II accumulation. The antibody utilized to assess LC3-II protein levels preferentially detects the LC3-II form of LC3. Measurement of LC3-II protein accumulation is an established method for the detection of autophagy (101,110). No LC3-II accumulation is detected at 24 h with the use of the isoprenoid pathway inhibitors. In contrast, LC3-II accumulation is detected at 48 h with the use of the lovastatin and bisphosphonates (zoledronate and DGBP). LC3-II accumulation is concentration-responsive with respect to both DGBP and zoledronate. Under the same condition, protein prenylation of Ras and Rap1a is assessed as described previously (Figure 24A). In addition, we utilized Rab6 to evaluate the status of proteins geranylgeranylated by GGTase II (Figure 24B). In order to assess Rab6 s prenylation status, cells were lysed in Triton X-114, which can undergo a phase separation above 20 C allowing for separation of amphiphilic (detergent

80 64 phase) from hydrophilic (aqueous phase) proteins (111). The detergent-rich fraction retains prenylated proteins, while unprenylated proteins are found in the aqueous phase (112); thus impairment of Rab6 geranylgeranylation in noted by the appearance of a band in the aqueous fraction of the western blots for Rab6. At 24 and 48 h, the impairment of Rab6 processing is noted with all isoprenoid pathway inhibitors. Bisphosphonate-induced LC3-II accumulation is dependent on GGPP depletion in PC3 cells. Isoprenoid pathway inhibitors were co-administered with exogenous isoprenoid pathway intermediates for 48 h in order to determine if LC3-II accumulation is dependent on the depletion of specific molecules within the isoprenoid pathway (Figure 24 C and D). The addition of mevalonate and GGPP, but not FPP completely prevents the effects of lovastatin on the induction of autophagy as measured by LC3-II levels. FPP addition does not prevent the effects of lovastatin due to the lack of isopentenyl diphosphate (IPP) to generate GGPP from FPP (Figure 1). GGPP, but not FPP, also completely prevents zoledronate-induced LC3-II accumulation. In addition, GGPP prevents DGBP-induced LC3-II accumulation. Notably, isoprenoid pathway inhibitor-induced impairment of Rap1a (a GGTase I substrate) geranylgeranylation was not prevented by GGPP addition under the conditions tested; while isoprenoid pathway inhibitor-induced impairment of Rab6 (a GGTase II substrate) geranylgeranylation was completely prevented by GGPP addition (Figure 24D). Bisphosphonates induce autophagic flux in PC3 cells. The accumulation of LC3- II can be caused by induction of autophagy as well as by disruption of autophagosomal processing (110). In order to confirm that bisphosphonate-induced GGPP depletion genuinely causes an induction of autophagy, experiments were performed to evaluate

81 65 autophagic flux (Figure 25). The lysosomal protease inhibitors (pepstatin A and E-64d) were employed to prevent the LC3-II degradation, allowing for analysis of autophagic flux. As shown, lovastatin, zoledronate, and DGBP induce LC3-II accumulation. Addition of protease inhibitors with each of the isoprenoid pathway inhibitors further enhances LC3-II accumulation suggesting that zoledronate and DGBP induce autophagy as opposed to disrupting autophagosomal processing. By blocking basal autophagosomal degradation, lysosomal inhibitors also increase LC3-II accumulation compared to control. Bisphosphonates also induce LC3-II accumulation in MDA-MB-231 but not in MDA-MB- 468 and HepG2 cells. The breast cancer cell lines MDA-MB-231 and MDA-MB-468 and the hepatocellular carcinoma HepG2 cell line were evaluated to determine if autophagic effects in PC3 cells were cell line-specific (Figure 26). Similar to PC3 cells, LC3-II accumulation is induced by each of the isoprenoid biosynthetic pathway inhibitors in MDA-MB-231 cells. In contrast, none of the inhibitors used result in detectable LC3-II formation in MDA-MB-468 or HepG2 cells under the conditions tested. Inhibition of either FTase or GGTase I does not induce LC3-II accumulation in PC3 cells. FTase and GGTase I inhibitors were utilized to determine whether direct impairment of protein farnesylation or protein geranylgeranylation could induce LC3-II accumulation (Figure 27). As in previous experiments, lovastatin serves as a positive control for LC3-II accumulation. After 48 h of treatment, GGTI-2133 impairs geranylgeranylation of Rap1a (GGTase I substrate), but not farnesylation of Ras (FTase substrate). In contrast, FTI-277 impairs Ras farnesylation, but not Rap1a geranylgeranylation. At longer exposure times, 10 µm FTI-277 does result in some

