Binding Structures and Potencies of Oxidosqualene Cyclase Inhibitors

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Binding Structures and Potencies of Oxidosqualene Cyclase Inhibitors with the Homologous Squalene-Hopene Cyclase Alexander Lenhart,† Dirk J. Reinert,† Johannes D. Aebi,‡ Henrietta Dehmlow,‡ Oliver H. Morand,‡ and Georg E. Schulz*,† Institut fu¨ r Organische Chemie und Biochemie, Albert-Ludwigs-Universita¨ t, Albertstrasse 21, Freiburg im Breisgau 79104, Germany, and Preclinical Research, Pharmaceuticals Division, Hoffmann-La Roche Ltd., Basel 4070, Switzerland Received December 17, 2002

The binding structures of 11 human oxidosqualene cyclase inhibitors designed as cholesterollowering agents were determined for the squalene-hopene cyclase from Alicyclobacillus acidocaldarius, which is the only structurally known homologue of the human enzyme. The complexes were produced by cocrystallization, and the structures were elucidated by X-ray diffraction analyses. All inhibitors were bound in the large active center cavity. The detailed binding structures are presented and discussed in the light of the IC50 values of these 11 as well as 17 other inhibitors. They provide a consistent picture for the inhibition of the bacterial enzyme and can be used to adjust and improve homology models of the human enzyme. The detailed active center structures of the two enzymes are too different to show an IC50 correlation. Introduction The enzyme oxidosqualene cyclase (OSC) converts oxidosqualene to lanosterol, forming the steroid scaffold in a single reaction.1,2 In a search for drugs that reduce plasma cholesterol levels in humans, the enzyme was established as an interesting target3-6 besides squalene synthase,7-9 squalene epoxidase,10 and HMG-CoA reductase.11,12 In contrast to the reductase, the downregulation of OSC has only minor consequences for the synthesis of other required isoprenoids.13 Moreover, OSC inhibition avoids the accumulation of toxic sterol intermediates that would emerge if catalytic steps further down the pathway of cholesterol biosynthesis were inhibited. Numerous inhibitors for OSC and for its bacterial counterpart squalene-hopene cyclase (SHC) have been reported.14 They can be grouped into irreversible inhibitors,15-17 reversible inhibitors with scaffolds resembling the substrate or reaction intermediates,18-22 and non-terpenoid inhibitors.3-6,23 Here, we focus on the last.4 The cyclization reactions of OSC and SHC involve the formation of several carbocations, among which the initial cation at one end of the hopenoid (and steroid) scaffold and the cation resulting from the last ring formation at the other end are suitable reference points.24,25 Since the first cation emerges through protonation by an acid, it was thought that a positive charge provided by an inhibitor may form an ion pair with the respective acidic residue. The other carbocation arising from the last ring closure should then be around 12 Å away. For bioavailability reasons, this second cation was not mimicked by a second cationic group but by a ketone carbon carrying a positive partial charge.25 With a tertiary amine for the initial cation this amino* To whom correspondence should be addressed. Phone: +49-761203-6058. Fax: +49-761-203-6161. E-mail: [email protected]. † Albert-Ludwigs-Universita ¨ t. ‡ Hoffmann-La Roche Ltd.

ketone combination was introduced in essentially all inhibitors examined here (Figure 1). Among the members of the (oxido)squalene cyclase family, only one species from a thermophilic bacterium could be crystallized and structurally elucidated.26,27 This squalene-hopene cyclase from Alicyclobacillus acidocaldarius is a dimer of 2 × 631 amino acid residues showing a peculiar monotopic association with the membrane and a catalytic center located in a large cavity at the subunit center. The cavity is connected through a channel with the membrane interior from where the cyclase picks the substrate squalene or oxidosqualene and to where it releases the product hopene or lanosterol, respectively. The channel contains a constriction formed by Phe166, Val174, Phe434, and Cys435. The active center location was established by mutations and by the binding structure of the inhibitor lauryl-dimethyl-aminoxide (LDAO) at the catalytic acid Asp376 of SHC.26 The sequences of human OSC and SHC have 17% identical residues and can be clearly aligned using sequence fingerprints. Consequently, the polypeptide chain folds, and thus, the general architectures of the active center cavities should be very similar. In light of this similarity, we analyzed 11 inhibitors designed for the human enzyme (Figure 1) with respect to their binding structures to the bacterial enzyme. All of them contain a tertiary amine with two small aliphatic substituents (allyl, methyl, and cyclopropyl) connected by a spacer (hexyloxy, but-2-enyl) to a couple of aromatic rings. The inhibitor binding modes are discussed in conjunction with the IC50 values for SHC and for OSC, making a wealth of data available for further studies. Results and Discussion Crystal Structures of Bound Inhibitors. All SHCinhibitor complex crystals belonged to form A′ with space group P3221 and nine SHC dimers in the unit cell, i.e., three SHC subunits per asymmetric unit. All analyses extended to about 2.8 Å resolution. All 11

