Squalene Monooxygenase – a Target for Hypercholesterolemic Therapy

Review

Squalene monooxygenase – a target for hypercholesterolemic therapy

Agnieszka Belter1 – 3 , Miroslawa Skupinska1,3 , monooxygenase inhibitors could become the hypocholester- Malgorzata Giel-Pietraszuk1 , Tomasz Grabarkiewicz2 , olemic agents of the future. Leszek Rychlewski3 and Jan Barciszewski1, * Keywords: 1 Institute of Bioorganic Chemistry , Polish Academy of cholesterol; drug design; epoxidation; Science, Noskowskiego 12/14, 61-704 Poznan , Poland hypercholesterolemia. 2 AdvaChemLab , Topazowa 8, 61-680 Poznan , Poland 3 BioInfoBank Institute , Limanowskiego 24A, 60-744 Poznan , Poland Introduction: squalene monooxygenase

* Corresponding author Squalene monooxygenase (squalene epoxidase, SE, EC e-mail: [email protected] 1.14.99.7), the enzyme of cholesterol synthesis pathway, was detected in rat liver microsomes in 1969 (Yamamoto and Bloch, 1970). Then it was discovered in fungi as an enzyme Abstract taking part in ergosterol synthesis and in plants, where it is a part of phytosterols synthesis pathways (Petranyi et al. , 1984 ; Squalene monooxygenase catalyzes the epoxidation of C-C Godio et al., 2007; Rasbery et al., 2007; Uchida et al., 2007; double bond of squalene to yield 2,3-oxidosqualene, the key He et al., 2008; Han et al., 2010). SE was also identifi ed in step of sterol biosynthesis pathways in eukaryotes. Sterols some bacteria which produce pentacyclic triterpens instead are essential compounds of these organisms and squalene of sterols, so-called hopanoids synthesized from squalene by epoxidation is an important regulatory point in their synthe- squalene-hopene cyclase (SHC) (EC 5.4.99.17) (Nakano et sis. Squalene monooxygenase downregulation in vertebrates al., 2003; Pearson et al. , 2003 ; Volkman , 2003 ; Nakano et al. , and fungi decreases synthesis of cholesterol and ergosterol, 2007). Several years of investigations aimed at fi nding inhibi- respectively, which makes squalene monooxygenase a potent tors of pathogenic fungi SE resulted in numerous antimycot- and attractive target of hypercholesterolemia and antifungal ics [e.g., terbinafi ne (Ryder , 1989 )], which are stiil in clinical therapies. Currently some fungal squalene monooxygenase use today (Berg and Plempel , 1989 ). inhibitors (terbinafi ne, naftifi ne, butenafi ne) are in clinical Squalene monooxygenase belongs to the fl avoprotein use, whereas mammalian enzymes ’ inhibitors are still under monooxygenase family, which catalyze a wide variety of investigation. Research on new squalene monooxygenase oxidative reactions (van Berkel et al., 2006; Joosten and van inhibitors is important due to the prevalence of hypercholes- Berkel , 2007 ). SE catalyzes squalene epoxidation. Reaction terolemia and the lack of both suffi cient and safe remedies. occurs in the presence of fl avin adenine dinucleotide (FAD), In this paper we (i) review data on activity and the structure molecular oxygen, nicotinamide adenine dinucleotide phos- of squalene monooxygenase, (ii) present its inhibitors, (iii) phate (NADPH), NADPH-cytochrome P-450 reductase (EC compare current strategies of lowering cholesterol level in 1.6.2.4), and the supernatant protein factor (Tai and Bloch , blood with some of the most promising strategies, (iv) under- 1972 ; Shibata et al. , 2001 ). In the fi rst step FAD is reduced to line advantages of squalene monooxygenase as a target for hydroperoxyfl avin, which is followed by electrophilic attack hypercholesterolemia therapy, and (v) discuss safety con- of oxygen from – OOH group on the double bond of squalene. cerns about hypercholesterolemia therapy based on inhibition As a result, 2,3-oxidosqualene is synthesized (Figure 1 ) of cellular cholesterol biosynthesis and potential usage of (Ghisla and Massey, 1989; Massey, 1994; Entsh and van squalene monooxygenase inhibitors in clinical practice. After Berkel , 1995 ; van Berkel et al., 2006). many years of use of statins there is some clinical evidence for There are several discrepancies between squalene and their adverse effects and only partial effectiveness. Currently other known fl avin monooxygenases (Joosten and van Berkel, they are drugs of choice but are used with many restrictions, 2007 ). Firstly, SE catalyzes epoxidation but not hydroxylation especially in case of children, elderly patients and women and does not contain cytochrome P450 as a prosthetic group of childbearing potential. Certainly, for the next few years, and does not bind NADPH directly. The electron transfer is statins will continue to be a suitable tool for cost-effective mediated by NADPH dependent cytochrome P450 reductase cardiovascular prevention; however research on new hypo- bound to microsomal membrane. Other fl avin monooxyge- lipidemic drugs is highly desirable. We suggest that squalene nases bind the nicotinamide cofactor directly (Sakakibara 1054 A. Belter et al.

O R SE R + + NADPH + O2 + NADP + H2O FAD

Figure 1 The epoxidation reaction catalyzed by squalene monooxygenase (SE).

et al. , 1995 ). These differences enhance the chances of design- Structure-activity relationship ing drugs selective enough to lower cholesterol synthesis but not disturbing other fl avin monooxygenases dependent processes. The alignment of amino acid sequence of squalene monooxygenses from different organisms shows a high sequence Specifi city of epoxidation reaction homo logy of 83 – 93 % among the mammalian enzymes and 30.2 – 35 % between fungal and mammalian SEs (Lee et al. , The reaction catalyzed by squalene monooxygenase is highly 2004 ). Some differences were found between enzymes of specifi c (Nagumo et al., 1995). Only terminal double bonds pathogenic fungi. For example squalene monooxygenase from of squalene are oxidized to 2,3-oxidosqualene and 2,3:22,23- Trichophyton tonsurans is homologous in 95 % , 75 % , 74 % dioxidosqualene subsequently. 5,6-Oxidosqualene and 10,11- and 65 % , with Trichophyton rubrum , Emericell nidulans , oxidosqualene are not observed in the reaction catalyzed Aspergillus fumigates and Aspergillus niger SEs, respectively by SE (van Duuren and Schmitt, 1960; Abe et al., 2007). (Table 1 ) (Motavaze et al., 2008). The high homology provides Oxidation of the terminal double bonds is characteristic for the possibility of fi nding an inhibitor with a broad spectrum of other enzymatic reactions (Lucas et al. , 2010 ). Zeaxanthin antifungal activity. However, signifi cant differences between epoxidase oxidizes double bonds only at the ends of zeax- mammalian and fungal squalene monooxygenases suggest they antin, despite the fact that this substrate, like squalene, con- have different sensitivities to various inhibitors (Ruckenstuhl et tains several internal double bonds which could be potentially al., 2008). Additionally, squalene monooxygenases of mammals epoxidized (Hieber et al. , 2000 ). and plants are more closely related to each other than fungal SE The activity of enzyme towards squalene analogs was also is to their mammalian counterparts, e.g., human SE shows 83 % checked (Abe et al. , 2007 ). A lack of one double bond (5,6- of homology to Arabidopsis thaliana SQE1 but only 36 % to dihydrosqualene, 10,11-dihydrosqualene) as well as two Saccharomyces cerevisiae and 25 % to Methylococcus capsula- double bonds (6,7,18,19-tetrahydrosqualene, 10,11,14,15- tet- tus SEs (Table 1 ) (Nakano et al., 2003). rahydrosqualene) of squalene do not preclude epoxidation of Todate, squalene monooxygenases from rat (Ono and Imai , terminal isoprene units (Figure 2 ). An analog with an additional 1985 ; Denner -Ancona et al., 1995 ), pig (Bai and Prestwich , methyl group or its depletion at the epoxidation site of squalene 1992; Denner-Ancona et al., 1995), liver microsomes, does not inhibit the reaction (Corey and Russey, 1966; van recombinant rat (Nagumo et al. , 1995 ; Sakakibara et al. , Tamelen and Heys , 1975 ) (Figure 3 ). For example 2-methylbu- 1995) and human (Laden et al., 2000) SEs have been tylene and propylene ends are still epoxidized, but fi ve times less purifi ed. These enzymes were unstable and tended to effi ciently than the 2-metylpropylene end. These results suggest form aggregates, even in the presence of detergents (Lee that the enzymatic epoxidation is not strongly infl uenced by et al., 2004), which are likely to impede the formation of steric hindrance due to methyl groups even in the close vicinity crystals. Until recently a three-dimensional (3D) structure of of the oxidation site (van Tamelen and Heys , 1975 ). SE had not yet been solved.

4 6 8 10 12 14 16 18 20 R = 579 11 13 15 17 19 21 Squalene 2 R 23 1322 24

SE 4 6 8 10 12 14 16 18 20 579 11 13 15 17 19 21 6,7-dihydrosqualene

1 R 23 2243 22 4 6 8 10 12 14 16 18 20 579 11 13 15 17 19 21 10,11-dihydrosqualene O

SE 4 6 8 10 12 14 16 18 20 579 11 13 15 17 19 21 10,11,14-15-tetrahydrosqualene 1 24 R 2 3 22 23 4 6 8 10 12 14 16 18 20 O O 579 11 13 15 17 19 21 6,7,18,19-tetrahydrosqualene

Figure 2 Squalene and its derivatives without one or two double bonds epoxidized by squalene monooxygenase. Squalene monooxygenase and hypercholesterolemic therapy 1055

C26H43 C26H43 2-methylopropylene end O

C H 26 43 SE, FAD C26H43

2-methylobutylene end NADPH, O2 O

C H C26H43 26 43 Propylene end O

Figure 3 Squalene, its derivatives with variations at its epoxidation site, and their epoxides. Extracted sites of epoxidation: 2-methylopropylene end of squalene, 2-methylobutylene end of (6E, 10E, 14E, 18E, 22E)-2,6,10,15,19,23-hexameth- ylpentacosa-2,6,10,14,18,22-hexaene and propylene end of (6E, 10E, 14E, 18E, 22Z)-2,6,10,15,19-pentamethyltetracosa-2,6,10,14,18,22-hexaene.

Table 1 Comparison of squalene monooxygenses ’ amino acid sequences from Homo sapiens (NP_003120.2), Rattus norvegicus (NP_058832.1), Arabidopsis thaliana (NP_564734.1), Saccharomyces cerevisiae (EDN61765.1), Mathylococcus capsulatus (YP_115265.1) with each other and from Trichophyton tonsurans ( ABP64796.1) with the enzyme from Trichophyton rubrum (AAQ18216.1), Emericell nidulans (ABD48706.1), Aspergillus fumigates (XP_001823492.1) and Aspergillus niger (CAG38355.1).

H. sapiens R. norvegicus A. thaliana S. cerevisiae M. capsulatus T. tonsurans

H. sapiens 1 0 0 8 3 4 3 3 6 2 5 – R. norvegicus 1 0 0 4 2 3 6 2 3 – A. thaliana 1 0 0 3 6 2 5 – S. cerevisiae 100 22 – M. capsulatus 100 – T. rubrum 95 E. nidulans 75 A. fumigates 74 A. niger 65 Numbers indicate percentage of identities between sequences.

Despite the low sequence similarity between squalene Site-directed mutagenesis of recombinant rat SE monooxygenases and other fl avoproteins, a signifi cant struc- (Ruckenstuhl et al., 2005, 2008) and photolabeling (Lee et tural homology exists among them (Lee et al., 2004). The al., 2000, 2004) of short 24 amino acids peptides experi- only fl avoprotein, for which a 3D structure has been solved, ments showed that the active site of enzyme is moderately is p-hydroxybenzoate hydroxylase (PHBH) (Schreuder et hydrophobic and located proximally to the FAD-binding al., 1989). Although PHBH and SEs share only about 20% motif. Conserved aromatic residues, through π -π interactions, of sequence identity, three highly conserved sequence motifs help in the correct folding of the π -electron rich, hydropho- are present in both enzymes (Ruckenstuhl et al. , 2005 ). All of bic substrate, and in this way affi rms the regio- and stereo- them participate in FAD binding. One of them, GxGxxG, is specifi city of an epoxidation reaction. Analysis of a series of responsible for binding ADP moiety of FAD and is a part of single amino acid mutants of rats’ squalene monooxygenase Rossmann fold, which is characteristic for numerous nucle- obtained for evaluation of the enzyme activity showed that otides binding proteins. The other motif has the highly con- almost all of them were less active than the wild-type enzyme served GD residues, which contact the 3 -OH of the ribose (Abe et al. , 2007 ). moiety of FAD. The third one takes part in interactions with ribitol moiety of FAD and is conserved in all fl avoproteins Squalene monooxygenase inhibitors (Eggink et al., 1995). The enzyme has no distinct binding domain of coenzyme NADPH (Wierenga et al., 1986; Eggink Antimycotic compounds et al. , 1995 ), which is typical for class A fl avoprotein monooxygenases (van Barkel et al., 2006). NADPH forms a transient Naftifi ne (1) (see Table 2) is the fi rst antifungal agent, which complex with SE, which disassociates immediately after FAD blocks biosynthesis of ergosterol, the crucial component of all reduction (van Barkel et al. , 2006 ). fungal cell membranes, by inhibiting epoxidation of squalene 1056 A. Belter et al.

Table 2 Antimycotic squalene monooxygenase inhibitors. Naftaﬁ ne and its derivatives (Georgopoulos et al. , 1981 ; Abe et al. , 1994 ).