82 66 detectable impairment of Rap1a geranylgeranylation (data not shown), suggesting some promiscuous activity of this compound for GGTase I. Treatment of cells with either GGTI-2133 or FTI-277 does not result in LC3-II accumulation, despite the effective impairment of prenylation normally facilitated by their respective target enzymes. Inhibition of autophagy enhances DGBP-induced reduction in MTT activity and DNA synthesis in PC3 cells. MTT assays and 3 H-thymidine incorporation assays were performed with zoledronate and DGBP combined with bafilomycin A1 at 48 h (Figure 28). Bafilomycin A1 is an inhibitor of autophagosome-lysosome fusion. Under the conditions tested, the combination of zoledronate and bafilomycin A1 did not significantly reduce MTT activity (Figure 28A) or DNA synthesis (Figure 28B) when compared to the single agents. In contrast, DGBP with bafilomycin A1 significantly decreased MTT activity (Figure 28A) and DNA synthesis (Figure 28B) when compared to each individual agent. Discussion It is well established that the statins and nitrogenous bisphosphonates deplete intermediates of the isoprenoid pathway (99). Many of the cellular effects of these agents have been attributed specifically to the depletion of GGPP (113,114). Other studies have further evaluated cellular consequences of specific GGPP depletion by DGBP (91,96). Recent work has suggested that statins can induce autophagy (103). In this work, the induction of autophagy in PC3 prostate cancer cells was assessed in response to GGPP depletion by zoledronate or DGBP. We have shown for the first time that treatment of cells with a NBP (zoledronate) or DGBP results in the induction of autophagy as measured by LC3-II accumulation.

83 67 Exogenous GGGP completely prevents the induction of LC3-II formation induced by bisphosphonates, suggesting that depletion of GGPP is the primary mechanism by which zoledronate and DGBP induce autophagy. Furthermore, GGPP did not prevent isoprenoid pathway inhibitor impairment of Rap1a (a GGTase I substrate) geranylgeranylation, but did prevent impairment of Rab6 (a GGTase II substrate) geranylgeranylation. This suggests that impairment of GGTase II substrates may be responsible for LC3-II accumulation in response to GGPP depletion. LC3-II accumulation can result from induction of autophagy or impaired autophagic processing (110). Lysosomal inhibitors were utilized to establish whether LC3-II accumulation was a result of autophagic induction or decreased autophagic flux by bisphosphonate drugs. Similar to the previously reported results with statins (103), the results herein suggest that accumulation of LC3-II was caused by induction of autophagy since the lysosomal inhibitors caused additional accumulation of LC3-II protein levels. To determine if autophagic induction induced by bisphosphonates was cell line specific to the PC3 prostate cancer cell line, we evaluated three additional cancer cell lines. As with PC3 cells, lovastatin, zoledronate, and DGBP can induce autophagy in MDA-MB-231 breast cancer cells as measured by accumulation of LC3-II. However, in HepG2 cells and MDA-MB-468 cells, LC3-II accumulation is not observed in the presence of lovastatin, zoledronate and DGBP. Other groups have published that statins do not induce autophagy in HepG2 cells (102). These results do not appear to be due to a lack of inducible autophagy, as there are published reports of both HepG2 and MDA- MB-468 being capable of autophagic induction (107,115).

84 68 Direct impairment of protein geranylgeranylation was evaluated for autophagic induction in PC3 cells. GGTI-2133 (GGTase I inhibitor) is not able to induce LC3-II accumulation despite effective impairment of geranylgeranylation. This suggests that while bisphosphonate-induced autophagy is due to the depletion of GGPP, it is not due to impairment of geranylgeranylation of proteins by GGTase I. These results suggest that GGPP depletion-induced autophagy may be the result of impairment of geranylgeranylation of GGTase II substrates. The data with Rab6 and GGPP correlates with this hypothesis. However, it cannot be ruled out that disruption of other processes dependent upon GGPP is responsible. Other studies have shown a novel GGTI when combined with a statin induced autophagy in STS-26T malignant peripheral nerve sheath tumor cells (104). These results may be a consequence of this novel GGTI inhibiting both GGTase I and II. Another study also using the STS-26T human malignant peripheral nerve sheath tumor cell line found that the combination of an FTI and lovastatin (which also resulted in inhibition of Rab geranylgeranylation) caused the formation of LC-II but did not increase autophagic flux, which the authors suggest resulted in an abortive autophagic program and non-apoptotic cell death (116). No induction of apoptosis was detected as measured by PARP cleavage in PC3 cells (data not shown); however, we did not directly measure cell death to determine if nonapoptotic cell death occurred. Specific impairment of geranylgeranylation of GGTase II substrates may be a mechanism by which GGPP depletion causes accumulation of LC3- II. However, the lack of commercially available reagents does not allow for direct examination of this hypothesis at this time. Previous studies have shown that farnesyl transferase inhibitors can induce autophagy in U2OS osteosarcoma cells (105). Our