10.1021/jm0211218 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/30/2003

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Figure 1. Chemical formulas of the applied inhibitors. Counterions are chloride (1, 3-5, 8, 10, 20, 25), fumarate (2, 6, 7, 9, 11-13, 16, 17, 19, 21-23), and bromide (14). The inhibitors are ordered and named according to their potencies for SHC. Inhibitors with established binding structures are marked with asterisks (/). The configuration at the spacer cyclopropyl group in 7, 8, and 10 is a trans, racemic mixture.

inhibitors were bound with high occupancies in the active center cavity, in agreement with their IC50 values ranging from 18 to 406 nM (Table 1). After an initial rigid body refinement, the (Fo - Fc) maps always showed density outlining the inhibitor at the 3σ level in all three subunits. Ten of the inhibitors analyzed contain a bromine; inhibitor 25 has a chlorine (Figure 1). The bromines were reliable guides for positioning the inhibitors because they caused peak heights of 10σ-20σ. The peak height for the chlorine was 9σ. Simulated annealing (Fo - Fc) omit maps were calculated after the structural refinement. Two of such omit maps are shown in Figures 2 and 3. All of them outlined the inhibitors very well, although the short allyl-, methyl-, and cyclopropyl substituents of the tertiary amine were not always supported by good density. In some bromophenyl groups the ortho carbons showed low densities, which is probably a technical problem because these carbons lie in the negative series termination density ring around the strongly scattering bromine substituent.28,29 Interactions between Inhibitors and SHC. The active center cavity of SHC is designed to accommodate squalene as well as to stabilize carbocation intermediates of the cyclization reaction. The inhibitors follow a common construction principle. Except for 15 and 28, all contain an alkyl-substituted tertiary amine giving rise to a positive charge under physiological and assay conditions (Figure 1). In all inhibitor complexes, this charge is located near the site of the initial squalene protonation (Table 2). It is stabilized by Asp374, Asp376, and Asp377, which together carry at least one negative

charge,27 and also by the suitably oriented dipoles of Tyr420 and Tyr609 and the π-systems of Trp312, Phe365, and Trp389.30,31 In the majority of the structures, the tertiary amine also contacts a sequestered water molecule, which in turn is connected to an acid. The π-systems of the aryl groups of polypeptide and inhibitors are involved in numerous interactions (Table 3). Apart from one possible π-π interaction between Phe365 and the spacer C-C double bond of 2, all of them are CH-π interactions.32,33 The π-systems of the inhibitor aryl moieties interact with CH groups of Ile261, Pro263, Phe129, Trp169, and Phe601, and several CH groups from the aryl moieties contact the π-system of Phe605. Moreover, CH-π contacts occur between the short alkyl substituents of the tertiary amine and Trp312 or Trp489, and some others occur between spacer CH groups and Phe365. Besides the electrostatic and the CH-π interactions, the inhibitors make a multitude of hydrophobic contacts. These involve for the most part aliphatic groups but also a number of polarized carbon atoms from the side chains of Arg127 and Ser307 and of the active center aspartates. The hydrophobic contacts are evenly distributed over all parts of the inhibitors (Table 3). The nonpolar interactions of the halogen substituents are numerous and therefore likely to make important contributions to the binding strength. Enzyme Kinetics and Inhibitor Potencies. Since the enzymes OSC and SHC pick their substrates from the membrane and release their products into the membrane, the kinetic parameters of the two enzyme assays are likely to depend on the chemical and physical

Oxidosqualene Cyclase Inhibitors

Journal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2085

Table 1. Inhibitor Potencies for SHC and OSC inhibitora 1* 2* 3* 4 5 6 7 8* 9* 10 11 12 13* 14 15 16 17* 18 19 20* 21* 22* 23* 24 25* 26 27 28

PDB codes

IC50(SHC) b (nM)

IC50(OSC) c (nM)

1H36 1O6Q 1H35

18 23 29 38 40 49 50 59 60 62 75 75 80 96 123 130 141 172 180 186 281 289 306 332 406 760 1200 2800

19.0 500 (83.3) 16.0 98 3.0 29 439 223 6.5 22 13.5 380 (79.0) 610 (86.0) 5.4 640 (86.5) 1860 (94.9) 1900(95.0) 4.1 39 3.5 29 19.6 2.9 240 (70.4) 11.3 48 71 36

1H37 1GSZ

1O6R

1H3B 1O6H 1H3A 1H39 1H3C 1O79

a Established binding structures are marked with asterisks (/). The data for inhibitor 9 have already been reported.35 b The detergent LDAO shows an IC50 value of 308 nM for SHC. The values for the inhibitors 2, 6, 11-13, 16-20, 24, 26, and 27 were taken from Weihofen.52 The errors are about (10%. c The values in parentheses are the residual activities (in %) of OSC at an inhibitor concentration of 100 nM. The IC50 value preceding such a percentage was calculated from the reading at 100 nM. The values for the inhibitors 9, 12-14, 17, 18, 20, 22, and 23 were taken from Dehmlow et al.53 The errors of the directly determined IC50 values range around (15%. The calculated IC50 values are model-dependent and therefore less accurate.