Compound Structural formula IC 50 or % inhibition (nm) for Candida albicans SE Core Substituent (R)

Naftiﬁ ne (1) 930 N R

Terbinaﬁ ne (2) 3 0

Butenaﬁ ne (3) 4 5

SDZ SBA 586 (4) 8

(5) 65 % (100 µm )

(6) 37 % (100 µ m )

(7) 4 0

(8) O 9 0

(9) 5 0

(10) 5 0 F

(11) 420

(Georgopoulos et al. , 1981 ; Ryder and Dupont , 1984 ; Ryder phenyl]methyl terbinafi ne (2), butenafi ne (3), and SDZ SBA et al., 1984; Kan et al., 1986). Currently it is used in the treat- 586 (4) respectively, have been obtained. They have a higher ment of infections caused by pathogenic fungi, e.g., C. albi- inhibitory activity than the starting compound and are rever- cans and Trichophyton rubrum (Abe et al. , 1994 ). It was a sible, non-competitive inhibitors of C. albicans squalene starting point for a series of successive squalene monooxy- monooxygenase with respect to squalene, FAD, NADH and genase inhibitors (2– 11) (Table 2 ). As a result of substitution NADPH (Ryder and Dupont, 1985; Abe et al., 1994). IC50 val- of cinnamyl group in naftifi ne with a 6,6-dimethyl-2-hepten- ues for mammalian SE are several orders of magnitude higher

4-ynyl, a 4-t-butylbenzyl, and a [4-(1-methyl-1-phentylethyl) than for the fungal enzyme (e.g., IC 50 for terbinaﬁ ne for Squalene monooxygenase and hypercholesterolemic therapy 1057

C. albicans = 30 n m, for rat > 100000 nm ) (Ryder et al., 1986). Generally, replacement of naphthalene moiety of terbinafi ne It suggests that during antimycotic therapy they do not (12-15) results in a loss of activity (Table 3 ) with one signifi - disturb the cholesterol biosynthesis pathway of the host. cant exception, SDZ 87-469 (12), in which the presence of To search the terbinafi ne ’ s structure-activity relationship its 3-chloro-7-benzo[b]thiophene group causes broadening of its derivatives were synthesized and their potential to inhibit antifungal activity (Favre and Ryder , 1997 ). The removal of squalene epoxidation were checked (Favre and Ryder , 1997 ). methyl group from the tertiary amine (16) or the substitutions

Table 3 Antimycotic squalene monooxygenase inhibitors. Terbinaﬁ ne and its derivatives (Ryder and Dupont, 1985; Abe et al., 1994).

Compound Structural formula IC50 (nm ) for Candida albicans SE Core Substituent (R)

Terbinaﬁ ne (2) 30

R SDZ 87 – 469 (12) 11 S

Cl (13) 3870

(14) 47 900

(15) 370

S (16) 2160

(17) 260

(18) 320

1058 A. Belter et al.

of nitrogen atom with oxygen (17) or carbon (18) remarkably showed that only methyl is effective at this position (Sawada decrease their affi nity for the enzyme (Table 3 ) (Favre and et al., 2004). N-ethyl derivative is more than two times stron- Ryder , 1996 ). ger than N-methyl and N-propyl analogs (Sawada et al. , Although terbinafi ne and its derivatives do not show any 2004 ). One of the most potent derivative is FR194738 (49). obvious structural similarities to squalene, FAD or NADPH, Compared to NB-598, it shows higher inhibitory activity, bio- it seems that they act as substrate analogs (Abe et al. , 1994 ). availability and stability (Table 5 ) (Sawada et al., 2004). It The point mutation studies of squalene monooxygenases inhibits cholesterol biosynthesis in HepG2 cells more potently from terbinafi ne resistant strains indicate amino acids criti- than the clinically used HMG-CoA reductase inhibitors (e.g., cal for terbinafi ne-enzyme recognition (Laber et al., 2003; simvastatin, fl uvastatin, pravastatin) (Sawada et al. , 2004 ). In Ruckenstuhl et al. , 2005, 2007 ). They are located at the studies carried out on dogs, FR194738 demonstrated an abil- C-terminus (Phe402, Phe420, Phe430 and Phe433 residues in ity of reducing triglyceride levels and plasma cholesterol in a Saccharomyces cerevisiae SE) and in the central part of the dose-dependent manner. LDL cholesterol level is decreased enzyme (Leu251) (Laber et al. , 2003 ). Based on a molecular more than HDL, resulting in an improved atherogenic index docking detailed mechanism of the SE, inhibition by terbi- (LDL/HDL ratio) (Sawada et al., 2004). The reduction in trig- nafi ne was proposed. According to that model the inhibitor lyceride level is due to reduced secretion of VLDL, triglycer- interacts with amino acids at C-terminus and the central part ide rich particles. It is well tolerated up to 100 mg/kg (Sawada of the enzyme (Nowosielski et al. , 2011 ). The naftalene ring et al. , 2002 ). It means that FR194738 could become a new interacts with residues in a small, highly hydrophobic cav- drug for hypercholesterolemia therapy. ity near the entrance to the substate’s binding pocket. Tert- Further investigations resulted in the discovery of a new butyl substituent of terbinafi ne points toward the center of class of potent mammalian squalene monooxygenase inhib- the enzyme blocking the main (one of two) entrance to the itors as silane derivatives of NB-598 [Table 4 , (52)– (72)] enzyme’ s catalytic site. This sustains the experimental evi- (Gotteland et al., 1995, 1997). IC 50 values between the phe- dence that terbinafi ne inhibits fungi SE in non-competitive nyl (53) and cyclohexyl (52) analog indicates the importance manner (Favre and Ryder , 1996 ; Nowosielski et al. , 2011 ). of an aromatic moiety directly attached to silicon (Gotteland et al., 1995). Additionally, 4-substitution of phenyl ring Anticholesterolemic agents with methoxy (54), thiomethyl (55), hydroxyl (56), thiol (57), carbamoyl (58), trifl uoroacetyl (59), ethynyl (60) moi- Allylamines The extension of the carbon skeleton between ety signifi cantly decrease, while fl uorine (61), methyl (62), the tertiary nitrogen and the naphthyl group or the substitution formyl (63), cyano (64), or vinyl (65) residues improve the of phenyl group with naphthyl group in terbinafi ne increase inhibitory potential of derivatives. Introduction of methyl specifi city of these compounds (19-23) toward mammalian (66), methoxy (67), trifl uoromethyl (68) and cyano (69) sub- SEs (Table 4) (Takezawa et al. , 1989 ). A similar effect stituents in position 2 of the phenyl ring also improve activ- was observed for the substitution of the N-methyl group ity. It is interesting that 3-methyl (70) substitution causes with ethyl (26) or propyl group (27) and the addition of almost complete loss of activity, although methyl in 2 and heteroaryl moiety at position 3 of the phenyl ring [Table 4 , 4 position of the ring increases affi nity of these derivatives (31)– (46)] (Takezawa et al., 1989, 1990). NB-598 (47) is into the enzyme (Gotteland et al., 1995). Further investiga- the fi rst compound obtained by chemical modifi cation of tions showed that ene-yne nitrogen substituent (66) can be aromatic moiety of terbinafi ne (Horie et al., 1991, 1993); replaced by an yne-yne side chain (72) without signifi cant it is a highly-potent, competitive and specifi c inhibitor of loss of activity. They resulted in (aryloxy)methylsilane deriv- mammalian squalene monooxygenase (Horie et al. , 1990 ). atives (71), (72). The clue is that they not only effi ciently This bis(thienyl) derivative strongly inhibits microsomal inhibit rat squalene monooxygenase in vitro, but they also squalene monooxygenase from rat and dog livers, and the block cholesterol biosynthesis in rats on oral administration (Gotteland et al. , 1995 ). EC values for these compounds enzyme from the HepG2 cell line with IC50 values of 4.4 nm , 50 2.0 n m, and 0.75 nm , respectively (Horie et al., 1990). It are relatively low, which made them promising candidates blocks cholesterol synthesis, but does not affect the synthesis for further hypercholesterolemic agents (Gotteland et al. , of free fatty acids, phospholipids, and triacylglycerols. In rats 1995 ). and beagle dogs, NB-598 markedly lowers total cholesterol and increases squalene levels in blood serum. Squalene derivatives An additional approach in the NB-598 is a strongly lipophilic molecule. Its poor solubility search for new potent competitive inhibitors of squalene correlates with poor permeation and absorption, which con- monooxygenase uses squalene analogs as a starting point. comitantly limits its bioavailability and its potential usage as Several squalene derivatives with different proximal groups a drug (Lipinski et al., 2001). A lipophilic property of NB-598 and with extended and truncated carbon skeletons have was improved by modifying the thienyl-thienyl structure and been tested however, until now few of them inhibit squalene introducing polar linker between terminal thiopene and aryl epoxidation (Table 6 ) (Sen and Prestwich , 1989b ; Sen et amine portion of the molecule [Table 4 , (48)– (51)] (Sawada al., 1990). The entire trisnorsqualenoid moiety is necessary et al., 2004). Ether derivative (49) is more potent than ester to achieve an inhibitory effect. Both addition and removal one (48) and NB-598 (Sawada et al. , 2004 ). Additionally, the of isoprene units in trisnorsqualenoid moiety, and even the substitution of geminal dimethyl moiety (49) with diethyl (51) removal of its terminal isopropylidene group cause a dramatic Squalene monooxygenase and hypercholesterolemic therapy 1059

Table 4 Anticholesterolemic squalene monooxygenase inhibitors. Terbinaﬁ ne ’ s derivatives (Takezawa et al., 1989; Gotteland, 1995).

Compound Structural formula IC 50 ( µ m )* for rat SE, **for human SE, ***for pig SE Core Substituent (R) Terbinaﬁ ne (2) > 1 0 0 *

R (19) 0 . 1 2 *

(20) 0 . 2 5 *

(21) 3 . 5 *

(22) 0 . 3 2 *

(23) 1 . 4 *

(24) H 6.8* R (25) CH 3 1 . 4 * (26) N CH2 CH 3 0 . 3 * (27) CH2 CH2 CH 3 0 . 2 7 * (28) CH2 CHCH2 2 . 1 * (29) CH2 CCH 9.6* (30) C H 3.9* O 3 6

(31) H 1.40*

(32) CH 3 0 . 6 6 * N (33) CH2 CH 3 0 . 3 6 * (34) CF3 3 . 1 0 * (35) OH 4.60* (36) Nitrile 0.51* (37) R Formyl6.80* O (38) Hydroxymethyl0.84* (39) Phenyl0.57* 1060 A. Belter et al.

Table 4 (Continued)

Compound Structural formula IC 50 ( µ m )* for rat SE, **for human SE, ***for pig SE Core Substituent (R) (40) 2-furyl 0.14* (41) 2-oxazolyl 1.20* (42) 2-thiazolyl 1.20* (43) 5-oxazolyl 0.063* (44) 5-thiazolyl 0.089* (45) 1-pyrrolyl 0.014* (46) 3-thienyl 0.011* NB-598 (47) 7 . 2 * * O

N S

R (48) O 2 3 * * O O FR194738 (49) O 5 . 4 * * O

(50) O 2 5 * * O (51) O 120** O

(52) 150***

R Si O

(53) 20***

(54) OCH3 0.87*** (55) SCH 3 1*** (56) N OH 8*** (57) SH 8*** R (58) CONH2 5***

(59) COCF 3 5*** (60) Si O CCH 1*** (61) F 6***

(62) CH3 0.12*** (63) CHO 0.1*** (64) CN 0.02***

(65) CHCH2 0.07***

(66) CH 3 0.03*** (67) OCH3 0.15*** (68) N CF3 0.07*** (69) CN 0.03*** R

Si O

Squalene monooxygenase and hypercholesterolemic therapy 1061

Table 4 (Continued)

Compound Structural formula IC 50 ( µ m )* for rat SE, **for human SE, ***for pig SE Core Substituent (R)

(70) CH 3 3***

N R

Si O

(71) R CH 3 0.09*** (72) OCH3 0.07***

Si O

*IC50 ( µm ) for rat SE; **IC50 ( µm ) for human SE; ***IC 50 ( µ m ) for pig SE.

Table 5 Pharmacokinetic parameters of NB-598 and FR194738 (Sawada et al., 2004).

SE inhibitory activity C max ( µ g/ml) T max (h) AUC0-8h BA ( % ) Dose (mg/kg) Plasma concentration

IC50 (nm ) ( µ g·h/ml) in dogs ( µ g/ml) Human Rat Dog 1 h 4 h 24 h

NB-598 7.2 43 36 0.329 1.67 1.392 16.5 10 < 0.05 < 0.05 < 0.05 FR194738 9.8 14 25 0.486 1.17 1.873 32.9 5 0.199 0.138 < 0.05 16 0.910 0.717 < 0.05

decrease in inhibitory potency [Table 6 , (74), (75)]. It means hindered analog (CH3 C = CF 2) did not inactivate the enzyme. that the terminal isoprene unit is important in a substrate It points at the orientation of difl uoro group in the active site binding to an enzyme. Trisnorsqualene alcohol (TNSA) (Sen and Prestwich , 1989a ). The time-dependent competition (73) is the fi rst effective inhibitor of vertebrate squalene with squalene, and structure-activity relationship data suggest monooxygenase (IC50 = 4 µ m for pig liver SE) (Table 6 ) (Sen that difl uoroolefi ns binds covalently to the enzyme (Moore et and Prestwich , 1989b ), which acts as a substrate analog, al. , 1992 ). The advantage of difl uoro compounds is that they mimicking a reactive intermediate of squalene epoxidation are active in mice after oral administration (Jarvi et al. , 1991 ). (Sen and Prestwich, 1989b). Compounds (76) and (77) also Several squalene analogs with substituted C-26 methyl inhibit squalene monooxygenase (the fi rst one 10-fold weaker group (83)–(85) are competitive inhibitors of squalene mono- than TNSA, the second one as potent as TNSA) (Table 6 ). It oxygenase (Table 6 ) (Bai , 1991 ). They act as substrates for is possible that the primary OH group of both of them and of the enzyme and are converted into 2,3- and 22,23-epoxides.