85 69 results did not show accumulation of LC3-II due to FTI-277 treatment in PC3 cells. Pan et al. speculate that the inhibition of Rheb farnesylation by farnesyl transferase inhibitors is responsible for autophagy induction (117). The difference between our and their data is likely attributable to cell line differences, as our data show results dependent upon cell line usage. Furthermore, it is possible that PC3 cells may be dysfunctional in the RhebmTOR arm of the autophagic pathway because in addition to a lack of FTI-induced autophagy, we also did not detect LC3-II accumulation upon treatment with rapamycin (data not shown). Rapamycin is an inhibitor of mtor that can induce autophagy. NBPs are currently used for treatment of metastatic bone cancers (118). The inhibition of autophagy is under intense evaluation with respect to anti-cancer applications (119). Therefore, zoledronate and DGBP were combined with bafilomycin A1, an inhibitor of autophagic function, to assess whether inhibition of autophagy would enhance the anti-proliferative effects of bisphosphonates. The addition of bafilomycin A1 with DGBP significantly decreased MTT activity and inhibited DNA synthesis greater than single agents alone. At the concentrations tested, bafilomycin A1 did not significantly decrease MTT activity or DNA synthesis when combined with zoledronate. While the reason for this distinction is unclear, it is possible that DGBP specific effects, such as FPP accumulation, contribute to this difference. This suggests that the combination of inhibitors of autophagy with GGDPS inhibitors should be further explored as a possible therapeutic strategy. Experimental Procedures Cell culture. PC3, MDA-MB-231, MDA-MB-468, and HepG2 cells were obtained from American Type Culture Collection (Manassas, VA). Cells were

86 70 maintained in Ham s F-12 (PC3), MEM (MDA-MB-231 and HepG2), and Leibovitz s L- 15 (MDA-MB-468) medium supplemented with 10 % fetal bovine serum at 5% CO 2 at 37 o C. Materials. Lovastatin, mevalonate, FPP, GGPP, pepstatin A, E-64d, bafilomycin A1, and GGTI-2133 were purchased from Sigma (St. Louis, MO). Zoledronate was obtained from Novartis (East Hanover, NJ). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) and FTI-277 were purchased from Calbiochem (San Diego, CA). Anti pan-ras was obtained from InterBiotechnology (Tokyo, Japan). Rap1a and αtub antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). LC3-II antibody was obtained from Abgent (San Diego, CA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit were from GE Healthcare, while antigoat was from Santa Cruz Biotechnology, Inc. The enhanced chemiluminescence (ECL) detection kit was obtained from GE Healthcare (Buckinghamshire, UK). Preparation of Cell Lysates. Cells were plated in T25 flasks and allowed to reach 50% confluence. Old media was then replaced with fresh media and relevant compounds added. All compounds were added simultaneously in experiments that required multiple agents in the same T25 flask. At the end of each experiment (24 or 48 h), media was removed and cells were washed twice in phosphate-buffered saline. Cells were collected by the trypsin method. Cell lysis. Cells were lysed in radioimmunoprecipitation buffer (150 mm NaCl, 50 mm Tris ph 7.4, 1 % sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma, 1X), sodium vanadate (1 mm), sodium fluoride (25 mm), and phenylmethylsulphonyl fluoride (1 mm). Lysates

87 71 were transferred to a 1.5 ml tube, vortexed several times over 30 minutes, and passed through a 27-gauge needle. Lysates were then centrifuged and supernatant transferred to a fresh 1.5 ml tube. All steps were performed at 4 o C. Triton X-114 separation. Based on the method of Bordier (111). For separation of prenylated and unprenylated Rab6, cells were lysed in ice cold TX114 lysis buffer (20 mm Tris ph 7.5, 150 mm NaCl, 1% Triton X-114). Cell lysate was passed through a 27 gauge needle and cleared by centrifugation at 12,000 x g for 15 minutes at 4 C, after which the supernatant was transferred to a new tube and incubated at 37 C for 10 minutes and then spun down at room temperature at 12,000 x g for 2 minutes. The aqueous (upper) phase was transferred to a new tube, and the lower detergent phase was diluted into buffer without Triton X-114 prior to electrophoresis. Western Blot Analysis. Protein concentrations were determined by the BCA assay and proteins were resolved on 12 or 15% gels western blotting was performed similar to as described in Chapter II Experimental Procedures. MTT Assay. 8 x 10 4 cells per well were allowed to adhere in 24-well plates overnight, treated the following day with the indicated compounds, and then MTT assays were performed as described in Chapter II Experimental Procedures. DNA Synthesis Assay. Cells (2,500 in 200 µl media) were plated in 96-well plates and allowed to adhere overnight. Cells were then treated with compounds as indicated in the figure legends, and DNA synthesis assays were performed as described in Chapter III Experimental Procedures. Statistical Analysis. The unpaired two-tailed Student s t-test was used to calculate statistical significance. P<0.01 was set as the level of significance.

88 Figure 22. Schematic of autophagy. 72

89 Figure 23. Isoprenoid pathway inhibitors interfere with protein prenylation, decrease MTT activity, and decrease DNA synthesis in PC3 cells. (A) Isoprenoid pathway 73 inhibitors interfere with protein prenylation. Cells were treated with several concentrations of isoprenoid pathway inhibitors (lovastatin, zoledronate, and DGBP) for 24 h. Cell lysis was followed by western blotting to assess protein prenylation. (B) Isoprenoid pathway inhibitors decrease MTT activity. (C) Isoprenoid pathway inhibitors decrease DNA synthesis. MTT and DNA synthesis assays were performed with indicated concentrations of lovastatin, zoledronate, and DGBP for 48 h. Error bars represent standard error, n = 3.