compositions of the reaction mixtures. Two detailed kinetic analyses were published for inhibitor 9 acting on SHC.34,35 The different assays used in these two studies resulted in quite different kinetic constants. However, in both cases the double-reciprocal Lineweaver-Burk plot showed a “mixed” inhibition in the most simple Michaelis-Menten interpretation. For one of the data sets,35 this result was confirmed by a nonlinear regression analysis. In both analyses, the data allowed within the margins of error a “noncompetitive” interpretation but were inconsistent with a “competitive” inhibition expected for noncovalent binding at the substrate position (Figures 2 and 3). This discrepancy would be resolved if the inhibitors were kinetically captured in the sequestered cavity, feigning a “noncompetitive” case.36 Capturing does not apply to the natural products hopene and lanosterol because these emerge from a strongly exergonic cyclization reaction providing ample energy to overcome the transport barrier. Unfortunately, IC50 measurements cannot resolve this discrepancy because the shape of an IC50 curve should not depend on the inhibition type, which merely changes the relation between IC50 and the kinetic constants. IC50 equals the inhibitor constant KI for a “noncompetitive” case but depends on the applied substrate concentration and the Michaelis constant according to IC50 ) KI(1 + [So]/KM) in a “competitive” case. For 9, the kinetic data35 yielded KM ) 50 µM and KI ) 60 nM when interpreted

as “noncompetitive”. A “competitive” approximation resulted in KM ) 25 µM and KI ) 32 nM, giving rise to IC50 ) 58 nM because [So] was 20 µM in the IC50 determinations. Consequently, in this case, the observed IC50 ) 60 nM (Table 1) is compatible with both interpretations. Comparison within a Group of Similar Inhibitors. The 28 analyzed inhibitors contain a group of 10 with a tertiary amine substituted by allyl (for 4, a cyclopropyl) and methyl groups as well as by a hexyloxy spacer, a central phenyl, and an unrestrained bromophenyl group. Except for 4, all modifications are at the central phenyl group that in most cases is extended to a bicyclic heteroaromate described by atoms X, Y, and Z (Table 4). The IC50 values of these inhibitors vary by a factor of 8 for SHC. With respect to the human OSC, they contain five of the most potent inhibitors with IC50 values below 10 nM but also two with low potency. The binding structures of five of these inhibitors were established. A superposition in Figure 4 shows that all of them were bound in similar conformations with tertiary amines that coincide within (0.5 Å. The allyl, methyl, and hexyloxy substituents were always bound in the same regions. Owing to the limited resolution, their conformational variances are not significant. The positional variation of the bromine substituent was only (0.2 Å, which is within the expected margins of error. The largest differences occurred at the central phenyl group and depended on X, Y, and Z (Table 4). The IC50 values increase by a factor of 2 between the first and second half of Table 4. The first half has either no additional ring or small five-membered rings, whereas the second half has bulkier five- and six-membered rings that slightly shift the bromophenyl group, pushing the bromine into close contact with Thr173 (Figure 4). The orientation of the bromophenyl with respect to the central phenyl group varies within the group of fivemembered rings and between five- and six-membered rings. It is critical because it determines the fit to the bent cavity surface. A comparison between inhibitor 9 with a fluorine as atom X and 14 without X shows that the fluorine tends to improve binding for SHC. The same tendency is observed for the couple 7 and 10. Since inhibitor 4 differs from 9 merely in an amine substituent, the lower IC50 of 4 indicates that the cyclopropyl group fits SHC better than the allyl group. The electrostatic interactions of the charged tertiary amine should be identical in all inhibitors (Table 2). Not so strong but differentiating between these inhibitors are the CH-π interactions and the extended hydrophobic contacts (Table 3). For OSC, the inhibitors of Table 4 show dramatic differences. Obviously, OSC requires large groups at positions X, Y, and Z, which may be due to the replacement of the sterically constraining Phe605 of SHC by a smaller cysteine in OSC.26,27,37 The high IC50 value connected to the cyclopropyl of 4 in relation to the allyl of 9 demonstrates that the respective OSC pocket is fine for an allyl but too narrow for a cyclopropyl group. The low potency for OSC of 12 and of 13 compared to 18 seems to be associated with the angle between the bromophenyl and the central phenyl group. For the two five-membered rings, this angle is wider than for the six-membered variant 18, possibly causing a clash of

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Figure 2. Stereoview of the active center of SHC with bound inhibitor 1 showing the highest potency for SHC (Figure 1, Table 1). The density is represented by the simulated-annealing omit (Fo - Fc) map calculated with the final model at the 5σ contour level. The given side chains outline the central cavity. Green side chains are at the channel constriction. Bound water molecules are blue. Hydrogen bonds are indicated as dotted lines.