TNSA adopt the same spatial orientation when they bond to IC50 values for the most potent inhibitors (83-85) for pig liver the enzyme, perhaps mimicking the transition state of epoxide SE amount to 10 µ m , 70 µ m and 109 µ m , respectively (Table formation (Sen and Prestwich , 1989b ). Other moderately 6 ) (Bai , 1991 ). active analogs are TNS hydroperoxide (78), TNS thiol (79), TNS cyclopropylamine (80) (Table 6 ) (Sen and Prestwich , Natural compounds and their derivatives Plants, fungi 1989a,b ). and higher animals are a wealthy source of pharmacologically Difl uoroolefi ns are another squalene derivative with SE valuable leading compounds. Over 300 plant extracts, inhibiting potential (81), (82) (Table 6 ) (Moore et al., 1992). mostly of herbs, which according to folk herbalism decrease Both difl uoroolefi n (81) and symmetrical tetrafl uoro ana- cholesterol level, were tested for SE inhibition activity logue (82) are recognized by the enzyme (Moore et al., 1992). (Gurib-Fakim, 2006). Extracts from Agrimonia pilosa

However, the homomethylene (CHC= CF 2 ) and sterically- (Agrimony) , Aleurites fordii (Tung tree), Euphorbia jolkini 1062 A. Belter et al.

Table 6 Anticholesterolemic squalene monooxygenase inhibitors.

Compound Structural formula IC 50 ( µm ) for pig SE Core Substituent (R)

TNSA (73) 4

(74) 1 0 0

(75) > 400

(76) OH 40 R

(77) CH2 CH2 O H 4

(78) CH2 O O H 4

(79) CH2 S H 3 0

(80) CH2 NH-c-C 3 H 5 2

(81) CHCF2 5 . 4

(82) CHCF2 4 . 5 R R

(83) CH2 O H 1 0

R (84) CHO 70

(85) CH2 NH 2 109 Squalene derivatives (Sen and Prestwich, 1989a,b; Sen et al., 1990; Bai, 1991; Jarvi et al., 1991; Moore et al., 1992).

(Euphorbia), Lagerstroemia indica (Crape myrtle) , Myrica have been synthesized (Abe et al., 2000a, 2001). Dodecyl gal- rubra (Chinese strawberry) (IC50 = 1 µ g/ml), Camellia late (DG) (92) showed the strongest inhibition toward recom- sinensis (Chinese camellia), Allium sativum (Garlic), Rheum binant rat and pig SEs (IC 50 = 0.061 µ m). It is about 10 times palmatum (Rhubarb), Cynara scolymus (Artichoke), Fraxinus more potent than previously mentioned, naturally occurring excelsior (European Ash), and Peumus boldus (Boldo) show EGCG (IC50 = 0.69 µm ) and over 1000 times more than the gal- a signifi cant inhibitory potential to SE (Abe et al. , 2000b,c ; lic acid (90) (IC50 = 73 µ m ). Gupta and Porter , 2001 ; Bundy et al. , 2008 ). There are a few putative mechanisms of EGCG’ s health- One of the most thoroughly investigated is extract obtained promoting activity. It exhibits a strong capacity for scavenging from green tea. Its major component (-)-epigallocatechin-3 exogenous H2 O 2 , reactive oxygen and nitrogen species (Frei -O-gallate (EGCG) (86) and a few other polyphenols, e.g., and Higdon , 2003 ) and also protects against them by activation (-)-epicatechin-3-O-gallate (ECG) (87), (-)-epigallocatechin of several cellular antioxidant mechanisms (Higdon and Frei , (EGC) (88), theasinensin A are very effi cient inhibitors of 2003 ). However, some data suggest that EGCG by itself gener- squalene epoxidation (Abe et al. , 2000c ) (Table 7). Other ates H2 O2 participating as mediator in cellular signaling, pro- good prospective natural galloyl compounds, such as gal- liferation, and differentiation (Forman et al. , 2004 ). Other data loyl fl avanols, galloyl proanthocyanidines, galloyl glucoses points out that EGCG causes the generation of superoxide radi- • - and ellagitannins have been found in other plant extracts. cal anions (O2 ), which promote production of hydroxyl radicals Several of them inhibit squalene epoxidation as effi ciently (· OH) in the Fenton reaction. Following this assumption, the as EGCG and they are also a starting point for the synthesis inhibitory effect could be a result of high reactivity of the afore- of new active compounds. Numerous gallic acid derivatives, mentioned radicals (Nakagawa et al., 2004; Elbling et al., 2005). such as alkyl gallates, gallolyl esters of isoprenoids, gallolyl Other reports indicate that EGCG specifi cally inhibits squalene amide and n-alkyl esters of hexahydroxydiphenol (HHDP)- monooxygenase (Abe et al., 2000a ) and telomerase (Noguchi et dicarboxylic acid containing n-alkyl gallate dimeric structure al., 2006 ), and specifi cally regulates the expression of VEGF, Squalene monooxygenase and hypercholesterolemic therapy 1063

matrix metalloproteinases, uPA, IGF-1, EGFR, cell cycle regu- and Neil, 1994; Stevinson et al., 2000) and cell culture stud- latory proteins and inhibits NFk B, PI3-K/Akt, Ras/Raf/MAPK ies (Gebhardt , 1993 ; Cho and Xu , 2000 ; Liu and Yeh , 2000 ) and AP-1 signaling pathways (Shankar et al., 2007). However, show that garlic extract signiﬁ cantly lowers the cholesterol it raises doubts as to whether one compound could regulate so level in plasma. Garlic compounds inhibiting squalene many different and unrelated factors. monooxygenase activity can be divided into three groups: Another noteworthy source of natural SE inhibitors is selenium compounds (Ip et al., 1992, 2000; McSheehy garlic. Data from animal studies (Chi et al. , 1982 ; Quereshi et al., 2000), tellurium compounds (Schroeder et al., 1967) et al., 1983), clinical trials (Adler and Holub, 1997; Bordia and allyl compounds (Gupta and Porter , 2001 ) (Table 8). et al. , 1998 ) , meta-analyses (Warshafsky et al. , 1993 ; Silagy Four selenium compounds [selenocystine (97), selenite (98),

Table 7 Anticholesterolemic squalene monooxygenase inhibitors.

Compound Structural formula Substituent (R/R1/R2) IC50 (µ m )

EGCG (86) OH R1= R2 = OH 0.69 OH

HO O R1

OH R2 O

OH OH ECG (87) R1= H 1.3 R2= OH EGC (88) R1= R2 = OH 3.2 EC (89) OH R = H > 1000 OH

HO O R

OH OH Gallic acid (90) OH -OH 73 OH R

OH OH (91) -O(CH2)7CH3 0.83 (92) -O(CH2)11CH3 0.061 (93) -O(CH2)15CH3 0.59 (94) -NH(CH2)11CH3 3 (95) 3.9

O (96) 0.57

O Natural compounds (Abe et al., 2000b). 1064 A. Belter et al.

selenium dioxide (99), methylselenol (100)] and three tellu- Table 8 Anticholesterolemic squalene monooxygenase inhibitors rium compounds [tellurite (101), tellurium dioxide (102), and from garlic extract (Gupta and Porter, 2001). dimethyltelluride (103)] inhibit SE with IC50 in the micromo- Compound Structural formula IC ( µ m ) lar range (Laden et al., 2000; Gupta and Porter, 2001; Laden 50 and Porter, 2001). The highest potency to inhibit SE are at Selenium compounds O NH 6 5 present: S-allylcysteine (104) (SAC, IC 50 = 110 µ m ), alliin ( 9 7 ) 2

(IC50 = 120 µ m ) from water-soluble fraction and 1,3-diallyl- Se OH HO Se trisulfane (105) (DATS, IC50 = 195 µm ) and 1,2-diallyldisul- fane (106) (DADS, IC = 400 µm ) from lipid-soluble fraction NH O 50 2 (Table 8 ) (Gupta and Porter, 2001). The removal of allyl group ( 9 8 ) HO OH 3 7 results in the loss of inhibitory activity of these compounds Se (Gupta and Porter, 2001). Monothiols and dithiols reverse O the inhibitory activity of selenite and methylselenol, which ( 9 9 ) o = S e = o 3 7 indicates that they react with sulfhydryl groups on protein. ( 1 0 0 ) = S e 9 5 The inhibitory activity of tellurium, selenocystine and garlic Tellurium compounds compounds containing allyl groups could be reversed only by ( 1 0 1 ) HO OH 1 7 Te dithiols which means that they bind to two vicinal cysteines (Gupta and Porter, 2001; Laden and Porter, 2001). However, O selectivity of allyl derivatives towards SE is low. There are ( 1 0 2 ) o = T e = o 1 7 several studies demonstrating that they interact with many ( 1 0 3 ) 0 . 1 Te other proteins (Hanther , 1968 ; Bjornstedt et al. , 1996 ; Barbosa Allyl compounds et al. , 1998 ; Park et al. , 2000 ). Moreover, allyl compounds ( 1 0 4 ) O 110 cross through the blood-brain barrier and inhibit SE in Schwann cells blocking myelin formation, leading as a conse- HO quence to peripheral segmental demyelination and paralysis + NH2 S (Wagner -Recio et al., 1991 ; Ammar and Couri , 1992 ; Stowe O et al., 1992). It excludes them as candidates for cures in mod- ( 1 0 5 ) S S 195 ern therapy. S ( 1 0 6 ) S 400 S Current strategies for lowering cholesterol level in blood Elevated LDL cholesterol levels are a result of perturba- tions in its metabolism (Toutouzas et al., 2010). Cholesterol There has been a long-standing debate regarding the main, homeostasis is a derivative of cholesterol intake, secretion, underlying causes of cardiovascular diseases. It started in resorption and its de novo synthesis (Yamamoto and Bloch , late 1950s, 10 years after the epidemiological approach of 1970; Brown and Goldstein, 1976) and is regulated at both cardiovascular disease was innovated. As a result, a cor- transcriptional and post-transcriptional levels (Goldstein and relation between elevated concentrations of plasma cho- Brown, 1990; Brown and Goldstein, 1997; Kathryn et al., lesterol and the development of coronary heart disease 2010). Understanding the mechanism of controlling choles- (CHD) became apparent (Kannel , 1995 ). It gave a birth to terol homeostasis enables us to choose new targets of hyper- the lipid hypothesis, which suggests that lowering choles- cholesterolemia therapy. terol reduces the risk of coronary events (Newland, 1976). Current strategies to control cholesterol levels in blood are Further investigations indicated that coronary heart dis- based on: (i) restriction of cholesterol intake from the food eases are associated mainly with low-density lipoprotein (diet) (Micallef and Garg , 2009 ), (ii) reduction of cholesterol (LDL) cholesterol, whereas high-density lipoprotein (HDL) and bile acids absorption from the gut (ion-exchange resins cholesterol is inversely correlated with CHD. In 1984, (Ginsberg , 1995 ), microsomal triglyceride transport protein the National Institute of Health (NIH) Coronary Primary (MTP) inhibitors (Cuchel et al. , 2007 ), Niemman-Pick C1 Like Prevention Trial approved that lowering elevated LDL cho- 1 protein inhibitors, (iii) inhibition of de novo cholesterol syn- lesterol with diet and drugs would reduce the risk of CHD thesis (statins, squalene synthase, 2,3-oxidosqualene cyclase, (NIH Consensus Development conference , 1985 ). Further squalene monooxygenase inhibitors) (Endo et al. , 1976 ; trials disclosed that a lowering of plasma total cholesterol Grundy , 1988 ), (iv) inhibition of LDL particles formation (pro- by 10 % and of LDL cholesterol by 40 mg/dl are followed by protein convertase subtilisin/kexin type 9 (PCSK9) antisense a 25 % and 20 % reduction in CHD incidence, respectively oligonucleotides) (Rizzo, 2010), (v) increasing the number (Baigent et al., 2005). Therefore, currently LDL levels are of LDL receptors (enhancers of LDL receptors synthesis and the most common marker in determining cardiovascular risk inhibitors of its degradation) (Boguslawski, 1993 ; Lagace et and LDL particles are the most desired target of hypercho- al. , 2006 ; Graham et al. , 2007 ; Shan et al. , 2008 ), and (vi) rais- lesterolemia therapy (Contoids et al., 2009; Sandeep and ing high-density lipoprotein levels (cholesteryl ester transfer Davidson , 2011 ). protein (CETP) inhibitors) (Barter et al., 2007). All of these are Squalene monooxygenase and hypercholesterolemic therapy 1065