90 74

91 75 Figure 24. Isoprenoid pathway inhibitor-induced LC3-II accumulation and impairment of GGTase-II protein geranylgeranylation, but not impairment of GGTase I geranylgeranylation is prevented by GGPP addition in PC3 cells. (A) Isoprenoid pathway inhibitors (lovastatin, zoledronate, and DGBP) impair protein geranylgeranylation by GGTase I and induce LC3-II accumulation. (B) Isoprenoid pathway inhibitors impair protein geranylgeranylation by GGTase II. Cells were treated with isoprenoid pathway inhibitors at indicated concentrations for 24 and 48 h (A and B). Cell lysis was followed by western blotting to assess protein levels. (C) GGPP addition prevents the accumulation of LC3-II, but does not prevent impairment of Rap1a geranylgeranylation by isoprenoid pathway inhibitors. (D) GGPP prevents the impairment of Rab6 prenylation by isoprenoid pathway inhibitors. Cells were treated with isoprenoid pathway inhibitors (1 µm lovastatin, 100 µm zoledronate, and 25 µm DGBP) in the absence or presence of exogenous isoprenoid pathway intermediates (500 µm mevalonate, 20 µm FPP, and 20 µm GGPP) for 48 h (C and D). Protein fractionation was followed by western blotting to assess protein levels. A=aqueous fraction (unprenylated), D=detergent fraction (prenylated).

92 76 A C

93 77 Figure 25. Bisphosphonates induce autophagic flux in PC3 cells. Cells were treated with isoprenoid pathway inhibitors: 1 µm lovastatin (Lov), 100 µm zoledronate (Zol), and 25 µm DGBP in the absence or presence of lysosomal protease inhibitors (10 µg/ml Pepstatin A and 10 µg/ml E64-d) for 48 h. Cell lysis was followed by western blotting to assess LC3-II protein levels.

94 78 Figure 26. Bisphosphonates induce LC3-II accumulation in MDA-MB-231 but not in MDA-MB-468 and HepG2 cells. Cells were treated with isoprenoid pathway inhibitors: lovastatin (Lov), zoledronate (Zol), and DGBP as indicated for 48 h. Cell lysis was followed by western blotting to assess protein levels.

95 79 Figure 27. Inhibition of either FTase or GGTase I does not induce LC3-II accumulation in PC3 cells. Cells were treated with isoprenoid pathway inhibitors: lovastatin (Lov), GGTI-2133, and FTI-277 at indicated concentrations for 48 h. Cell lysis was followed by western blotting to assess protein levels.

96 80 Figure 28. Inhibition of autophagy enhances DGBP-induced reduction in MTT activity and DNA synthesis in PC3 cells. (A) MTT assay. (B) DNA synthesis assay. PC3 cells were treated with 50 µm zoledronate and 25 µm DGBP in the presence or absence of 5 nm bafilomycin A1 for 48 h. Error bars represent standard error, n = 3, ns = not significant, p<0.01 **.

97 81 CHAPTER V: INHIBITORS OF MYCOBACTERIUM ISOPRENOID BIOSYNTHESIS Abstract The Mycobacterium species is characterized by an elaborate cell envelope. Decaprenyl diphosphate synthase catalyzes the synthesis of the 50-carbon isoprenoid decaprenyl diphosphate, which is critical for formation of multiple aspects of the cell wall. Herein, it is determined that bisphosphonates inhibit Mycobacterium tuberculosis E,Z-FPP synthase and decaprenyl diphosphate synthase, and structure activity relationships are evaluated. Utilizing Mycobacterium smegmatis as a surrogate for drug sensitivity, we find that compound 25 (Figure 35) has no effect on growth when used alone. However, when combined with ethambutol, an agent capable of increasing membrane permeability, growth inhibition is enhanced compared to ethambutol alone. The addition of exogenous decaprenyl diphosphate prevents the enhanced growth inhibition, providing evidence that the amplified growth inhibition is due to bisphosphonate-induced depletion of decaprenyl diphosphate by in vivo inhibition of decaprenyl diphosphate synthase. In addition, exogenous decaprenyl diphosphate is able to prevent to growth inhibition induced by SQ109, a novel isoprenoid-containing antituberculosis compound currently in clinical trials with an unknown mechanism of action. Introduction Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), and is estimated to have infected over 1/3 of the world population (120). In most cases, TB infection becomes latent, and individuals can remain asymptomatic for their lifetime. However, there is a 5-10% lifetime chance that the infection can become active TB, and