Figure 3. Stereoview of inhibitor 17 bound to the active center of SHC. The density is represented by the simulated-annealing omit (Fo - Fc) map calculated with the final model at the 5σ contour level. The given side chains outline the central cavity. The channel constriction residues are green. Bound water molecules are blue. Hydrogen bonds are indicated as dotted lines.

the bromophenyl group with a tryptophan of OSC26,37 that assumes the position of the less bulky Phe129 of SHC (Figure 4). Second Binding Site for the Bromophenyl Group. Inhibitors 17 and 23 differ only with respect to the isothiazol annelation either to the bromophenyl or to the central phenyl group, respectively, which changes the spatial requirement and the conformational freedom of the aryl arrangement. The binding structure of 17 shows that the annelated bromophenyl group rotates 180° with respect to the group of five inhibitors depicted in Figure 4, bringing the bromine into a pocket lined

by Phe129, Phe437, and Arg127 (Figure 3). It has been suggested that this pocket contains the water molecules likely to participate in the protonation or hydration of the final cation of the SHC reaction.27 Similar interactions of a bromophenyl group with arginine and lysine side chains have been observed before.38,39 The superposition of 17 with 23 in Figure 5 clarifies this difference and shows slight displacements of Phe437, Phe601, and Phe605, indicating some flexibility of the cavity walls. In conclusion, 17 revealed a second binding mode in SHC with increased potency when compared to its direct counterpart 23.



Table 2. Polar Interactions of the Tertiary Aminea minimum distanceb (Å)

maximum distanceb (Å)

median distanceb (Å)

5.1 3.0 4.2 3.9 2.4 4.4 4.4 4.6 4.3

6.4 6.3 5.1 4.9 4.3 6.1 5.6 5.3 5.9

5.8 4.6 4.7 4.3 3.0 5.0 5.1 4.9 5.2

Trp312-π Asp376-Oδ Asp377-Oδ Asp374-Oδ waterc Phe365-π Tyr609-Oη Trp489-π Tyr420-Oη

a The amino acid residues are ordered from the top to the bottom of the active center cavity. b All 12 established binding structures are considered. c This water was assigned in all inhibitors except for 2, 21, and 23.

Table 3. CH-πa and Hydrophobic Interactionsb between Inhibitors and SHC Polypeptidec inhibitor 1

2

3

8

9

13

Trp312 1,- -,1 Asp376 -,1 Asp377 -,1 Asp374 -,1 -,1 -,2 -,1 -,1 Phe365 -,3 -,3 1,1 1,1 -,1 Trp489 -,3 1,3 -,1 2,1 -,3 -,2 Ser307 -,1 Leu607 -,1 Ile261 1,- -,1 Trp169 -,1 -,2 Phe601 1,- -,2 2,- 2,2 -,6 -,2 Phe605 -,2 -,3 -,1 -,2 -,1 Pro263 1,- 1,- 1,- -,1 -,1 1,Ala170 -,2 -,2 Phe437 -,2 -,2 -,2 -,1 -,1 Thr173 Phe129 -,3 -,1 -,2 -,2 2,- 2,1

17

20

21

-,1 -,2 -,1 -,1 -,1 1,3 1,3 -,5 -,2 -,1 -,2 -,1 -,1 -,1 1,- -,1 1,-,1 -,3 2,- -,2 2,- -,3 1,1 1,- 1,2 1,1 -,2 -,4 -,1 -,2 -,2 2,- -,1

22

23

25

-,1 -,1 -,1 1,-,1 -,2 -,1 1,1 1,-,2 -,1 1,1 -,4 1,1 1,2 -,2 -,2 -,2 -,1 2,- -,2

1,-,2 -,1 -,1 1,-,1 -,1 1,-,2 1,1 -,3 -,2 -,1 2,-

a CH-π interactions were assigned according to the criteria of Brandl et al.33 The number of these interactions is indicated to the left the comma. b The number of hydrophobic contacts between pairs of atoms with distances less than 4 Å is indicated to the right of the comma. c The residues are ordered from the top to the bottom of the active center cavity.

Table 4. Comparison of Inhibitors with Hexyloxy Spacer and Unrestrained Bromophenyl Group

inhibitorsa 4 9* 12 13* 14 18 19 20* 22* 23*

X F F O O CH2 NH CH N-Me S

atomb Y / / / O CH

Z

IC50(SHC) (nM)

IC50(OSC) (nM)

O O N CH O N N N N N

38 60 75 80 96 172 180 186 289 306

98 6.5 380c 610c 5.4 4.1 39 3.5 19.6 2.9

a Known structures are marked with an asterisk (/). For all compounds, R denotes allyl-hexyloxy-methyl-amine except for 4 where the allyl group is replaced by a cyclopropyl. b If X and Z are not connected, Y is marked by a slash. c Estimate as specified in Table 1.

With OSC, the pair 17 and 23 shows a dramatic difference. The former does not bind efficiently, whereas the latter has the highest potency of all (Table 1).