sophisticated but none of them fully cover patients’ demands blocks cholesterol absorption by 54 % and increases the fecal (Katsnelson, 2010 ). There is constant need for new strategies. excretion of neutral sterols by 72% cholesterol without affect- Thorough analysis of strengths and weaknesses of current strat- ing the absorption of triglycerides, bile acids and fat-soluble egies could help to provide direction for further investigations. vitamins (Knopp et al., 2001; van Heek et al., 2001; Sudhop An increase in the availability of new food choices and in et al. , 2002 ). After 2 weeks of treatment (10 mg ezetimide/day) energy intake, sedentary lifestyle, and poor eating habits has LDL-cholesterol decreased by 20.4 % (Sudhop et al. , 2002 ). A led to an increase in obesity and associated metabolic dis- weak correlation between the percent of reduction of LDL- eases. Dietary habits are changing, particulary in regards to cholesterol and cholesterol absorption is the result of boosted the intake of sugar-rich foods and drinks and animal fats (Pan (by 89 % on ezetimide relative to placebo) cholesterol synthe- et al. , 2011 ). Daily cholesterol consumption is approximately sis, which compensates the inhibition of cholesterol absorp- 500 mg and it is even two times higher than average in devel- tion (Sudhop et al., 2002). This rise in cholesterol synthesis oping countries (Callejo Gimenez et al., 2003). Cardiovascular may explain why ezetimide does not reduce cardiovascular prevention must therefore target sedentary lifestyle, excess events (Landmesser et al., 2005; Kastelein et al., 2008). weight, and favor low-calorie, low-fat diet and natural com- However, administration of ezetimibe and its analogs with pounds with potential cardiovascular benefi ts, such as phy- statins (inhibitors of cholesterol synthesis) could give more tosterols, omega-3 fatty acids (Butt et al. , 2009 ; Micallef and benefi ts. It is a highly acclaimed treatment option for patients Garg , 2009 ; Smart et al. , 2011 ). Therapeutic education holds with hypercholesterolemia (Ballantyne et al. , 2003 , Yubin a growing and complementary role in the public health sys- et al. , 2011 ). tem (Baudet et al., 2011). Limitation of cholesterol intake is Statins are the most common and strongest drugs of the primary strategy of regulating cholesterol levels in blood, modern anti-hypercholesterolemic therapy. They inhibit however it is diffi cult to implement to therapy mainly because HMG-CoA reductase, a key enzyme from the cholesterol bio- of deep-rooted bad eating habits. synthesis pathway (Stancu and Sima, 2001). A reduction of The reduction of bile acids resorption from the intestine is de novo cholesterol synthesis results in reduced intracellular another aim of the modern antihypercholesterolemic therapy. cholesterol levels. It causes upregulation of the LDL-receptor Approximately 50 % of consumed cholesterol is absorbed (Reihner et al. , 1990 ), and eventually lowers LDL-cholesterol (Heinemann et al., 1993). Ion-exchange resins, the fi rst hyper- in blood, with an average of 33% , and even by 50% in the case cholesterolemic drugs, trap bile acids and then, are excreted of atorvastatin (80 mg/day) and rosuvastatin (20– 40 mg/day) from body as a complex. It interrupts enterohepatic circula- (LaRosa et al. , 2005 ; Drexel , 2009 ). Some studies suggest that tion of cholesterol and its derivates and as a consequence benefi ts of a treatment with statins may not be entirely due to lowers LDL-cholesterol by 10 – 15 % (Black , 2002 ). They their effects on LDL cholesterol. It is postulated that statins have no major effect on HDL-cholesterol and triglycerides, possess anti-infl ammatory, antioxidant, and antithrombotic however, there is evidence of their moderate benefi cial effect activity (Liao , 2005 ; Ray et al. , 2006 ; Sadowitz et al. , 2010 ). on the cardiovascular endpoint (The Lipid Research Clinics Long statins administration boosts the expression of Coronary Primary Prevention Trial results, 2002). The main HMG-CoA reductase and other enzymes from the choles- disadvantage of resins is a low tolerability because of gastro- terol synthesis pathway, making it necessary to increase the intestinal side effects (Drexel , 2009 ). dose of cure to achieve the therapeutic effect (Brown and There is some evidence that microsomal triglyceride Goldstein, 1979 , 1980 ; Grundy , 1988 ). Inhibition of HMG- transport protein (MTP) could be the target of future therapy CoA reductase results in mevalonate depletion (Naoumova et (Hussain et al., 2011). It is an endosomal protein, which cata- al., 1996), which disturbs the synthesis of isoprenoid deriva- lyzes the assembly of cholesterol, triglycerides, and apoliporo- tives, such as dolichol, ubiquinone (Co-enzyme Q10), heme tein to very low-density lipoprotein (VLDL) or chylomicrons. A, isopentenyl adenosine and prenylated proteins (Figure 4) Inhibition of MTP in the intestine blocks fat absorption and (Schafer and Rine, 1992 ; Ghirlanda et al., 1993). It responds in the liver reduces hepatic secretion of VLDL. It causes sig- with severe side effects, such as, myalgias, muscle cramps, nifi cant LDL cholesterol and triglycerides reductions, but also polyneuropathy myositis and myopathy with incidence of causes hepatic steatosis and other gastrointestinal adverse rhabdomyolysis, neuropathy, and cognitive defects, as well effects, due to a hepatic accumulation of triglycerides (Cuchel as liver and kidney disorders (Edison and Muenke, 2004; et al., 2007; Rizzo, 2010). Still, AEGR-733 and lomitapide, Armitage , 2007 ). Cerivastin as one of the most promising sta- small molecule MTP inhibitors, are being used in some cases tins has caused the deaths of 35 patients and was withdrawn in patients who cannot achieve therapeutic treatment on from the market (Igel et al. , 2001 ). Due to side effects statins the available regimens or suffer from intolerance to statins are not recommended for patients with liver, renal and heart (Davidson , 2009 ). The regulation of MTP takes place at tran- failures, for children, elderly people and pregnant women scriptional, post-transcriptional and post-translational levels (Edison and Muenke , 2004 ; Armitage , 2007 ). by macronutrients, hormones and other factors and could be The administration of ezetimibe with statins helps to suf- an option to reduce plasma lipids (Hussain et al. , 2011 ). fi ciently reduce LDL-cholesterol with lower doses of statins. Ezetimibe, a cholesterol absorption inhibitor, is another For example, an application of 10 mg of ezetimibe to atorvas- promising drug candidate. It is glucuronidated in intestine and tatin allows a reduction in the statin dose from 80 to 10 mg/day liver, localized at the brush border of the small intestine, where (Ballantyle et al., 2003). Therapy combining mode of action of it binds to Niemman-Pick C1 Like 1 protein. This protein these remedies (inhibition of cholesterol synthesis by statin and 1066 A. Belter et al.

cholesterol absorption across the intestinal wall by ezetimibe) inhibitors of its degradation) (Lagace et al. , 2006 ; Grahan et provides substantial incremental reduction in total and LDL- al., 2007; Shan et al., 2008) and other cholesterol transporters cholesterol and triglycerides (Ballantyle et al., 2003). It may (ATP-binding cassette proteins) (Quazi and Molday, 2011), reduce statin doses in clinical practice, which is extremely impor- and raising high-density lipoprotein levels [cholesteryl ester tant taking into account the severe side effect of these agents. transfer protein (CETP) inhibitors (Barter et al., 2007; Kuang- Inhibition of cholesterol synthesis at the stage beyond Yuh, et al., 2011 )]. Most of them are still under investigation. HMG-CoA reductase, downstream of farnesyl pyrophos- Recently miRNAs (miR-33, miR122, miR-370) involved phate is another approach. It could spare isoprenoid synthe- in post-transcriptional regulation of lipid metabolism genes sis, avoid mevalonate depletion, and in turn, statin-derived have been identifi ed. It has opened new possibilities for treat- adverse effects. It is also desirable to block the enzyme, which ment of hypercholesterolemia (Kathryn et al. , 2010 ). is located in the pathway upstream lanosterol biosynthesis in order to avoid the accumulation of intermediate products which are toxic in high concentration. These requirements Safety of hypercholesterolemia therapy are fulfi lled by three enzymes: squalene synthase (Ugawa et al. , 2000 ), 2,3-oxidosqualene cyclase (van Sickle et al. , Therapy based on inhibitors of cholesterol biosynthesis 1992 ) and squalene monooxygenase. through the prism of cellular functions of cholesterol (Simons Squalene synthase catalyzes the reductive dimerization and Ehehalt , 2002 ) and disorders of its synthesis (Hermann , of farnesyl-diphosphate to squalene, thereby diminishes 2003 ), raises some doubts and concerns. Cholesterol is a key hepatic cholesterol synthesis and upregulates LDL receptors. component of cell membranes and the precursor of all steroid However, the safety profi le of currently known squalene syn- hormones and bile acids. It can be converted to oxycholes- thase inhibitors is of concern. They cause elevation of several terol that can act as a regulatory signaling molecule involved liver enzymes levels, probably because farnesyl-diphosphate in numerous developmental processes (Mann and Beachy , is converted into toxic farnesol-derived dicarboxylic acid 2000 ). Additionally, intermediates of cholesterol biosynthesis (Ugawa et al. , 2000 ). It is unlikely that this enzyme will be a pathway are tied to a variety of important cellular functions, target for future drugs (Davison, 2009). especially pre-squalene isoprenoid intermediates, where An advantage of 2,3-oxidosqualene cyclase as a target depletions cause numerous severe dysfunctions as mentioned for hypercholesterolemia therapy is that partial inhibition of above. It confi rms that inhibition of cholesterol synthesis in that enzyme causes accumulation of 2,3-oxidosqualene and the post-squalene half of the pathway, e.g., at the stage of 2,3:22,23-dioxidosqualene. They are converted into 24,25- squalene epoxidation could give better therapeutic effect than epoxycholesterol, which decreases the transcription level using statins. However, defects in post-squalene cholesterol of HMG-CoA reductase gene and stimulates degradation of biosynthesis could cause some dysfunctions as well. Seven this enzyme. Numerous 2,3-oxidosqualene cyclase inhibitors disorders involving post-squalene enzymes ’ defects have have been found (van Sickle et al. , 1992 ), however, none of been described recently – Smith-Lemli-Opitz syndrome, des- them have been approved as clinical drugs for the treatment mosterolosis, X-linked dominant chondrodysplasia punctate, of hypercholesterolemia because of the toxicological issues CHILD syndrome, lathosterolosis, Greenberg skeletal dyspla- (Watanabe et al., 2010 ). sia and Antley-Bixler syndrome (Hermann, 2003). The patho- Among the enzymes from cholesterol synthesis pathway genesis of these disorders results mainly from accumulation squalene monooxygenase seems to be the best candidate for of toxic sterol intermediates above each enzyme block and a target of hypercholesterolemia therapy. SE inhibition does abnormal feedback regulation of earlier steps in the pathway, not cause the cytotoxic effect, typical for the inhibition of including the synthesis of key isoprenoid compounds, not from enzymes from cholesterol biosynthesis pathway downstream reduced plasma cholesterol levels. It is thought, that lowered lanosterol synthesis (Fernandez et al., 2005). Cholesterol- below average plasma cholesterol levels do not respond for dependent degradation of squalene monooxygenase proves symptoms, such as growth and mental retardation, poor feed- that this enzyme is a control point in cholesterol synthesis, ing in infancy, skeletal abnormalities, cholestatic liver dis- beyond HMG-CoA reductase (Gill et al. , 2011 ). ease, renal anomalies, congenital sensorineural hearing loss, The effective therapy of reduction cholesterol level should structural CNS malformations, and embryonic lethality typi- combine several approaches. It should be as effective as statins cal for some of these disorders (Hermann, 2003). In prelimi- in the reduction of cholesterol de novo synthesis, but devoid nary experiments, it has been shown that cholesterol levels of their side effects. The best way to meet these requirements in tissues in embryos bearing defects related with cholesterol is to inhibit the squalene monooxygenase activity or silence synthesis near the time of death are normal, presumably as its gene transcription supported by other approaches such as, a result of its placental transport from the mother (Caldas diet, inhibition of cholesterol and bile acids absorption, reduc- et al., 2005, Hermann, unpublished). It confi rms the validity tion of LDL particles formation by antisense oligonucleotides of assumptions that therapy based on inhibitors of cholesterol directed against proprotein convertase subtilisin/kexin type 9 synthesis could be safe. That is because cellular cholesterol (PCSK9) (Frank-Kemenetsky et al. , 2008 ; Rizzo , 2010 ) or depletion is a result of diminished cholesterol synthesis and against apolipoprotein B-100 (apoB) – mipomersen (Crooke is supplemented by cholesterol from plasma. It helps to main- et al. , 2005 ; Patel and Hegele , 2010 ), increasing number of tain tissues in proper conditions, and reduce cholesterol levels LDL receptors (enhancers of LDL receptors synthesis and in plasma, which allows a therapeutic effect to be achieved. Squalene monooxygenase and hypercholesterolemic therapy 1067

S CoA

O 2-acetyl-CoA

HMG-CoA reductase O S CoA HO OH O HMG-CoA

O OH HO OH Mevalonic acid

OPPi Isopentenyl adenosine (tRNA) Isopentenyl pyrophosphate

OPPi Geranyl pyrophosphate

Prenylated proteins

Ubiquinone OPPi Fernesyl pyrophosphate Heme A

Squalene synthase Dolichol

Squalene

Squalene monooxygenase

O 2,3-oxidosqualene

Oxidosqualene cyclase

OH Lanosterol

OH Cholesterol

Figure 4 Main steps in cholesterol biosynthesis pathway. Enzymes catalyzing successive stages of cholesterol synthesis pathway, important from the point of hypercholesterolemia therapy are distinguished. On the right: derivatives of mevalonic acid. 1068 A. Belter et al.