98 82 when left untreated there is a high mortality rate (120). The long required treatment course for TB infection causes difficulty with medicinal compliance, which partially contributes to the resistance of these microorganisms to antibiotics. Multi-drug resistant (MDR) strains of tuberculosis are resistant to the both of the front line drugs, isoniazid and rifampicin. The problem of drug resistance in M. tuberculosis has even progressed to warrant an additional classification of drug resistance, known as extensively drugresistant (XDR). With this rationale, it is clear that the development of novel therapeutics is necessary. Mycobacteria utilize the non-mevalonate (also known as the 2-C-methyl-Derythritol 4-phosphate, MEP; Figure 4) pathway to produce the 5-carbon core isoprenoid, isopentenyl diphosphate (IPP). IPP and its isomer dimethyl allyl diphosphate (DMAPP) can then be combined to produce the 10 carbon geranyl diphosphate (GPP) by a recently identified GPP synthase (Rv0989c) (121). The 15 carbon ω-e,z-farnesyl diphosphate (FPP) is formed from the incorporation of another isoprene unit from IPP onto GPP by ω- E,Z-FPP synthase (Rv1086) (122). Decaprenyl diphosphate synthase (Rv2361c), then stereospecifically adds multiple isoprene units from IPP onto ω-e,z-fpp resulting in formation of ω-e,poly-z-decaprenyl diphosphate (C 50 PP) ( ). Interestingly, decaprenyl diphosphate synthase was identified as essential from a large-scale transposon mutagenesis screen, while ω-e,z-fpp synthase was not (125). This discrepancy may be because decaprenyl diphosphate synthase can also utilize the substrate of ω-e,z-fpp synthase, GPP, as a substrate to produce decaprenyl diphosphate (123), and/or because it can also utilize ω-e,e-fpp as a substrate, and Mycobacterium tuberculosis also contains an ω-e,e-fpp synthase (Rv3398c) (126). Decaprenyl phosphate, functions as a glycosyl

99 83 carrier lipid involved in the formation the arabinoglactan, peptidoglycan, and mycolic acid components of the mycobacterial cell wall (127,128) (Figure 29). Decaprenyl phosphate also plays a role in the formation of the glycolipid known as lipoarabinomannan (LAM), which in M. tuberculosis is a virulence factor involved in modulation of host immune response (129) Our laboratory (73,95) and others have successfully utilized bisphosphonates to inhibit various isoprenoid diphosphate-utilizing enzymes. A major focus of this approach has been on the inhibition of human isoprenoid biosynthetic enzymes, although some bacterial isoprenoid biosynthetic enzymes such as E. coli undecaprenyl diphosphate synthase (53) and Staphylococcus aureus dehydrosqualene synthase (55) have also been targeted. This study was designed to determine if bisphosphonates could inhibit the mycobacterial cell wall biosynthetic enzymes ω-e,z-fpp synthase and decaprenyl diphosphate synthase. SQ109, a novel anti-tuberculosis compound containing an isoprene chain with an unknown mechanism of action is also evaluated as a possible isoprenoid biosynthetic pathway inhibitor. SQ109 is an isoprenoid-containing molecule under investigation as an antituberculosis agent (130). In a screen of over 63,000 compounds based on the pharmacophore of ethambutol, SQ109 was among the most potent compounds identified at disrupting cell wall biosynthesis as measured by fluorescence of a GFP protein under the control of a promoter known to be sensitive to inhibitors of cell wall biosynthesis (130). SQ109 emerged as a lead compound due to its potency in vitro and in vivo against M. tuberculosis, its selectivity for M. tuberculosis over human cells, and its efficacy in mouse models of disease (131). SQ109 also localizes considerably to the lung, the most

100 84 common organ of tuberculosis infection (132,133). In addition, SQ109 displays synergy with the major clinical anti-tuberculosis compounds (134). While this compound is currently in phase II clinical trials (135,136), the mechanism of action of SQ109 is unknown. It retains potent activity in ethambutol-resistant strains, suggesting a distinct target from ethambutol (131). Studies attempting to elucidate the mechanism of action utilizing strains resistant to the compound have proved unsuccessful, which is promising in respect to potential as a drug used against bacteria prone to resistance, but has increased the difficultly in determining the mechanism (137). Results In vitro inhibitors of ω-e,z-fpp synthase. M. tuberculosis recombinant ω-e,z- FPP synthase was purified (Figure 30) and the basic enzyme kinetics were evaluated (Figure 31). Due to the formation of ω-e,z-fpp as the product of ω-e,z-fpp synthase, it was hypothesized that farnesyl-containing bisphosphonates would inhibit this enzyme, and that potency would depend upon the stereochemistry of the farnesyl group. Specifically, it was hypothesized that the isomer most similar to the natural product would be the most potent. All possible mono-farnesyl bisphosphonate stereoisomers (73) were tested for inhibition of ω-e,z-fpp synthase activity in vitro (Figure 32). 2E,6Efarnesyl bisphosphonate (20), 2E,6Z-farnesyl bisphosphonate (21), 2Z,6E-farnesyl bisphosphonate (22), 2Z,6Z-farnesyl bisphosphonate (23) all inhibit ω-e,z-fpp synthase with IC 50 values determined to be 240, 340, 370 and 240 nm, respectively (Table 3). The NBP, zoledronate, had no inhibitory activity toward ω-e,z-fpp synthase, even at 100 µm.