Actually, this pair emphasizes the main mechanistic difference between OSC and SHC.2 In SHC, the last carbocation at the lower end of the cavity is protonated by a water molecule that was replaced in the peculiar binding mode of 17. Certainly OSC lacks such a water molecule and hence this “second” binding mode because during the reaction the carbocation after the last ring closure has to be stabilized in a nonpolar environment from where it then has to move backward to the center of the steroid scaffold in the following methyl rearrangements. Tertiary Amine Dominance Revealed by Spacer Length. The seven inhibitors 1, 2, 5, 6, 11, 16, and 21 contain a short butenyloxy spacer in contrast to the inhibitors with hexyloxy spacers of Table 4. The butenyloxy spacer has only five bonds without rotational restraint compared with the eight of hexyloxy, providing an entropic bonus of about 7 kJ/mol.40-43 Three of these short spacer inhibitors have been analyzed with respect to their structures. Among them, inhibitor 2 has the long spacer counterpart 13 (Figure 1) that is also structurally established. The binding structures of these two inhibitors were superimposed in Figure 6, showing the tertiary amine at the same position and the aryl groups of 2 displaced toward the top of the cavity allowing Ile261 and Pro263 to interact with the central aryl group and Phe129 as well as Phe437 with the bromo substituent. The analyses were confirmed by the unambiguous bromine positions. Compound 2 with the short spacer binds by a factor of 3.5 more strongly than its long spacer counterpart 13 (Table 1). This corresponds to a free energy difference of 3.4 kJ/mol that could be amply provided by the 7 kJ/mol entropy bonus associated with the mobility reduction upon binding. The same amine position was observed in the other two short spacer inhibitors 1 (Figure 2) and 21 (see below), confirming the dominance of the electrostatic interactions over the other binding forces. The importance of the charged amine is aided by the alkylation that appreciably reduces the free energy of desolvation, increasing the net binding effect.44-46 In SHC, the amine forms an ionic pair with one of the aspartates residue and it is further stabilized by the suitably oriented dipoles of Tyr420, Tyr609, and Tyr612 as well as by cation-π interactions with Trp312, Phe365, and Trp489 (Table 2). Inhibitor 21 binds with its tertiary amine at the usual position, but its methylated indazole group was rotated by 180° with respect to the benzofuran of 2 (Figure 7). This rotation does not occur in 22, which is the long spacer counterpart (except for a fluorine) of 21 because the methylated indazole finds space in the lower part of the cavity in the binding conformation of all hexyloxy compounds (Figure 4). The flipped indazole of 21 undergoes CH-π interactions with Ile261, and its fluoro substituent finds a nonpolar pocket formed by Leu36, Ala306, and Leu607. Moreover, bromophenyl makes CH-π interactions with Phe601 and Phe605. Still, 21 has a much lower SHC potency than 2, indicating that SHC repels a short spacer combined with a spacious central aryl group. The short spacer inhibitor 1 has the highest potency for SHC. Its tertiary amine binds at the common position (Figure 2) and its benzophenone moiety is flexible enough to adjust to a suitable conformation

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Figure 4. Stereoview of a superposition of inhibitors 9 (gray), 13 (pink), 20 (green), 22 (blue), and 23 (black) bound to the active center of SHC. The side chains of the residues outlining the central cavity are given. All five inhibitors have allyl, methyl, and hexyloxy substituents at the tertiary amine as listed in Table 4.

Figure 5. Stereoview of a superposition of inhibitors 17 (blue) and 23 (gray) bound to the active center of SHC. These compounds differ by the annelation position of the five-membered ring at one or the other side of the aromatic system.

interacting with Pro263 and Phe601. The binding mode resembles the nearly equipotent SHC inhibitor 2, which also has a short spacer. The cyclopropyl of 1 is changed to an allyl in its direct counterpart 5 (Figure 1). An IC50 comparison reveals that the cyclopropyl seems to fit SHC better than the allyl, which is in agreement with a CH-π interaction between the cyclopropyl and Trp312 (Figure 2). For OSC, however, the comparison between 1 and 5 shows that cyclopropyl is detrimental for the potency, confirming the interpretation of the long spacer pair 4 and 9 (see above). The short spacer pair 2 with a benzofuran and 11 with a thiophen differs only by the oxygen-sulfur exchange. The sulfur decreases the SHC potency by a factor of 3.3 presumably because of steric hindrance at its contacting partners Ser307 and Leu607 (Figure 6). Surprisingly,

OSC accepts the thiophen but repels the benzofuran (Table 1) presumably because the bromine of the benzofuran inhibitor 2 clashes with the tryptophan of OSC that is likely to substitute Phe129 of SHC.26,37 Spacers with Cyclopropyl and Phenyl Groups. The spacers of 7, 8, and 10 contain cyclopropyl groups and the spacers of 3, 15, 25, and 28 have phenyl groups (Figure 1). In both cases the degrees of freedom are reduced when compared to hexyloxy or butenyloxy spacers so that the potencies should increase if no steric hindrance was introduced. Among this group, the binding structures of 3, 8, and 25 have been determined. Inhibitor 25 differs from the majority of the presented inhibitors but is a close analogue of OSC inhibitor BIBX793 with an IC50 of 4 nM. Its binding mode is similar to that of 22; however, its bulky piperidine causes CH-π



Figure 6. Stereoview of a superposition of inhibitors 2 (blue) and 13 (gray) bound to the active center of SHC. These inhibitors differ only with respect to the spacer between the tertiary amine and the aromatic ring system. The amine positions are identical, demonstrating the dominance of their interactions.