Substrate accumulation is a major danger in enzyme inhi- cellular membranes, precursor of oxysterols, steroid hormones bition therapy. Inhibition of squalene monoxygenase results and bile acids. It regulates embryogenesis and development in squalene accumulation. Although some reports suggest (Xu et al. , 2005 ). Cellular cholesterol is strictly controlled by that excessive intake of squalene (20 g/kg/day) causes neural multiple pathways (Chang et al. , 2006 ; Goldstein et al. , 2006 ). damage in rats (Gajkowska et al. , 1999 ), and lipoid pneumo- The disturbance of its synthesis causes several severe dysfunc- nia in a human patient (Asnis et al., 1993), there is generally tions. Up until now, eight distinct inherited disorders as a result thought that squalene has a positive effect on human health of defects in the isoprenoid/cholesterol biosynthetic pathways (Kelly , 1999 ). About 60 % of dietary squalene is absorbed have been identifi ed (Waterham, 2006). Patients show a broad and up to 90 % of it is transported in serum (Strandberg et al. , spectrum of severe symptoms, which are a result of abnor- 1990 ). Even though a high intake of dietary squalene (200 – mally increased/decreased levels of intermediate metabolites, 400 mg/day in the Mediterranean countries in comparison but not reduced cholesterol levels (Waterham, 2006). to USA – 30 mg/day) increases its concentration in plasma High cholesterol concentration has been recognized as and a part of its pool is converted into cholesterol, a positive one of the most important predictors of arthrosclerosis and effect of high intake of squalene on LDL and triglycerides coronary heart disease. The far-reaching consequences of (TG) levels has been proved (Strandberg et al., 1990). It was dyslipidemia have led to heart disease, diabetes, obesity, the shown that consumption of 860 mg squalene daily for 7 – 30 metabolic syndrome, stroke, and non-alcoholic fatty liver days increased its concentration 17 times in blood. However, disease (Pearson, 2004). Additionally, accumulation of cho- the cholesterol level was affected only slightly. Longer ther- lesterol and other lipids in late endosomes/lysosomes causes apy, up to 20 weeks, reduced cholesterol, LDL and TG con- neuronal degradation, and it is known as Niemann-Pick dis- centrations of 17 % , 22 % and 5 % , respectively (Chan et al. , ease type C (Arora et al., 2010). So the main goal for thera- 1996). Squalene signifi cantly enhances the fecal excretions peutic intervention is normalization of cholesterol levels in of cholesterol, its non-polar derivatives and bile acids. At the the blood. There is abundant evidence that the reduction of same time it causes, due to negative feedback regulation, an cholesterol concentration lowers morbidity and mortality inhibition of HMG-CoA reductase activity (Strandberg et al. , from the mentioned diseases (Downs et al. , 1998 ). 1990 ). Finally, squalene potentiates the cholesterol-lowering Currently, the most popular drugs for treatment of effect of statins, e.g., pravastatin (Chan et al. , 1996 ). hypercholesterolemia, which have dominated the lipids Squalene has an excellent safety profi le (Christian, 1982; therapy market, are statins, HMG-CoA reductase inhibi- Fox , 2009 ). Injected squalene (21 mg) into over million cases tors. Although they are effective in most cases, they cause (including infants) showed very low incidence rate of side several adverse effects as well. Studies on new approaches effects (mostly pain at injection site) (Ketomaki et al., 2004). of lowering cholesterol levels are highly desired. There is Nearly 10 years ago antibodies to squalene were detected in some evidence that squalene monooxygenase could be the the blood of veterans of the Gulf War (Gronseth , 2005 ). As target of future hypercholesterolemia therapy. Inhibitors a result a discussion about the safety of using squalene as of this enzyme act further downstream in the cholesterol an adjuvant of vaccines arose. However, further clinical tri- pathway than statins, which prevent the disturbance in als proved that squalene is poorly immunogenic (Lippi et al., the synthesis of mevalonate derivatives, such as dolichol, 2001). Squalene’ s safety profi le as a vaccine adjuvant is well ubiquinone, heme A, and isoprenylated proteins. It avoids documented (Schultze et al. , 2008 ). the side effects caused by their depletion which is typical A serum pool of squalene is distributed ubiquitously in tis- for therapy with statins. sue (Strandberg et al., 1990). The greatest concentration of Up until now several potent squalene monooxygenase squalene is in the skin, where it functions as a quencher of inhibitors, e.g., allylamines [FR194738, NB-598, theirs singlet oxygen, protecting it against exposure to UV and other aryloxy(methylsilane)], squalene analogs (trisnorsqualene sources of radiation (Kelly, 1999). As a result of prolonged, alcohol), and several natural, selenium and tellurium com- high-dose oral administration it could also accumulate in the pounds, have been found. They decrease LDL levels in a dose- liver (Kamimura et al. , 1989 ). Metabolic studies of squalene dependent manner in HepG2 cells, L6 myoblasts, rat, hamster, in tissues indicate that about 80 % of cellular squalene is met- and dogs similarly or even greater than statins (Hidaka et al., abolically inactive and deposited in a neutral lipid droplet, 1991 ; Chugh et al. , 2003 ). Additionally, they decrease tri- while the remaining 20 % is bound to the microsomal mem- glyceride and increase HDL levels and do not affect HMG- branes. Additionally, only 10% of the newly synthesized CoA reductase activity (Chugh et al. , 2003 ). However, in the squalene is used in cholesterol synthesis (Tilvis et al. , 1982 ). case of a high dose of tellurium compounds, their benefi cial Therefore, squalene accumulated in response to squalene features in SE inhibition were quenched by an unsatisfac- monooxygenase inhibition is non-toxic for cells even at high tory safety profi le. Their increased ( > 0.1 % of diet) prolonged concentration (Chan et al. , 1996 ). exposure causes demyelination of the pheripheral nervous system, possibly due to decreased cholesterol synthesis, but also by downregulation of mRNA level of P0 protein being Conclusions a crucial component of myelin (Katsuno et al., 2009). Due to the blood-brain barrier the level of brain cholesterol is inde- Cholesterol plays a crucial role in all eukaryotic cells (Bloch, pendent from fl uctuations of the pool circulating in blood as a 1979 ). In vertebrates, it is an important structural component of result of diet or medication. Hence, it is possible to decrease Squalene monooxygenase and hypercholesterolemic therapy 1069

its level in serum without changes in the brain. It is extremely Asnis, D.S., Saltzman, H.P., and Melchert, A. (1993). Shark oil pneu- important taking into account the fact that low cholesterol monia. An overlooked entity. Chest 103 , 976 – 977. amount in the brain is supposed to be responsible for a range Bai, M. (1991). Purifi cation and Characterization of Squalene of neurodegenerative disorders (Katsuno et al., 2009). This Epoxidase, PhD Thesis, State University of New York, Stony SE inhibitor showed dermatosis-like toxicity in dogs, possi- Brook, USA. Bai, M. and Prestwich, G.D. (1992). Inhibition and activation of por- bly due to the accumulation of squalene in skin cells (Horie cine squalene epoxidase. Arch. Biochem. Biophys. 293 , 305 – 313. et al., 1991). Additionally, antibodies to squalene have been Baigent, C., Keech, A., Kearney, P.M., Blackwell, L., Buck, G., detected in the blood of veterans of the Gulf War (Gronseth, Pollicino, C., Kirby, A., Sourjina, T., Peto, R., Collins, R., et al. 2005 ), however, eventually allegations of safety concerns (2005). Cholesterol Treatment Trials ’ (CTT) Collaborators, effi - were rejected (Lippi et al. , 2010 ). Taking into account the cacy and safety of cholesterol lowering treatment: prospective healthy properties of squalene there is a chance that squalene meta-analysis of data from 90056 participants in 14 randomised monooxygenase could be an alternative approaches to old trials of statin. Lancet 366 , 1267 – 1278. targets in hypercholesterolemia therapy (Chugh et al., 2003). Ballantyne, C.M., Houri, J., Notarbartolo, A., Melani, L., Lipka, Previous results hold promise for the prevention of cardio- L.J., Suresh, R., Sun, S., LeBeaut, A.P., Sager, P.T., and Veltri, vascular diseases with squalene monooxygenase inhibitors, E.P. (2003). Ezetimibe Study Group, effect of ezetimibe coad- however, clinical trials need to be carried out. ministrated with atorvastatin in 628 patients with primary hypercholesterolemia: a prospective, randomized, double-blind trial. Circulation 107 , 2409 – 2415. Barbosa, N.B.V., Rocha, J.B.T., Zeni, G., Emanullei, T., Beque, M.C., Acknowledgments and Braga, A.L. (1998). Effect of organic forms of selenium on δ-aminolevulinate dehydrogenase from liver, Sidney, and brain This work was supported by European Commission grant within 7 of adult rats. Toxicol. Appl. Pharmacol. 149 , 243 – 253. Framework Project (OxyGreen; KBBE-2007-212281) and the Polish Barter, P.J., Caulfi eld, M., Eriksson, M., Grundy, S.M., Kastelein, J.J., Ministry of Education and Science. Komajda, M., Lopez-Sendon, J., Mosca, L., Tardif, J.C., Waters D.D., et al. (2007). Effects of torcetrapib in patients at high risk for coronary events. N. Engl. J. Med. 357 , 2109 – 2122. References Baudet, M., Daugareil, C., and Ferrieres, J. (2011). Cartiovascular disease prevention and life hygiene modifi cations. Ann. Abe, I., Abe, T., Lou, W., Masuoka, T., and Noguchi, H. (2007). Cardiol. Angeiol. (Paris). Epub ahead of print. Doi:10.1016/j. Site-directed mutagenesis of conserved aromatic residues in rat ancard.2011.05.007. squalene epoxidase. Biochem. Biophys. Res. Commun. 352 , Berg, D. and Plempel, M. (1989). Inhibitors of fungal sterol syn- 259 – 263. thesis: squalene epoxidation and C14-demethylation. J. Enzyme Abe, I., Kashiwagi, Y., Noguchi, H., Tanaka, T., Ikeshiro, Y., and Inhib. 3 , 1 – 11. Kashiwada, Y. (2001). Ellagitannins and hexahydroxydiphenoyl Bjornstedt, M., Odlander, B., Kuprin, S., Klaesson, H.E., and esters as inhibitors of vertebrate squalene epoxidase. J. Nat. Prod. Holmgren, A. (1996). Selenite incubated with NADPH and 64 , 1010 – 1014. mammalian thioredoxin reductase fi elds selenie, which inhibits Abe, I., Seki, T., and Noguchi, H. (2000a). Potent and selective lipoxygenase and changes the electron spin resonance spectrum inhibition of squalene epoxidase by synthetic galloyl esters. of active site iron. Biochemistry 35 , 8511 – 8516. Biochem. Biophys. Res. Commun. 270 , 137 – 140. Black, D.M. (2002). Gut-acting drugs for lowering cholesterol. Curr. Abe, I., Seki, T., Noguchi, H., and Kashiwada, Y. (2000b). Galloyl Atheroscler. Rep. 4 , 71 – 75. esters from rhubarb are potent inhibitors of squalene epoxi- Bloch, K.E. (1979). Speculations on the evolution of sterol structure dase, a key enzyme in cholesterol biosynthesis. Planta Med. 66 , and function. CRC Crit. Rev. Chem. 7 , 1 – 5. 753 – 756. Boguslawski, W. (1993). Structure and function of LDL receptors Abe, I., Seki, T., Umehara, K., Miyase, T., Noguchi, H., Sakibara, in familial hypercholesterolemia. Postepy Hig. Med. Dosw. 47 , J., and Ono, T. (2000c). Green tea polyphenols: novel and 55 – 65. potent inhibitors of squalene epoxidase. Biochem. Biophys. Res. Bordia, A., Verma, S.K., and Srivastava, K.C. (1998). Effect of Commun. 268 , 767 – 771. garlic (Allium sativum) on blood lipids, blood sugar, fi brinogen Abe, I., Tomesch, I., Wattanasin, S., and Prestwich G.D. (1994). and fi brinolytic activity in patients with coronary artery disease. Inhibitors of squalene biosynthesis and metabolism. Nat. Prod. Prostaglandins Leukot Essent. Fatty Acids 58 , 257 – 263. Rep. 11 , 279 – 302. Bundy, R., Walker, A.F., Middleton, R.W., Wallis, C., and Simpson, Adler, A.J. and Holub, B.J. (1997). Effect of garlic and fi sh-oil sup- H.C.R. (2008). Artichoke leaf extracts (Cynara scolymus ) plementation on serum lipid and lipoprotein concentrations in reduces plasma cholesterol in otherwise healthy hypercholes- hypercholesterolemic men. Am. J. Nutr. 65 , 445 – 450. terolemic adults: a randomized, double blind placebo controlled Ammar, E.M. and Couri, D. (1992). Acute toxicity of sodium sel- trial. Phytomedicine 15 , 668 – 675. enite and selenomethionine in mice after ICV or IV administra- Brown, M.S. and Goldstein, J.L. (1979). A receptor-mediated path- tion. Neurotoxicology 2 , 383 – 386. way for cholesterol metabolism. Science 191 , 150 – 154. Armitage, J. (2007). The safety of statins in clinical practice. Lancet Brown, M.S. and Goldstein, J.L. (1980). Multivalent feedback regu- 370 , 1781 – 1790. lation of HMG-CoA reductase, a control mechanism coordinating Arora, S., Beaudry, C., Bisanz, K.M., Sima, C., Kieler, J.A., and isoprenoid synthesis and cell growth. J. Lipid Res. 21 , 505 – 517. Azorsa, D.O. (2010). A High-content rnai-screening assay to Brown, M.S. and Goldstein, J.L. (1997). The SREBP pathway: regu- identify modulators of cholesterol accumulation in Niemann- lation of cholesterol metabolism by proteolysis of a membrane- Pick type C cells. Assay Drug Dev. Technol. 8 , 295 – 320. bound transcription factor. Cell 89 , 331 – 340. 1070 A. Belter et al.