101 In vitro inhibitors of decaprenyl diphosphate synthase. Recombinant decaprenyl diphosphate synthase was purified (Figure 33) and the basic enzyme kinetics were 85 analyzed (Figure 34). Compounds (Figure 35) were screened against M. tuberculosis decaprenyl diphosphate synthase in vitro at 10 µm and compounds inhibiting more than 50% of enzyme activity were further tested using concentration-response curves to generate IC 50 values (Table 4). The nitrogenous bisphosphonate zoledronate was tested up to 100 µm and did not inhibit decaprenyl diphosphate synthase. Monogeranyl bisphosphonate 1 inhibited decaprenyl diphosphate synthase activity to approximately 50% at 10 µm. The IC 50 value of compound 15 was determined to be greater than 10 µm and the IC 50 of compound 24 was determined to be approximately 5 µm. Compound 25 was more potent with an IC 50 value of 1 µm. Compound 26, a prodrug analog of compound 25 containing pivaloyloxymethyl (POM) groups attached to the bisphosphonate esters and a methyl ester at the carboxylic acid displayed no inhibition at 10 µm. The concentrations of the substrates IPP (Figure 37A) and E,E-FPP (Figure 37B) were varied in the presence or absence of compound 25 and double reciprocal plots were generated for analysis. With respect to both substrates, competitive-type inhibition is not consistent with the double-reciprocal graphs. Novel trans-stilbene containing bisphosphonates (Figure 36) were also tested for in vitro inhibition of decaprenyl diphosphate synthase. A mono-stilbene containing bisphosphonate (28) has an IC 50 value of ~10µM, while 29 and 30 have IC 50 values greater than 10 µm. Compound 31 is the most potent compound identified to date with an IC 50 value of 0.88 µm.

102 86 Impairment of Mycobacterium smegmatis growth. A blast search was performed to identify the putative M. smegmatis decaprenyl diphosphate synthase, and subsequently the polyprenyl diphosphate synthase proteins of M. smegmatis and M. tuberculosis were aligned using ClustalW ( The sequences are found to be approximately 90% similar at the protein level (Figure 38). The addition of the bisphosphonate 25 had no effect by itself on M. smegmatis growth when tested at 250 µm (Figure 39). The combination of ethambutol with compound 25 significantly decreased growth compared to ethambutol alone. Addition of decaprenyl diphosphate significantly prevented the growth inhibitory effects of compound 25 when combined with ethambutol (Figure 39). Compound 26 is similar to the lead in vitro compound 25, although a methyl ester is attached to its carboxylate and the bisphosphonate portion of the compound contains POM groups. Due to this compound disrupting the optical density of the growth media; after growth in liquid culture, M. smegmatis were plated onto agar-containing plates and colony-forming units were counted. Compound 26 is able to impair M. smegmatis in this assay at concentrations similar to the control isoniazid (Figure 40). M. smegmatis was also treated with 15 µm SQ109, a novel anti-tuberculosis compound with an unknown mechanism of action (Figure 41). M. smegmatis growth was impaired by 15 µm SQ109, and 30 µm decaprenyl diphosphate or decaprenyl monophosphate was able to prevent SQ109-mediated growth inhibition (Figure 42). Decaprenol at 30 µm was unable to prevent SQ109-mediated growth inhibition. Commercially available smaller isoprenoid diphosphates at 30 µm (omega-e,e-fpp,

103 87 GGPP, GPP, IPP, and combinations thereof) were also co-treated with SQ109, but are unable to prevent SQ109-mediated growth inhibition. Discussion Due to the formation of ω-e,z-fpp as the product of ω-e,z-fpp synthase, it was hypothesized that farnesyl-containing bisphosphonates would inhibit ω-e,z-fpp synthase dependently of the stereochemistry of the farnesyl group. It was expected that the closest analog to ω-e,z-fpp (2Z,6E-farnesyl bisphosphonate) would be the most potent. However, all farnesyl bisphosphonates were inhibitory with relatively similar IC 50 values, though the all cis (2Z,6Z; 23) and all-trans (2E,6E; 20) farnesyl bisphosphonates appeared to be slightly more potent than the mixed cis- and trans-type farnesyl bisphosphonates (21 and 22). Interestingly, these farnesyl bisphosphonate analogs also inhibit human GGDPS with a broad range of potency (73). The 2E,6E-farnesyl bisphosphonate is a relatively potent inhibitor, while 2Z,6Z-farnesyl bisphosphonate is a poor inhibitor of GGDPS and did not impair protein geranylgeranylation of K562 cells at 50 µm (73). These results suggest that specific inhibition of mycobacterial ω-e,z-fpp synthase over human GGDPS can be obtained. Mono-geranyl bisphosphonate 1 was a modest inhibitor of decaprenyl diphosphate synthase. Compound 15 contains an additional primary amine at the 9- position and this addition diminished activity. Compound 24 further builds upon this scaffold, and contains an aniline group. Compound 24 displays enhanced activity compared to compound 1. Compound 25 is further modified with a negatively charged carboxylic acid at the ortho position on the aniline ring, and potency is enhanced further. Compared to compound 25, compound 26 contains a methyl ester instead of a free