Figure 7. Stereoview of a superposition of inhibitors 2 (black) and 21 (gray) bound to the active center of SHC. These inhibitors differ with respect to the aryl systems and their substituents.

interactions with Phe365 and Trp412 and it displaces the tertiary amine by about 2 Å from the common position (Table 2). The carbamate nitrogen of 25 superimposes the indazol nitrogens of 22 displayed in Figure 4, and the chlorine of 25 is at the bromine position of 22. The potency of 25 for SHC is much lower than for OSC. Remarkably, the amine oxidation of 25, giving rise to inhibitor 15, increased the potency for SHC conceivably because the piperidine had shifted the amine from the charge-stabilizing position into a region better suited for the aminoxide dipole. For OSC, however, this particular oxidation greatly decreases the potency (Table 1). Other aminoxide inhibitors have been reported, for instance, LDAO (Table 1) and the N-oxide of azasqualene.20

Inhibitors 3 and 8 contain bromobenzophenone moieties together with phenyl and cyclopropyl spacers and have low IC50 values for SHC. Their binding conformations resemble those of other bromobenzophenone inhibitors as illustrated in the superposition with 1 and 9 in Figure 8. The biphenyl group of 3 shows a 75° twist between the phenyl rings and the bromobenzophenone. It fits the cavity as in the other cases. The allyl and cyclopropyl substituents of 8 make CH-π interactions with Trp489 as in 1, and a water molecule connects the tertiary amine with the charged couple Asp374-Asp377. Obviously, the allylcyclopropyl moiety is well suited for SHC but diminishes binding to OSC (Table 1).

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Figure 8. Stereoview of the superposition of the inhibitors with a benzophenone moiety bound to the active center of SHC. These are 1 (blue), 3 (black), 8 (green), and 9 (gray). The hopene model (pink) is given as a reference.27

Conclusion The structure of SHC clarified the initial protonation by Asp376 and revealed the stabilization of the final carbocation by the π-electrons of Phe601 and Phe605.27,47 The position of the final carbocation can be derived from the hopene model depicted in Figure 8. The binding structures of the aminoketone inhibitors now showed that the tertiary amine is always located close to Asp376 (Table 2) and the ketone carbon and its equivalents are close to Phe601 and Phe605, confirming the original proposal. However, the contribution of the ketone cannot be crucial considering that inhibitor 14 with a ketone is only slightly more potent than 13 with an electronrich benzofuran. The comparisons between short and long spacer variants demonstrated further that the binding contribution of the tertiary amine dominated over all others. With a given amine position, the influence of the remainder could be checked in a more rational way. However, this rational approach failed for 17, which was bound in a hitherto neglected pocket of SHC (Figure 3), and failed also for 21, the central aryl group of which is rotated by 180° (Figure 7). Unfortunately, the amino acid sequence differences between the OSC and SHC residues lining the cavity (Tables 2 and 3) amount to 47%, abolishing any correlation between the potencies despite similar binding modes. In general, the OSC cavity seems to be spatially more restricted than the SHC cavity. The presented data should help to build an accurate active center model of human OSC because structure and activity data can now be combined and the applied inhibitors used as additional restraints. Recently, a model of rat OSC23 and the first model of human OSC used in a mechanistic study were published.37 Improving the latter on the basis of the reported data should result in more potent OSC inhibitors. Experimental Section Preparation of SHC and of the SHC-Inhibitor Complex Crystals. The SHC gene was incorporated into a high copy plasmid and expressed in E. coli JM105 as described.27

The purification process involved cell disintegration in a French press, centrifugation, and two chromatographic steps using ion exchange (Q-Sepharose-FF) and size-exclusion (Superdex-200) materials. The yield was about 4 mg of pure SHC per liter of culture. The enzyme was stored at a concentration of 25 mg/mL in 20 mM Tris-HCl, pH 8.0, 0.6% (w/v) octyltetraoxyethylene (C8E4), 10 mM β-mercaptoethanol, and 200 mM NaCl at 4 °C. The SHC-inhibitor complexes were produced in solution and subsequently crystallized. For this purpose, a 16 mg/mL enzyme solution was incubated for 15 min at 55 °C in 5 mM Tris-HCl, pH 8.0, 0.6% (w/v) C8E4 together with a 1.5- to 10-fold molar excess of the respective inhibitor. The high temperature approaches the activity maximum of SHC and is necessary for inhibitor binding but cannot be applied in crystal-soaking experiments. The ligated SHC could only be crystallized in the crystal form A′ that belongs to space group P3221 with three subunits of the dimeric enzyme in the crystallographically asymmetric unit and diffracts X-rays to around 2.7 Å resolution.27 The superior tetragonal crystal form B with one subunit per asymmetric unit27 could not be reproduced. Voluminous crystals up to a length of 1400 µm grew over several weeks at 20 °C in hanging drops containing 5-8 mg/mL ligated enzyme, 50 mM sodium citrate, pH 4.8, 50 mM NaCl, 3-9% (v/v) poly(ethylene glycol)-600, and 0.3% (w/v) C8E4. A cryoprotocol could not be established. X-ray Data Collection and Analyses. All data were collected on a rotating anode X-ray generator (model RU200B, Rigaku) equipped with a wire-frame area detector (model X-1000, Nicolet-Bruker) at room temperature using one to five crystals for each inhibitor. The data were merged and processed with programs XDS,48 SCALA, and TRUNCATE.49 A total of 11 SHC-inhibitor complexes were analyzed to about 2.8 Å resolution. The data collection results are summarized in Table 5. The unit cell dimensions were distributed around a ) b ) 141.0 Å and c ) 244.3 Å with standard deviations of 0.3 and 0.6 Å, respectively, in agreement with the values 141.0 and 243.8 Å of the unligated enzyme crystals.27 The structures were solved by the difference Fourier technique using the established structure of the unligated enzyme in the same crystal form A′.27 After adjustment for small packing differences by an initial rigid body refinement with the program CNS,50 the (Fo - Fc) density maps outlined the bound inhibitor in all cases. The inhibitor models were built using the program SYBYL and fitted into the density. The structure refinements with the program CNS resulted in reasonable R factors (Table 5). During refinement, NCS restraints were applied for polypeptides and inhibitors. The bound water molecules were not restrained. The crystallographic temperature factors were