Butt, M.S., Sultan, M.T., Butt, M.S., and Iqbal, J. (2009). Garlic: Eggink, G., Engel, H., Vriend, G., Terpsta, P., and Witholt, B. (1995). nature ’ s protection against physiological threats. Crit. Rev. Food Rubredoxin reductase of Pseudomonas oleovorans . Structural Sci. Nutr. 49 , 538 – 551. relationship to other fl avoprotein oxidoreductases based on one Caldas, H., Cunningham, D., Wang, X., Jiang, F., Humphries, L., NAD and two FAD fi ngerprints. J. Mol. Biol. 212 , 135 – 142. Kelley, R.I., and Herman, G.E. (2005). Placental defects are Elbling, M., Weiss, R.M., Teufelhofer, O., Uhl, M., Knasmueller, associated with male lethality bare patches and striated embryos S., Schulte-Hermann, R., Berger, W., and Micksche, M. (2005). defi cient in the NAD(P)H steroid dehydrogenase-like (NSDHL) Green tea extract and (-)-epigallocatechin-3-gallate, the major enzyme. Mol. Genet. Metab. 84 , 48 – 60. tea catechin, exert oxidant but lack antioxidant activities. FASEB Callejo Gimenez, E., Iglesias Bonilla, P., Lapetra Peralta, J., Santos J. 19 , 807 – 809. Lozano, J.M., Mayoral Sanchez, E., and Lopez Lopez, B. (2003). Endo, A., Kuroda, M., and Tanzawa, K. (1976). Competitive inhi- [Dietary habits of the population of an urban health district]. bition of 3-hydroxy-3-methyglutaryl coenzyme A reductase by Aten. Primaria 31 , 421 – 427. ML-236A and ML-236B fungal metabolites, having hypocholes- Chan, P., Tomlinson, B., Lee, C.B., and Lee, Y.S. (1996). Effective- terolemic activity. FEBS Lett. 72 , 323 – 326. ness and safety of low-dose pravastatin and squalene, alone and Entsh, B. and van Berkel, W.J.H. (1995). Structure and mechanism in combination, in elderly patients with hypercholesterolemia. of para-hydroxybenzoate hydroxylase. FASEB J. 9 , 476 – 483. J. Clin. Pharmacol. 36 , 422 – 427. Favre, B. and Ryder, N.S. (1996). Characterization of squalene Chang, T.Y., Chang, C.C., Ohgami, N., and Yamauchi, Y. (2006). epoxidase activity from the dermatophyte trichophyton rubrum Cholesterol sensing, traffi cking, and esterifi cation. Annu. Rev. and its inhibition by terbinafi ne and other antimycotic agents. Cell Dev. Biol. 22 , 129 – 157. Antimicrob. Agents Chemother. 40 , 443 – 447. Chi, M.S., Koh, E.T., and Stewart, T.J. (1982). Effect of garlic on lipid Favre, B. and Ryder, N.S. (1997). Differential inhibition of fungal metabolism in rats fed cholesterol or lard. J. Nutr. 112 , 241 – 248. and mammalian squalene epoxidase by the benzylamine SDZ Cho, B.H.S. and Xu, S. (2000). Effects of allyl mercaptan and various SBA 586 in comparison with the allylamine terbinafi ne. Arch. allium-derived compounds on cholesterol synthesis and secretion Biochem. Biophys. 340 , 265 – 269. in Hep-G2 cells. Comp. Biochem. Physiol. C 126 , 195 – 201. Fernandez, C., Martin, M., Gomez-Coronado, D., and Lasuncion, Christian, M.S. (1982). Final report on the safety assessment of M.A. (2005). Effects of distal cholesterol biosynthesis inhibitors squalene and squalene. Int. J. Toxicol. 1 , 37 – 56. on cell proliferation and cell cycle progression. J. Lipid Res. 46 , Chugh, A., Ray, A., and Gupta, J. (2003). Squalene epoxidase as hypo- 920 – 929. cholesterolemic drug target revisited. Prog. Lipid Res. 42 , 37 – 50. Forman, H.J., Fukuto, J.M., and Torres, M. (2004). Redox signaling: Contoids, J.H., McConnell, J.P., Sethi, A.A., Csako, G., Devaraj, S., thiol chemistry defi nes which reactive oxygen and nitrogen spe- Hoefner, D.M, and Warnick, G.R. (2009). Apolipoprotein B and cies can act as second messengers. Am. J. Physiol. Cell Physiol. cardiovascular disease risk: position statement from the AACC 287 , C246 – C256. Lipoproteins and Vascular Diseases Division Working Group on Fox, C.B. (2009). Squalene emulsions for parenteral vaccine and Best Practices. Clin. Chem. 55 , 407 – 419. drug delivery. Molecules 14 , 3286 – 3312. Corey, E.J. and Russey, W.E. (1966). Metabolic fate of 10,11-dihy- Frank-Kemenetsky, M., Grefhorst, A., Anderson, N.N., Racie, T.S., drosqualene in sterol-producing rat liver homogenate. J. Am. Bramlage, B., Akinc, A., Butler, D., Charisse, K., Dorkin, R., Chem. Soc. 88 , 4751 – 4752. Fan, Y., et al. (2008). Therapeutic RNAi targeting PCSK9 acutely Crooke, R.M., Graham. M.J., Lemonidis, K.M., Whipple, Ch.P., lowers plasma cholesterol in rodents and LDL cholesterol in non- Koo, S., and Perera, R.J. (2005). An apolipoprotein B antisense human primates. PNAS 105 , 11915 – 11920. oligonucleotide lowers LDL cholesterol in hyperlipidemic mice Frei, B. and Higdon, J.V. (2003). Antioxidant activity of tea poly- without causing hepatic steatosis. J. Lipid Res. 46 , 872 – 884. phenols in vivo: evidence from animal studies. J. Nutr. 133 , Cuchel, M., Bloedon, L.T., Szapary, P.O., Kolansky, D.M., Wolfe, 3275S – 3284S. M.L., Sarkis, A., Millar, J.S., Ikewaki, K., Siegelman, E.S., Gajkowska, B., Smialek, M., Ostrowski, R.P., Piotrowski, P., and Gregg, R.E., et al. (2007). Inhibition of microsomal triglycer- Frontczak-Baniewicz, M. (1999). The experimental squalene ide transfer protein in familiar hypercholesterolemia. N. Engl. J. encephaloneuropathy in the rat. Exp. Toxicol. Pathol. 51 , 75 – 80. Med. 356 , 148 – 156. Gebhardt, R. (1993). Multiple inhibitory effects of garlic extracts on Davidson, M.H. (2009). Novel nonstatin strategies to lower low-den- cholesterol biosynthesis in hepatocytes. Lipids 28 , 613 – 619. sity lipoprotein cholesterol. Curr. Atheroscler. Rep. 11 , 67 – 70. Georgopoulos, A., Petranyi, G., Mieth, H., and Drews, J. (1981). In Denner-Ancona, P., Bai, M., Lee, H.K., Abe, I., and Prestwich, G.D. vitro activity of naftifi ne, a new antifungal agent. Antimicrob. (1995). Purifi cation of pig and rat squalene epoxidase by affi nity Agents Chemother. 19 , 386 – 389. chromatography. Bioorg. Med. Chem. Lett. 5 , 481 – 486. Ghirlanda, G., Oradei, A., Manto, A., Lippa, S., Uccioli, L., Caputo, Downs, J.R., Clearfi eld, M., Weis, S., Whitney, E., Shapiro, D.R., Beere, S., Greco, A.V., and Littarru, G.P. (1993). Evidence of plasma P.A., Langendorfer, A., Stein, E.A., Kruyer, W., and Gotto, A.M.Jr. CoQ10-lowering effect by HMG-CoA reductase inhibitors: a (1998). Primary prevention of acute coronary events with lovas- double-blind, placebo-controlled study. J. Clin. Pharmacol. 33 , tatin in men and women with average cholesterol levels: results 226 – 229. of AFCAPS/TexCAPS. Air Force/Texas Coronary Atherosclerosis Ghisla, S. and Massey, V. (1989). Mechanism of fl avoprotein-cata- Prevention Study. J. Am. Med. Assoc. 279 , 1615 – 1622. lyzed reactions. Eur. J. Biochem. 181 , 1 – 17. Drexel, H. (2009). Statins, fi brates, nicotinic acid, cholesterol absorp- Gill, S., Stevenson, J., Kristiana, I., and Brown, A.J. (2011). tion inhibitors, anion-exchange resins, omega-3 fatty acids: Cholesterol-dependent degradation of squalene monooxyge- which drugs for which patients ? Fundam. Clin. Pharmacol. 23 , nase, a control point in cholesterol synthesis beyond HMG-CoA 687 – 692. reductase. Cell Met. 13 , 260 – 273. Edison, R.J. and Muenke, M. (2004). Central nervous system and Ginsberg, H.N. (1995). Update on the treatment of hypercholester- limb anomalies in case reports of fi rst-trimester statin exposure. olemia, with a focus on HMG-CoA reductase inhibitors and com- N. Engl. J. Med. 350 , 1579 – 1582. bination regimens. Clin. Cardiol. 18 , 307 – 315. Squalene monooxygenase and hypercholesterolemic therapy 1071

Godio, R.P., Fouces, R., and Martin, J.F. (2007). A squalene epoxi- Horie, M., Hayashi, M., Satoh, T., Hotta, H., Nagata, Y., and Ishida, dase in involved in biosynthesis of both the antitumor compound F. (1993). An inhibitor of squalene epoxidase, NB-598, sup- clavic acid and sterols in the Basidomycete H. sublateritium . presses the secretion of cholesterol and triacylglycerol and simul- Chem. Biol. 14 , 1334 – 1346. taneously reduces apolipoprotein B in HepG2 cells. Biochim. Goldstein, J.L. and Brown, M.S. (1990). Regulation of the Biophys. Acta 1168 , 45 – 51. mevalonate pathway. Nature 343 , 425 – 430. Hussain, M.M., Nijstad, N., and Franceschini, L. (2011). Regulation Goldstein, J.L., DeBose-Boyed, R.A., and Brown, M.S. (2006). of microsomal triglyceride transfer protein. Clin. Lipidol. 6 , Protein sensors for membrane sterols. Cell 124 , 35 – 46. 293 – 303. Gotteland, J.-P., Brunel, I., Gendre, F., Desire, J., Delhon, A., Igel, M., Sudhop, T., and von Bergmann K. (2001). Metabolism Junquero, D., Oms, P., and Halazy, S. (1995). (Aryloxy)meth- and drug interactions of 3-hydroxy-3-methylglutaryl coenzyme ylsilane derivatives as new cholesterol biosynthesis inhibi- A-reductase inhibitors (statins). Eur. J. Clin. Pharmacol. 57 , tors: synthesis and hypercholesterolemic activity of a new 357 – 364. class of squalene epoxidase inhibitors. J. Med. Chem. 38 , Ip, C., Lisk, D.J., and Stoewsand, G.S. (1992). Mammary cancer 3207 – 3216. prevention by regular garlic and selenium-enriched garlic. Nutr. Gotteland, J.-P., Dax, C., and Halazy, S. (1997). Design and synthe- Cancer 17 , 279 – 286. sis of new potential photoaffi nity labels for mammalian squalene Ip, C., Birringer, M., Block, E., Kotrebai, M., Tyson, J.F., Uden, P.C., epoxidase. Bioorg. Med. Chem. Lett. 7 , 1153 – 1156. and Lisk, D.J. (2000). Comparative speciation infl uences com- Graham, M.J., Lemonidis, K.M., Whipple, C.P., Subramaniam, A., parative activity of selenium-enriched garlic and yeast in mam- Monia, B.P., Crooke, S.T., and Crooke, R.M. (2007). Antisense mary cancer prevention. J. Agric. Food Chem. 48 , 2062 – 2070. inhibition of proprotein convertase subtilisin/kexin type 9 Jarvi, E.T., McCarthy, J.R., and Edwards, M.L. (1991). Eur. Pat. reduces serum LDL in hyperlipidemic mice. J. Lipid Res. 48 , Appl. EP 0448934A2. 763 – 768. Joosten, V. and van Berkel, W.J.H. (2007). Flavoenzymes. Curr. Gronseth, G.S. (2005). Gulf war syndrome: a toxic exposure ? A sys- Opin. Chem. Biol. 11 , 195 – 202. tematic review. Neurol. Clin. 23 , 523 – 540. Kamimura, H., Koga, N., Oguri, K., Yoshimura, H., Inoue, H., Sato, Grundy, S.M. (1988). HMG-CoA reductase inhibitors for treatment K., and Ohkubo, M. (1989). Studies on distribution, excretion of hypercholesterolemia. N. Engl. J. Med. 319 , 24 – 33. and subacute toxicity of squalene in dogs. Fukuoka Igaku Zasshi Gupta, N. and Porter, T.D. (2001). Garlic and garlic-derived com- 80 , 269 – 280. pounds inhibit human squalene monooxygenase. J. Nutr. 131 , Kan, V.L., Henderson, D.K., and Bennett, J.E. (1986). Bioassay 1662 – 1667. for SF 86-327, a new antifungal agent. Antimicrob. Agents Gurib-Fakim, A. (2006). Medicinal plants: traditions of yesterday Chemother. 30 , 628 – 629. and drugs of tomorrow. Mol. Aspects Med. 27 , 1 – 93. Kannel, W.B. (1995). Clinical misconceptions dispelled by epide- Han, J.-Y., In, J.-G., Kwon, Y.-S., and Choi, Y.-E. (2010). Regulation miological research. Circulation 92 , 3350 – 3360. of ginsenoside and phytosterol biosynthesis by RNA interferences Kastelein, J.J., Akdim, F., Stroes, E.S., Zwinderman, A.H., Bots, of squalene epoxidas gene in Panax ginseng. Phytochemistry 71 , M.L., Stalenhoef, A.F., Visseren, F.L., Sijbrands, E.J., Trip, 36 – 46. M.D., Stein, E.A., et al. (2008). Simvastatin with or without Hanther, H.E. (1968). Selenotrisulfi des. Formation by the reaction of ezetimibe in familial hypercholesterolemia. N. Engl. J. Med. thiols with selenious acid. Biochemistry 7 , 2898 – 2905. 358 , 1431 – 1443. He, F., Zhu, Y., He, M., and Zhang, Y. (2008). Molecular cloning Kathryn, J.M., Katey, J.R., Yajaira, S., and Carlos F.H. (2010). and characterization of the gene encoding squalene epoxidase in MicroRNAs and cholesterol metabolism. Trends Endocrinol. Panax notogineseng . DNA Seq. 19 , 270 – 273. Metabol. 21 , 699 – 706. Heinemann, T., Axtmann, G., and von Bergmann, K. (1993). Katsnelson, A. (2010). Good news for ‘ good ’ cholesterol. Nature Comparison of intestinal absorption of cholesterol with different 468 , 354. plant sterols in man. Eur. J. Clin. Invest. 23 , 827 – 831. Katsuno, M., Adachi, H., and Sobue, G. (2009). The case for choles- Hermann, G.E. (2003). Disorders of cholesterol biosynthesis: proto- terol. Nature Medicine 15 , 253 – 254. typic metabolic malformation syndromes. Hum. Mol. Gen. 12 , Kelly, G.S. (1999). Squalene and its potential clinical uses. Altern. R75 – R88. Med. Rev. 4 , 29 – 36. Hidaka, Y., Hotta, H., Nagata, Y., Iwasawa, Y., Horie, M., and Ketomaki, A., Gylling, H., and Miettinem, T.A. (2004). Removal of Kamei, T. (1991). Effect of a novel squalene epoxidase inhibitor, intravenous intralipid in patients with familial hypercholester- NB-598, on the regulation of cholesterol metabolism in HepG2 olemia during inhibition of cholesterol absorption and synthesis. cell. J. Biol. Chem. 266 , 13171 – 13177. Clin. Chim. Acta 344 , 83 – 93. Hieber, A.D., Bugos, R.C., and Yamamoto, H.Y. (2000). Plant Knopp, R., Bays, H., and Manion, C. (2001). Effect of ezetimibe lipocalins: violaxanthin de-epoxidase and zeaxanthin epoxidase. on serum concentrations of lipids-soluble vitamins. Atheroscler. Biochim. Biophys. Acta 1482 , 84 – 91. Suppl. 2 , 90. Higdon, J.V. and Frei, B. (2003). Tea catechins and polyphenols: Kuang-Yuh, C., Anish, P., and Prediman, K.S. (2011). Progress in health effects, metabolism, and antioxidant functions. Crit. Rev. HDL-based therapies for atherosclerosis. Curr. Atheroscler. Rep. Food Sci. Nutr. 43 , 89 – 143. 13 , 405 – 412. Horie, M., Tsuchiya, Y., Hayashi, M., Iida, Y., Iwasawa, Y., Nagata, Laber R., Fuchsbichler, S., Klobunikova, V., Schweighofer, N., Y., Sawasaki, Y., Fukuzumi, H., Kitani, K., and Kamei, T. (1990). Pitters, E., Wohlfarter, K., Lederer, M., Landl, K., Ruckenstuhl, NB-598: a potent competitive inhibitor of squalene epoxidase. Ch., Hapala, I. et al. (2003). Molecular mechanism of resistance J. Biol. Chem. 265 , 18075 – 18078. to terbinafi ne in Saccharomyces cerevisiae. Antimicrob. Agents Horie, M., Sawasaki, Y., Fukuzumi, H., Watanabe, K., Iizuka, Y., Chemother. 47 , 3890 – 3900. Tsuchiya, Y., and Kamei, T. (1991). Hypolipidemic effects of Laden, B.P., Tang, Y. and Porter, T.D. (2000). Cloning, hetero- NB-598 in dogs. Arteriosclerosis 88 , 183 – 192. logous expression, and enzymological characterization of 1072 A. Belter et al.