104 88 carboxylate group and also contains POM groups masking the negative charge of the bisphosphonate head group. Compound 26 had no inhibitory activity as tested. This is likely because of the loss of negative charge, which would abolish the interaction with Mg 2+ in the active site. Since this compound impaired M. smegmatis growth, it would be likely that the addition of the methyl group at the carboxylate of 25 is not responsible for the loss of in vitro activity. The mono-stilbene containing bisphosphonate 28 was also found to modestly inhibit decaprenyl diphosphate synthase, and again addition of a second R group as either a methyl (29) or geranyl (30) diminishes activity. The collective results suggest that with the current inhibitor scaffolds, it is rational to focus on synthesis of novel mono-substituted bisphosphonates, but not di-substituted. Interestingly, compound 31, containing a nitro group at the para position of the phenyl ring distal from the bisphosphonate enhanced activity in comparison to the parent compound (28). The two most potent bisphosphonates (25 and 31) both contain hydrophilic groups followed by a negative charge containing substituent. It may be worthwhile that novel compounds synthesized for inhibition of decaprenyl diphosphate synthase retain these attributes. Compound 25 was the lead inhibitor of decaprenyl diphosphate synthase. The more potent compound 31 was identified later and has not yet been evaluated in kinetic or bacterial assays. The double-reciprocal plots generated for kinetic analysis were not consistent with competitive-type inhibitors, as was anticipated. Noncompetitive inhibitors are particularly intriguing for use as bacterial enzyme inhibitors, as they are not out-competed by the ensuing accumulation of substrate that results from enzyme inhibition.

105 89 M. smegmatis has been utilized as a genetic workhorse in place of M. tuberculosis, due to its lack of pathogenesis and faster doubling time. In addition, studies have demonstrated that M. smegmatis may serve as a surrogate model for MDR strains of M. tuberculosis for use in the design of anti-mycobacterial drugs (138). Sequence alignment confirmed a high similarity between M. smegmatis and M. tuberculosis polyprenyl synthases, suggesting that inhibitors of the M. tuberculosis enzyme would like inhibit the M. smegmatis enzyme. With the aforementioned points in mind, M. smegmatis is utilized as a model to determine if the lead decaprenyl diphosphate synthase inhibitor could impair mycobacterial growth. Inhibitors of decaprenyl diphosphate synthase were selected for use in bacterial assays because this enzyme (but not omega- E,Z-FPP synthase) has been identified as essential (125). Compound 25 did not inhibit M. smegmatis growth when tested as a monotreatment. This result was perhaps predictable, as bisphosphonates generally have a high charge to mass ratio, resulting in difficulty traversing cell membranes (77). In addition, the complex cell envelope of mycobacteria provides a natural antibiotic resistance to many drugs (56). These results suggest that bisphosphonate salts may be unable to enter mycobacterial cells. Another study using bisphosphonates as inhibitors of a S. aureus enzyme also found that bisphosphonates were apparently unable to enter into these bacterial cells (55). Interestingly, a phosphosulfonate analog of the lead bisphosphonate molecule was used successfully in bacterial and mouse studies (55). Ethambutol is an inhibitor of arabinogalactan biosynthesis in mycobacteria and functions in part by disrupting the cell envelope resulting in increased permeability to other drugs (139). The combination of ethambutol with compound 25 appears to allow for entry of the

106 90 bisphosphonate, as this combination displayed significantly impaired growth inhibition compared to ethambutol alone. To determine if this combined effect was indeed due to bisphosphonate-induced depletion of decaprenyl diphosphate, bacteria were co-treated with decaprenyl diphosphate (Figure 39). The addition of decaprenyl diphosphate was able to prevent the effect of the compound 25 when combined with ethambutol, suggesting that decaprenyl diphosphate depletion is the reason for bisphosphonateinduced enhancement of ethambutol growth inhibition. Decaprenyl diphosphate depletion is likely due to inhibition of decaprenyl diphosphate synthase, as this enzyme was inhibited in vitro by compound 25. The POM groups on compound 26 mask the negative charge of the bisphosphonate and also greatly increase the lipophilicity of the compound (77). Compound 26 is able to impair M. smegmatis growth at concentrations comparable to isoniazid. Although, it should be noted that compared to M. tuberculosis, M. smegmatis is relatively resistant to isoniazid (140). A POM prodrug of a compound that did not inhibit decaprenyl diphosphate synthase in vitro had no effect on M. smegmatis growth (data not shown), supporting the presumption that the effects on cell growth are not solely due to the POM groups. These results suggest that while bisphosphonate salts may be unable enter M. smegmatis, prodrug strategies may provide a mechanism for circumventing the difficulty of transversing the mycobacterial cell envelope. M. smegmatis growth is inhibited by 15 µm SQ109 (Figure 42), although M. smegmatis is relatively resistant to SQ109 compared to M. tuberculosis (134). Interestingly, treatment of M. smegmatis with SQ109 and decaprenyl diphosphate or decaprenyl monophosphate is able to prevent SQ109-induced inhibition of M. smegmatis