Table 5. X-ray Data Collection and Refinement Statisticsa complex with inhibitor no. of reflns redundancy completeness (%) Rsym (%) I/σI data for refinement Rcryst (%) Rfree (%) water molecules

1

2

3

8

9

13

17

20

21

22

23

25

148 698 2.6 82 8.4 7.4 57 131 22.1 25.8 267

209 837 3.8 80 8.8 6.3 55 265 20.1 23.7 260

216 866 3.8 82 8.9 7.0 56 775 21.9 25.8 210

180 971 3.2 81 9.4 6.8 56 318 20.5 24.8 281

156 593 2.9 79 10.0 5.0 54 200 20.3 25.9 255

157 336 2.5 81 7.4 7.9 61 932 19.3 23.6 277

242 782 4.0 88 9.8 3.7 59 971 20.6 25.0 208

175 699 3.2 80 7.2 8.2 55 010 17.4 23.3 341

124 813 2.4 78 11.3 4.9 52 980 24.8 27.0 242

124 826 2.3 80 12.5 4.3 54 870 22.5 25.5 256

76 483 1.5 73 11.7 4.5 45 709 25.8 28.1 191

134 820 3.0 79 13.7 4.4 52 186 24.0 27.3 237

a All data were collected between about 40 and 2.8 Å resolution (data set 13 to 2.7 Å). The average completeness of the last shell was 53%. Inhibitor 9 is included for comparison.35 For the 12 structures, the average real space correlation between the inhibitor molecules and their densities was 92.7% ( 2.4%. The average B factors of the protein, inhibitor, and water molecules were 46 ( 8, 43 ( 12, and 29 ( 7, respectively.

refined in groups consisting of the main chain and side chain moieties of each residue. Coordinates and X-ray Diffraction Data. These are deposited in the Protein Data Bank under accession codes 1H35, 1H36, 1H37, 1H39, 1H3A, 1H3B, 1H3C, 1O6H, 1O6Q, 1O6R, and 1O79 (Table 1). The data for 9 had been deposited under 1GSZ.35 Activity Assay for SHC. For IC50 determination, the assay of Feil et al.51 has been modified. The substrate stock emulsion was produced by ultrasonic homogenization of a 200 µM squalene and 2% (w/v) taurodeoxycholate solution. It was stored at -20 °C and homogenized again before usage. The desired amount of the dissolved (ethanol/water) inhibitor was vacuum-dried in a 2 mL polyethylene vial in order to avoid ethanol poisoning of SHC. Subsequently, the inhibitor was dissolved in 100 µL of 1 M sodium citrate, pH 6.0, 100 µL of substrate stock emulsion, and 790 µL of water, and the reaction was started by adding 0.5 µg of the enzyme. After 30 min at 60 °C, the reaction was stopped by adding 800 µL of cold 2-propanol. The educt and the products were extracted with 1.2 mL of hexane. The hexane phase was separated, vacuum-dried, and dissolved in 30 µL of hexane/2-propanol and analyzed by capillary gas chromatography with a DB-1 (25 m × 0.32 mm) column using a temperature gradient between 273 and 315 °C, hydrogen as carrier, and split injection (1:5). The analytes were quantified with a flameionization detector. Activity Assay for OSC. Human liver microsomes were prepared as described,4 resulting in 0.8 mg/mL microsomal proteins in buffer P (100 mM sodium phosphate, pH 7.4). The substrate stock solution contained [14C]-R,S-monooxidosqualene (12.8 mCi/mmol) in buffer P with 1% (w/v) bovine serum albumin (BSA) and up to 5% ethanol. The inhibitors were dissolved in DMSO and diluted to the desired concentration in buffer P with 1% BSA. The reaction was started by adding 20 µL of the substrate solution to a mixture of 40 µL of microsomal protein solution with 20 µL of inhibitor solution. The final concentrations were 0.4 mg/mL microsomal proteins, 30 µM substrate, the chosen concentration for the inhibitor, 0.5% (w/v) BSA, less than 0.1% DMSO, and less than 2% ethanol. The assays were incubated for 60 min at 37 °C and were then stopped by the addition of 0.6 mL of 10% KOH in methanol, 0.7 mL of water, and 0.1 mL of hexane/diethyl ether (1:1, v/v) containing as nonradioactive carriers 25 µg of R,Smonooxidosqualene and 25 µg of lanosterol. The mixture was stirred, complemented by 1 mL of hexane/ether, again stirred, and centrifuged. The upper phase was recovered, and the lower phase was again extracted with hexane/ether, separated, and combined with the first extraction. The extract was dried under nitrogen, resuspended in 50 µL of hexane/ether, and analyzed by thin-layer chromatography with hexane/ether as the eluent. The monooxidosqualene and lanosterol bands were then quantitated by 14C counting in a phosphorimager (AmershamPharmacia). The ratio of lanosterol to monooxidosqualene plus lanosterol was taken as the yield of the reaction.