human squalene monooxygenase. Arch. Biochem. Biophys. 374 , gene in dermatophyte pathogen Trichophyton tonsurans . Iran. 381 – 388. Biomed. J. 12 , 55 – 58. Laden, B.P. and Porter, T.D. (2001). Inhibition of squalene monoox- Nagumo, A., Kamei, T., Sakakibara, J., and Ono, T. (1995). ygenase by tellurium compounds: evidence of interaction with Purifi cation and characterization of recombinant squalene epoxi- vicinal sulfhydryls. J. Lipid Res. 42 , 235 – 240. dase. J. Lipid Res. 36 , 1489 – 1497. Lagace, T.A., Curtis, D.E., Garuti, R., McNutt, M.C., Park, S.W., Nakagawa, H., Hasumi, K., Woo, J.T., Nagai, K., and Wachi, M. Prather, H.B., Anderson, N.N., Ho, Y.K., Hammer, R.E., and (2004). Generation of hydrogen peroxide primarily contributes Horton, J.D. (2006). Secreted PCSK9 decreases the number of to the induction of Fe(II)-dependent apoptosis in Jurkat cells by LDL receptors in hepatocytes and in livers of parabiotic mice. (-)-epigallocatechin gallate. Carcinogenesis 25 , 1567 – 1574. J. Clin. Invest. 116 , 2995 – 3005. Nakano, Ch., Motegi, A., Sato, T., Onodera, M., and Hoshino, T. Landmesser, U., Bahlmann, F., Mueller, M., Spiekermann, S., (2007). Sterol biosynthesis by a prokaryote: fi rst in vitro iden- Kirchhoff, N., Schulz, S., Manes, C., Fischer, D., de Groot, K., tifi cation of gene encoding squalene epoxidase and lanosterol Fliser, D., et al. (2005). Simvastatin versus ezetimibe: pleiotro- synthase from Methylococcus capsulatus . Biosci. Biotechnol. pic and lipid-lowering effects on endothelial function in humans. Biochem. 71 , 2543 – 2550. Circulation 111 , 2356 – 2363. Naoumova, R.P., Marais, A.D., Mountney, J., Firth, J.C., Rendell, LaRosa, J.C., Grundy, S.M., Waters, D.D., Shear, C., Barter, P., N.B., Taylor, G.W., and Thompson, G.R. (1996). Plasma Fruchart, J.C., Gotto, A.M., Greten, H., Kastelein, J.J., Sheperd, mevalonic acid an index of cholesterol synthesis in vivo, and J., et al. (2005). Treating to New Targets (TNT) Investigators, responsiveness to HMG-CoA reductase inhibitors in hamilial intensive lipid lowering with atorvastatin in patients with stable hypercholesterolemia. Altherosclerosis 119 , 203 – 213. coronary disease. N. Engl. J. Med. 352 , 1425 – 1435. Newland, H. (1976). The lipid hypothesis. Ann. Intern. Med. 85 , Lee, H.K., Denner-Ancona, P., Sakakibara, J., Ono, T., and 826. Prestwich, G.D. (2000). Photoaffi nity labelling and site-di- NIH Consensus Development conference. Lowering blood cho- rected mutagenesis of rat squalene epoxidase. Arch. Biochem. lesterol to prevent heart disease. NIH Consensus Development Biophys. 381 , 43 – 52. Conference Statement (1985). Nutr. Rev. 43 , 283 – 291. Lee, H.K., Zheng, Y.F., Xiao, X.Y., Bai, M., Sakakibara, J., Ono, Noguchi, M., Yokoama, M., Watanabe, S., Uchiyama, M., Nakao, T., and Prestwich, G.D. (2004). Photoaffi nity labelling identifi es Y., Hara, K., and Iwasaka, T. (2006). Inhibitory effect of the tea the substrate-binding site of mammalian squalene epoxidase. polyphenol, (-)-epigallocatechin gallate, on growth of cervical Biochem. Biophys. Res. Commun. 315 , 1 – 9. adenocarcinoma cell lines. Cancer Lett. 234 , 135 – 142. Liao, J.K. (2005). Pleiotropic effects of statins. Annu. Rev. Nowosielski, M., Hoffmann, M., Wyrwicz, L.S., Stepniak, Pharmacol. Toxicol. 45 , 89 – 118. P., Plewczynski, D.M., Lazniewski, M., Ginalski, K., and Lipinski, C.A., Lombardo, F., Dominy, B.W., and Feeney, P.J. (2001). Rychlewski, L. (2011). Detailed mechanism of squalene epoxi- Experimental and computational approaches to estimate solubil- dase inhibition by terbinafi ne, J. Chem. Inf. Model 51 , 455 – 462. ity and permeability in drug discovery and development settings. Ono, T. and Imai, Y. (1985). Squalene epoxidase from rat liver Adv. Drug Deliv. Rev. 46 , 3 – 26. microsomes. Methods Enzymol. 110 , 375 – 380. Lippi, G., Targher, G., and Franchini, M. (2010). Vaccination, Pan, W.H., Wu, H.J., Yeh, C.J., Chuang, S.Y., Chang, H.Y., Yeh, squalene and anti-squalene antibodies: facts or fi ction ? Eur. J. N.H., and Hsieh, Y.T. (2011). Diet and health trends in Taiwan: Intern. Med. 21 , 70 – 73. comparison of two nutrition and health surveys from 1993 – 1996 Liu, L. and Yeh, Y.-Y. (2000). Inhibition of cholesterol biosynthe- and 2005 – 2008. Asia Pan. J. Clin. Nutr. 20 , 238 – 250. sis by organosulfur compounds derived from garlic. Lipids 35 , Park, H.S., Park, E., Kim, M.S., Ahn, K., Kim, I.Y., and Choi, E.J. 197 – 203. (2000). Selenite inhibits c-Jun N-terminal kinase/stress activated Lucas, D., Goulitquer, S., Marienhagen, J., Fer, M., Dreno, Y., protein kinase (JNK/SAPK) through a thiol redox mechanism. J. Schwaneberg, U., Amet, Y., and Corcos, L. (2010). Stereoselec- Biol. Chem. 275 , 2527 – 2531. tive epoxidation of the last double bond of polyunsaturated fatty Patel, N. and Hegele, R.A. (2010). Mipomersen as a potential acids by human cytochromes P450. J. Lipid Res. 51 , 1125 – 1133. adjunctive therapy for hypercholesterolemia. Expert. Opin. Mann, R.K. and Beachy, P.A. (2000). Cholesterol modifi cation of Pharmacother. 11 , 2569 – 2572. proteins. Biochim. Biophys. Acta 1529 , 188 – 202. Pearson, A., Budin, M., and Brocks, J.J. (2003). Phylogenetic and Massey, V. (1994). Activation of molecular oxygen by fl avins and biochemical evidence for sterol synthesis in the bacterium fl avoproteins. J. Biol. Chem. 269 , 22459 – 22462. Gemmata obscuriglobus. Proc. Natl. Acad. Sci. USA 100 , McSheehy, S., Yang, W., Pannier, F., Szpunar, J., Lobinski, R., 15352 – 15357. Auger, J., and Potin-Gautier, M. (2000). Speciation analysis of Pearson, T.A. (2004). The epidemiologic basis for population-wide selenium in garlic by two dimensional high-performance liquid cholesterol reduction in the primary prevention of coronary chromatography with parallel inductively coupled plasma mass artery disease. Am. J. Cardiol. 94 , 4F – 8F. spectrometric and electrospray tandem mass spectrometric detec- Petranyi, G., Ryder, N.S., and St ü tz, A. (1984). Allylamine deriva- tion. Anal. Chim. Acta 421 , 147 – 153. tives: new class of synthetic antifungal agents inhibiting fungal Micallef, M.A. and Garg, M.L. (2009). Beyond blood lipods: photos- squalene epoxidase. Science 224 , 1239 – 1241. terols, statins and omega-3 polyunsaturated fatty acid therapy for Quazi, F. and Molday, R.S. (2011). Lipid transport by mammalian hyperlipidemia. J. Nutr. Biochem. 20 , 927 – 939. ABC proteins. Essays Biochem. 50 , 265 – 290. Moore, W.R., Schatzman, G.L., Jarvi, E.T., Gross, R.S., and Quereshi, A.A., Abuirmeileh, N., Din, Z.Z., and Elson, C.E. (1983). McCarthy, J.R. (1992). Terminal difl uoro olefi n analogs of Burger, W.C. Inhibition of cholesterol and fatty acid biosynthesis squalene are time-dependent inhibitors of squalene epoxidase. J. in liver enzymes and chicken hepatocytes by polar fractions of Am. Chem. Soc. 114 , 360 – 361. garlic. Lipids 18 , 343 – 348. Motavaze, K., Namvar, Z., Emami, M., Noorbakhsh, F., and Rezaie, Rasbery, J.M., Shan H., LeClair, R.J., Norman, M., Matsuda, S.P.T., S. (2008). Molecular characterization of a squalene epoxidase and Bartel, B. (2007). Arabidopsis thaliana squalene epoxidase Squalene monooxygenase and hypercholesterolemic therapy 1073