107 91 growth. Decaprenol, the alcohol, was unable to prevent SQ109-mediated growth inhibition. These results suggest that SQ109 may impair the synthesis or processing of decaprenyl diphosphate or phosphate. Smaller isoprenoid or combinations thereof, when co-treated with SQ109, were unable to prevent SQ109-induced inhibition of M. smegmatis growth. However, due to this being a negative effect, it is unknown if the smaller isoprenoids were able to cross the mycobacterial membrane and enter cells. SQ109 did not appear to inhibit M. tuberculosis E,Z-FPP synthase or decaprenyl diphosphate synthase in vitro (data not shown). However, SQ109 is metabolized by liver microsomes to numerous derivatives (133), so its action as a prodrug inhibitor of either of these enzymes cannot be ruled out. It is also possible that SQ109 impairs menaquinone biosynthesis and that the decaprenyl monophosphate and diphosphate can promiscuously incorporate into menaquinone, if SQ109 disrupts its synthesis. In particular, MenA is responsible for adding a trans-polyprenyl (C45) onto 1,4-dihydroxy-2-naphthoate to form demethylmenaquionone, which is then processed by MenG to form menaqunone (141). Menaquinone is important for the electron transport chain and ATP synthesis. In conclusion, bisphosphonates were identified that inhibit M. tuberculosis ω-e,z- FPP synthase and decaprenyl diphosphate synthase in vitro. An initial lead inhibitor of decaprenyl diphosphate synthase was identified (25) with an in vitro IC 50 of 1 µm, although subsequently a more potent compound was discovered (31). Compound 25 is incapable of altering growth of the surrogate M. smegmatis when used alone, but is able to potentiate growth inhibition of the cell wall-disrupting compound ethambutol. Compound 26 was also able to impair M. smegmatis growth, likely due to the POM groups facilitating entry of this compound. Future studies should involve the synthesis

108 92 and evaluation of various analogs of bisphosphonates such as phosphonosulfonates or bisphosphonate prodrugs to circumvent the cell entry issues (54,75). Furthermore, novel in vitro inhibitors of decaprenyl diphosphate synthase can now be synthesized based on the current lead compounds to increase potency and further elaborate in vitro structure activity relationships. Experimental Procedures Materials. All trans FPP was obtained from Sigma. Decaprenol ( t 3 -c 6 -OH, C 50 OH), decaprenyl monophosphate ( t 3 -c 6 -P, C 50 P), and decaprenyl diphosphate ( t 3 -c 6 -PP, C 50 PP) were obtained from the laboratory of Dr. Ewa Swiezewska at the Polish Academy of Sciences (Warsaw, Poland). The bisphosphonates used were synthesized previously (72,142). [ 14 C]-IPP (55 mci/mmol) was obtained from Perkin Elmer. Protein purification. A plasmid containing N-terminal His-tagged ω-e,z-farnesyl diphosphate (Rv1086) or decaprenyl diphosphate synthase (Rv2361c) was provided by the laboratory of Dr. James Naismith at St Andrews University (St Andrews, United Kingdom). The plasmids were transformed into E. coli DE3 BL21 star (Invitrogen; Carlsbad, California). Bacteria were then grown to log phase and expression was induced by addition of 0.1 mm IPTG overnight at room temperature. Cells were then pelleted by centrifugation and then resuspended in 50 mm Tris ph 8 containing 300 mm NaCl, 10 mm imidazole, and 1 mm phenylmethanesulphonylfluoride (PMSF). Cells were lysed using 1 mg/ml lysozyme for 30 minutes at room temperature. Lysate was filtered and loaded onto a His Select (Sigma) nickel affinity resin column, washed, and eluted with 50 mm Tris ph 8 buffer containing 300 mm NaCl and 250 mm imidazole

109 93 according to the manufacturer instructions, except 0.1% Triton-X-100 was added to final washes and included in the elution buffer. In vitro enzyme assays. Enzyme assays were typically performed in 20 µl reactions containing 50 mm Tris-HCl ph 7.9 buffer, 1 mm MgCl 2, 0.15% Triton-X-100, 1 mm DTT. For ω-e-z-fpp synthase, 46 ng recombinant enzyme, 100 µm GPP and 30 µm IPP were used unless otherwise noted. For decaprenyl diphosphate synthase, 30 ng recombinant enzyme and unless otherwise noted 100 µm FPP and 30 µm 14 C-IPP were used. Note the FPP used in the in vitro assay was all-trans FPP, which was utilized due to its commercial availability, and ability to be utilized by decaprenyl diphosphate synthase (123). Compounds were added prior to substrate addition and allowed to preincubate for 10 min at 37 C, after which substrates were added and reactions were allowed to proceed for 10 min at 37 C. Products were extracted with 1-butanol and washed, and radioactivity in the butanol fraction was quantitated using a liquid scintillation counter. In vivo Mycobacterium smegmatis growth assays. Mycobacterium smegmatis (ATCC 607) was obtained from American Type Culture Collection and cultured in 7H9 broth (Sigma) containing ADC (Sigma), 0.5% glycerol, and 0.05% Tween-80. Bacteria were grown overnight and then diluted, aliquoted, and grown in 96 well plates in the presence of indicated drugs with shaking at 37 C. Growth was measured by optical density at 650 nm using a spectrophotometer. Alternatively, when indicated, aliquots were spotted onto 7H9 agar plates, allowed to grow for 48 h and colony-forming units (CFU) were counted and normalized per ml.

110 94 Figure 29. Schematic of mycobacterial cell wall. Included is an abbreviated biosynthetic pathway for decaprenyl diphosphate, and the major components of the cell wall.

111 95 ω-e,z-fpp synthase Figure 30. Coomassie Blue stained SDS-PAGE of purified mycobacterial ω-e,z-fpp synthase. Protein standards are labeled at 37 and 25 kda. The predicted molecular mass of recombinant ω-e,z-fpp synthase is 29.4 kda.