Acknowledgment. We thank Drs. Jean Ackermann, Yu-Hua Ji, and Synese Jolidon for the chemical syntheses of some of the inhibitors as well as Wilhelm Weihofen for measuring several IC50 values for SHC. References (1) Abe, I.; Rohmer, M.; Prestwich, G. D. Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes. Chem. Rev. 1993, 90, 2189-2206. (2) Wendt, K. U.; Schulz, G. E.; Corey, E. J.; Liu, D. R. Enzyme mechanisms for polycyclic triterpene formation. Angew. Chem., Int. Ed. 2000, 39, 2812-2833. (3) Mark, M.; Mu¨ller, P.; Maier, R.; Eisele, B. Effects of a novel 2,3oxidosqualene cyclase inhibitor on the regulation of cholesterol biosynthesis in HepG2 cells. J. Lipid Res. 1996, 37, 148-158. (4) Morand, O. H.; Aebi, J. D.; Dehmlow, H.; Ji, Y. H.; Gains, N.; Lengsfeld, H.; Himber, J. Ro48-8071, a new 2,3-oxidosqualene: lanosterol cyclase inhibitor lowering plasma cholesterol in hamsters, squirrel monkeys, and minipigs: comparison to simvastatin. J. Lipid Res. 1997, 38, 373-390. (5) Eisele, B.; Budzinski, R.; Mu¨ller, P.; Maier, R.; Mark, M. Effects of a novel 2,3-oxidosqualene cyclase inhibitor on cholesterol biosynthesis and lipid metabolism in vivo. J. Lipid Res. 1997, 38, 564-575. (6) Brown, G. R.; Hollinshead, D. M.; Stokes, E. S. E.; Clarke, D. S.; Eakin, M. A.; Foubister, A. J.; Glossup, S. C.; Griffiths, D.; Johnson, M. C.; McTaggart, F.; Mirrlees, D. J.; Smith, G. J.; Wood, R. Quinuclidine inhibitors of 2,3-oxidosqualene cyclaselanosterol synthase: optimization from lipid profiles. J. Med. Chem. 1999, 42, 1306-1311. (7) Brown, G. R.; Clarke, D. S.; Foubister, A. J.; Freeman, S.; Harrison, P. J.; Johnson, M. C.; Mallion, K. B.; McCormick, J.; McTaggart, F.; Reid, A. C.; Smith, G. J.; Taylor, M. J. Synthesis and activity of a novel series of 3-biarylquinuclidine squalene synthase inhibitors. J. Med. Chem. 1996, 39, 2971-2979. (8) Pandit, J.; Danley, D. E.; Schulte, G. K.; Mazzalupo, S.; Pauly, T. A.; Hayward, C. M.; Hamanaka, E. S.; Thompson, J. F.; Harwood, H. J. Crystal structure of human squalene synthase. A key enzyme in cholesterol biosynthesis. J. Biol. Chem. 2000, 275, 30610-30617. (9) Hiyoshi, H.; Yanagimachi, M.; Ito, M.; Ohtsuka, I.; Yoshida, I.; Saeki, T.; Tanaka H. Effect of ER-27856, a novel squalene synthase inhibitor, on plasma cholesterol in rhesus monkeys: comparison with 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors. J. Lipid Res. 2000, 41, 1136-1144. (10) Sawada, M.; Matsuo, M.; Hagihara, H.; Tenda, N.; Nagayoshi, A.; Okumura, H.; Washizuka, K.; Seki, J.; Goto, T. Effect of FR194738, a potent inhibitor of squalene epoxidase, on cholesterol metabolism in HepG2 cells. Eur. J. Pharmacol. 2001, 431, 11-16. (11) Istvan, E. S.; Deisenhofer, J. Structural mechanism for statin inhibition of HMG-CoA reductase. Science 2001, 292, 11601164. (12) Illingworth, D. R.; Tobert, J. A. HMG-CoA reductase inhibitors. Adv. Protein Chem. 2001, 56, 77-114. (13) Gru¨nler, J.; Ericsson, J.; Dallner, G. Branch-point reactions in the biosynthesis of cholesterol, dolichol, ubiquinone and prenylated proteins. Biochim. Biophys. Acta 1994, 1212, 259-277. (14) Cattel, L.; Ceruti, M. Inhibitors of 2,3-oxidosqualene cyclase as tools for studying the mechanism and function of the enzyme. Crit. Rev. Biochem. Mol. Biol. 1997, 33, 353-373.

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JM0211218

Binding Structures and Potencies of Oxidosqualene Cyclase Inhibitors

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