1 is essential for root and seed development. J. Biol. Chem. 282 , Schroeder, H.A., Buckman, J., and Balassa, J.J. (1967). Abnormal 17002 – 17013. trace elements in man: tellurium. J. Chronic Dis. 20 , 147 – 161. Ray, K.K., Cannon, C.P., and Ganz, P. (2006). Beyond lipid lower- Schultze, V., D ’ Agosto, V., Wack, A., Novicki, D., Zorn, J., and ing: what have we learned about the benefi ts of statins from the Hennig, R. (2008). Safety of MF59 adjuvant. Vaccine 26 , acute coronary syndromes trials ? Am. J. Cardiol. 98 , 18P – 25P. 3209 – 3222. Reihner, E., Rudling, M., Stahlberg, D., Berglund, L., Ewerth, S., Sen, S.E. and Prestwich, G.D. (1989a). Squalene analogues contain- Bjorkhem, I., Einarsson, K., and Angelin, B. (1990). Infl uence of ing isopropylidene mimics as potential inhibitors of pig liver pravastatin, a specifi c inhibitor of HMG-CoA reductase, on hepatic squalene epoxidase and oxidosqualene cyclase. J. Med. Chem. metabolism of cholesterol. N. Engl. J. Med. 323 , 224 – 228. 32 , 2152 – 2158. Rizzo, M. (2010). Lomitapide, a microsomal triglyceride trans- Sen, S.E. and Prestwich, G.D. (1989b). Trisnorsqualene alcohol, a fer protein inhibitor for the treatment of hypercholesterolemia. potent inhibitor of vertebrate squalene epoxidase. J. Am. Chem. IDrugs 13 , 102 – 111. Soc. 111 , 1508 – 1510. Ruckenstuhl, C., Eidenberger, A., Lang, S., and Turnowsky, F. Sen, S.E., Wawrzenczyk, C., and Prestwich, G.D. (1990). Inhibition (2005). Single amino acid exchange in FAD-binding romains of of vertabrate squalene epoxidase by extended and truncated ana- squalene epoxidase of Saccharomyces cerevisiae lead to either logues of trisnorsqualene alcohol. J. Med. Chem. 33 , 1698 – 1701. loss of functionality or terbinafi ne sensitivity. Biochem. Soc. Shan, L., Pang, L., Zhang, R., Murgolo, N.J., Lan, H., and Hedrick, Trans. 33 , 1197 – 1201. J.A. (2008). PCSK9 binds to multiple receptors and can be func- Ruckenstuhl, C., Lang, S., Poschenel, A., Eidenbergger, A., Barol, tionally inhibited by EGF-A peptide. Biochem. Biophys. Res. P.K., Kohut, P. Hapala, I., Gruber, K., and Turnowsky, F. (2007). Commun. 375 , 69 – 73. Characterization of squalene epoxidase of Saccharomyces cer- Shankar, S., Ganapathy, S., and Srivastava, R.K. (2007). Green tea evisiae by applying terbinafi ne-sensitive variants. Antimicrob. polyphenols: biology and therapeutic implications in cancer. Agents Chemother. 51 , 275 – 284. Front. Biosci. 12 4881 – 4899. Ruckenstuhl, C., Poschenel, A., Possert, R., Baral, P.K., Gruber, Shibata, N., Arita, M., Misaki, Y., Dohmae, N., Takio, K., Ono, T., K., and Turnowsky, F. (2008). Structure-function correlation Inoue, K., and Arai, H. (2001). Supernatant protein factor, which of two highly conserved motifs in Saccharomyces cerevi- stimulates the conversion of squalene to lanosterol, is a cytosolic siae squalene epoxidase. Antimicrob. Agents Chemother. 52 , squalene transfer protein and enhances cholesterol biosynthesis. 1496 – 1499. Proc. Nucl. Acid Sci. USA 98 , 2244 – 2249. Ryder, N.S. (1989). Mechanism of action of terbinafi ne. Clin. Exp. Silagy, C., and Neil, A. (1994). Garlic as a lipid lowering agent – a Dermatol. 14 , 98 – 100. metaanalysis. J. R. Coll. Physicians Lond. 28 , 39 – 45. Ryder, N.S. and Dupont, M.-C. (1984). Properties of a particulate Simons, K. and Ehehalt, R. (2002). Cholesterol, lipid rafts, and dis- squalene epoxidase from Candida albicans. Biochem. Biophys. ease. J. Clin. Invest. 110 , 597 – 603. Acta 794 , 466 – 471. Smart, N.A., Marshall, B.J., Daley, M., Boulos, E., Windus, J., Ryder, N.S. and Dupont, M.C. (1985). Inhibition of squalene epoxi- Baker, N., and Kwok, N. (2011). Low-fat diets for acquired dase by allylamine antimycotic compounds. A comparative hypercholesterolemia. Cochrane Database Syst. Rev. 16 , study of the fungal and mammalian enzymes. Biochem. J. 230 , CD007957. 765 – 770. Stancu, C. and Sima, A. (2001). Statins: mechanism of action and Ryder, N.S., Frank, I., and Dupont, M.-C. (1986). Ergosterol biosyn- effects. J. Cell Mol. Med. 5 , 378 – 387. thesis inhibition by the thiocarbamate antifungal agents tolnaftate Stevinson, C., Pittler, M.H., and Ernst, E. (2000). Garlic for treating and tolciclate. Antimicrob. Agents Chemother. 31 , 858 – 860. hypercholesterolemia. Ann. Intern. Med. 133 , 420 – 429. Ryder, N.S., Seidl, G., and Troke, P.F. (1984). Effect of antimycotic Stowe, H.D., Eavey, A.J., Granger, L., Halstead, S., and Yamini, B. drug naftifi ne on growth of and sterol biosynthesis in Candida (1992). Selnium toxicosis in feeder pigs. J. Am. Vet Med. Assoc. albicans . Antimicrob. Agents Chemother. 25 , 483 – 487. 201 , 292 – 295. Sadowitz, B., Seymour, K., Costanza, M.J., and Gahtan, V. (2010). Statin therapy – part II: clinical considerations for cardiovascular Strandberg, T.E., Tilvis, R.S., and Miettinen, T.A. (1990). Metabolic disease. Vasc. Endovasc. Surg. 44 , 421 – 433. variables of cholesterol during squalene feeding in humans: compar- Sakakibara, J., Watanaba, R., Kanai, Y., and Ono, T. (1995). ison with cholestyramine treatment. J. Lip. Res. 31 , 1637 – 1643. Molecular cloning and expression of rat squalene epoxidase. Sudhop, T., Lutjohann, D., Kodal, A., Igel, M., Tribble, D.L., Shah, J. Biol. Chem. 270 , 17 – 20. S., Perevozskaya, I., and von Bergmann, K. (2002). Inhibition Sandeep, S.M. and Davidson H.M. (2011). The incremental value of intestinal cholesterol absorption by ezetimibe in humans. of lipids and infl ammatory biomarkers in determining residual Circulation 106 , 1943 – 1948. cardiovascular risk. Curr. Atheroscler. Rep. 13 , 373 – 380. Tai, H.H. and Bloch, K. (1972). Squalene epoxidase of rat liver. Sawada, M., Matsuo, M., and Seki, J. (2002). Inhibition of choles- J. Biol. Chem. 247 , 3767 – 3773. terol synthesis causes both hypercholesterolemia and hypocho- Takezawa, H., Hayashi, M., Iwasawa, Y., Hosoi, M., Iid, Y., Tsuchiya, lesterolemia in hamsters. Biol. Pharm. Bull. 25 , 1577 – 1582. Y., Horie, M., and Kamei, T. (1989). European Patten Application Sawada, M., Washizuka, K., and Okumura, H. (2004). Synthesis EP 0318860A2. and biological activity of a novel squalene epoxidase inhibitor, Takezawa, H., Hayashi, M., Iwasawa, Y., Hosoi, M., Iid, Y., Tsuchiya, FR194738. Bioorg. Med. Chem. Lett. 14 , 633 – 637. Y., Horie, M., and Kamei, T. (1990). European Patten Application Schafer, W.R. and Rine, J. (1992). Protein prenylation: genes, EP 0395768A1. enzymes, targets, and functions. Annu. Rev. Genet. 26 , 209 – 237. The Lipid Research Clinics Coronary Primary Prevention Trial Schreuder, H.A., Prick, P.A., Wierenga, R.K., Vriend, G., Wilson, results. (2002). I. Reduction in incidence of coronary heart dis- K.S., Hol, W.G., and Drenth, J. (1989). Crystal structure of the ease. J. Am. Med. Assoc. 251 , 351 – 364. p-hydroxybezoate hydroxylase-substrate complex refi ned at Tilvis, R., Kovanen, P.T., and Mietten, T.A. (1982). Metabolism of 1.9A resolution. Analysis of the enzyme-substrate and enzyme- squalene in human fat cells. Demonstration of a two-pool system. product complexes. J. Mol. Biol. 208 , 679 – 696. J. Biol. Chem. 257 , 10300 – 10305. 1074 A. Belter et al.

Toutouzas, K., Drakopoulou, M., Skoumas, I., and Stefanadis, C. Wagner-Recio, M., Toews, A.D., and Morell, T. (1991). Tellurium (2010). Advancing therapy for hypercholesterolemia. Expert. blocks cholesterol synthesis by inhibiting squalene metabo- Opin. Pharmacother. 11 , 1659 – 1672. lism: preferential vulnerability to this metabolic block leads Uchida, H., Sugiyama, R., Nakayachi, O., Takemura, M., and to peripheral nervous system demyelization. J. Neurochem. Ohyama, K. (2007). Expression of the gene for sterol-biosyn- 57 , 1891 – 1901. thesis enzyme squalene epoxidase in parenchyma cells of the oil Warshafsky, S., Kamer, R.S., and Sivak, S.L. (1993). Effect of garlic plant, Euphorbia tirucalli . Planta 226 , 1109 – 1115. on total serum cholesterol. A meta-analysis. Ann. Intern. Med. Ugawa, T., Kakuta, H., Moritani, H., Matsuda, K., Ishihara, T., and 119 , 599 – 605. Yamaguchi, M. (2000). YM-53601, a novel squalene synthase Watanabe, T., Umezawa, Y., Tkahashi, Y., and Akamatsu, Y. (2010). inhibitor, reduces plasma cholesterol and triglyceride levels in Novel pyrrole- and 1,2,3-triazole-based 2,3-oxidosqualene several animal species. Br. J. Pharmacol. 131 , 63 – 70. cyclase inhibitors. Bioorg. Med. Chem. Lett. 20 , 5807 – 5810. van Barkel, W.J.H., Kamerbeek, N.M., and Fraaije, M.W. (2006). Waterham, H.R. (2006). Defects of cholesterol biosynthesis. FEBS Flavoprotein monooxygenases, a diverse class of oxidative bio- Lett. 580 , 5442 – 5449. catalysts. J. Biotechnol. 124 , 670 – 689. Wierenga, R.K., Terpstra, P., and Hol, W.G. (1986). Prediction of the van Duuren, B.L. and Schmitt, F.L. (1960). Epoxidation and cycliza- occurrence of the ADP-binding beta alpha beta-fold in proteins, tion of squalene. J. Org. Chem. 25 , 1761 – 1765. using an amino acid sequence ﬁ ngerprint. J. Mol. Biol. 187 , van Heek, M., Farley, C., Compton, D.S., Hoos, L., and Davis, 101 – 107. R.S. (2001). Ezetimibe selectively inhibits intestinal cholesterol Xu, F., Rychnovsky, S.D., Belani, J.D., Hobbs, H.H., Cohen, J.C., absorption in rodents in the presence and absence of exocrine and Rawson, R.B. (2005). Dual roles for cholesterol in mamma- pancreatic function. Br. J. Pharmacol. 134 , 409 – 417. lian cells. Proc. Natl. Acad. Sci. USA. 102, 14551 – 14556. van Sickle, W.A., Angelastro, M.R., Wilson, P., Cooper, J.R., Yamamoto, S., and Bloch, K. (1970). Studies of squalene epoxidase Marquart, A., and Flanagan, M.A. (1992). Inhibition of choles- of rat liver. J. Biol. Chem. 245 , 1670 – 1674. terol synthesis by cyclopropylamine derivatives of squalene in Yubin, W., Haiqian W., Wenlong, H., Huibin, Z., and Jinpei, Z. human hepatoblastoma cells in culture, Lipids 27 , 157 – 160. (2011). Synthesis and biological evaluation of ezetimibe analogs van Tamelen, E.E. and Heys, J.R. (1975). Letter: enzymatic epoxida- as possible cholesterol absorption inhibitors. Lett. Drug Des. tion of squalene variants. J. Am. Chem. Soc. 97 , 1252 – 1253. Discov. 8 , 500 – 505. Volkman, J.K. (2003). Sterols in microorganisms. Appl. Microbiol. Biotechnol. 60 , 495 – 506. Received August 10, 2011; accepted October 8, 2011

Jan Barciszewski received his Malgorzata Giel-Pietraszuk PhD in 1974 in organic chem- received her PhD in bio- istry from the Adam Mickie- chemistry from the Institute wicz University in Poznan, of Bioorganic Chemistry Pol- Poland. Since 1993 he has ish Academy of Sciences in been Professor of Biology at Poznan in 1998, working with the Institute of Bioorganic Professor Mirosława Bacisze- Chemistry of the Polish Acad- wska on the interaction of emy of Sciences in Poznan. nucleic acids with proteins. He has worked at the Insti- She worked with Professor tute of Molecular Biology in Matthias Sprinzl at the Univer- Moscow, Aarhus University sity of Bayreuth on the inter- (Denmark), Zurich University action of IF2 with ribosome. (Switzerland), the National Cancer Center in Tokyo (Japan), Currently she pursues research interests on the effect of high the Institute of Molecular and Cellular Biology in Strasbourg hydrostatic pressure on conformation of ribonucleic acids. (France), and at the Free University of Berlin with Volker A. Erdmann. His research focuses on the structure and function of nucleic acids and their interactions with proteins, catalytic RNAs, and the development of nucleic acid tools for the treatment of brain tumors and other diseases. Squalene monooxygenase and hypercholesterolemic therapy 1075

Leszek Jerzy Rychlewski Tomasz Grabarkiewicz is a obtained the MBBS in co-founder and Chief Execu- 1998 at the Charite, Hum- tive Offi cer of AdvaChem- boldt Universitas in Berlin. Lab, a start-up research and Between 1996 and 1998 he development company dedi- was a Research Fellow at cated to providing innovative the Scripps Research Insti- and cost-effective products tute, La Jolla, CA, USA. and services to the pharma- Between 1998 and 1999 he ceutical and fi ne chemical was employed as a Postdoc- industries. He obtained his toral Researcher at the Uni- PhD in computational chem- versity of California, San istry in 2008 from the Adam Diego, CA, USA. In 2001 he Mickiewicz University in Poznan, Poland. His research inter- founded the bioinformatics lab at the BioInfoBank Institute ests span computer aided drug design, medicinal and biophys- in Poznan. ical chemistry as well as asymmetric catalysis and synthesis of functional molecules. Agnieszka Belter graduated in biotechnology in 2008 at Miroslawa Skupinska gradu- the Adam Mickiewicz Uni- ated in molecular biology in versity in Poznan, Poland. 2004 at the Adam Mickiewicz She has gained professional University in Poznan, Poland. experience at BioInfoBanki She has gained professional Institute and AdvaChemLab. experience in drug develop- Now as a PhD student she ment working at Jerini AG is focusing on drug develop- (Shire) in Berlin, Germany ment: specifi cally on cata- and at BioInfoBank Institute lytic RNAs for the treatment in Poznan. Currently she is of brain tumors, the inhibitors a PhD student studying the of human squalene monooxy- inhibitors of bacterial amin- genase and the inhibitors of oacyl-tRNA synthetases and bacterial aminoacyl-tRNA synthetases. designing therapeutic proteins against targeted cancer cells.