Design of new benzoxazole-2-thione-derived inhibitors of Streptococcus pneumoniae hyaluronan lyase: structure of a complex with a 2-phenylindole

Glycobiology, Aug 2006

The bacterial hyaluronan lyases (Hyals) that degrade hyaluronan, an important component of the extracellular matrix, are involved in microbial spread. Inhibitors of these enzymes are essential in investigation of the role of hyaluronan and Hyal in bacterial infections and constitute a new class of antibiotics against Hyal-producing bacteria. Recently, we identified 1,3-diacetylbenzimidazole-2-thione and related molecules as inhibitors of streptococcal Hyal. One of such compounds, 1-decyl-2-(4-sulfamoyloxyphenyl)-1-indol-6-yl sulfamate, was co-crystallized in a complex with Streptococcus pneumoniae Hyal and its structure elucidated. The resultant X-ray structure demonstrates that this inhibitor fits in the enzymatic active site via interactions resembling the binding mode of the natural hyaluronan substrate. X-ray structural analysis also indicates binding interactions with the catalytic residues and those of a catalytically essential hydrophobic patch. An IC50 value of 11 µM for Hyal from Streptococcus agalactiae (strain 4755) qualifies this phenylindole compound as one of the most potent Hyal inhibitors known to date. The structural data suggested a similar binding mode for N-(3-phenylpropionyl)-benzoxazole-2-thione. This new compound’s inhibitory properties were confirmed resulting in discovery of yet another Hyal inhibitor (IC50 of 15 µM). These benzoxazole-2-thiones constitute a new class of inhibitors of bacterial Hyals and are well suited for further optimization of their selectivity, potency, and pharmacokinetic properties.

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Design of new benzoxazole-2-thione-derived inhibitors of Streptococcus pneumoniae hyaluronan lyase: structure of a complex with a 2-phenylindole

Glycobiology Design of new benzoxazole-2-thione-derived inhibitors of Streptococcus pneumoniae hyaluronan lyase: structure of a complex with a 2-phenylindole Daniel J. Rigden 0 2 Alexander Botzki 1 Masatoshi Nukui 0 R. Brandon Mewbourne 0 Ejvis Lamani 0 Stephan Braun 1 Erwin von Angerer 1 Günther Bernhardt 1 Stefan Dove 1 Armin Buschauer 1 Mark J. Jedrzejas 0 0 Children's Hospital Oakland Research Institute , Oakland, CA 94609 1 Institute of Pharmacy, University of Regensburg , 93040 Regensburg , Germany 2 School of Biological Sciences, University of Liverpool , Crown Street, Liverpool L69 7ZB , UK The bacterial hyaluronan lyases (Hyals) that degrade hyaluronan, an important component of the extracellular matrix, are involved in microbial spread. Inhibitors of these enzymes are essential in investigation of the role of hyaluronan and Hyal in bacterial infections and constitute a new class of antibiotics against Hyal-producing bacteria. Recently, we identified 1,3diacetylbenzimidazole-2-thione and related molecules as inhibitors of streptococcal Hyal. One of such compounds, 1-decyl-2(4-sulfamoyloxyphenyl)-1-indol-6-yl sulfamate, was co-crystallized in a complex with Streptococcus pneumoniae Hyal and its structure elucidated. The resultant X-ray structure demonstrates that this inhibitor fits in the enzymatic active site via interactions resembling the binding mode of the natural hyaluronan substrate. X-ray structural analysis also indicates binding interactions with the catalytic residues and those of a catalytically essential hydrophobic patch. An IC50 value of 11 mM for Hyal from Streptococcus agalactiae (strain 4755) qualifies this phenylindole compound as one of the most potent Hyal inhibitors known to date. The structural data suggested a similar binding mode for N-(3-phenylpropionyl)-benzoxazole-2-thione. This new compound's inhibitory properties were confirmed resulting in discovery of yet another Hyal inhibitor (IC50 of 15 mM). These benzoxazole-2-thiones constitute a new class of inhibitors of bacterial Hyals and are well suited for further optimization of their selectivity, potency, and pharmacokinetic properties. antimicrobials/hyaluronidase/molecular mechanism/pneumococci/spreading factor Introduction The glycosaminoglycan hyaluronan (hyaluronic acid [HA]) was first isolated from the vitreous humor of bovine eyes *These authors contributed equally to this work. 1To whom correspondence should be addressed; e-mail: 2To whom correspondence should be addressed; e-mail: (Meyer and Palmer, 1934) and is a major component of the extracellular matrix (ECM) of animal tissues. This strictly linear polymer consists of repeating disaccharide units of (β,1–4)-D-glucuronic acid-(β,1–3)-N-acetyl-D-glucosamine. It has been identified in the capsule of some bacteria and in essentially all vertebrates where it is present in significant quantities in skin (dermis and epidermis), brain, and the central nervous system (Laurent and Fraser, 1992). HA is degraded by hyaluronidases, a class of enzymes with two main families, the eukaryotic hyaluronoglucosaminidases (EC and the prokaryotic hyaluronan lyases (Hyals) (EC (Kreil, 1995; Coutinho and Henrissat, 1999). The best characterized of the bacterial enzymes are Streptococcus pneumoniae and Streptococcus agalactiae Hyals (Jedrzejas and Chantalat, 2000; Jedrzejas, 2002b). Both enzymes degrade HA at the β-1,4-glycosidic linkage between D-glucuronic acid and N-acetyl-D-glucosamine. The product of this elimination reaction is the unsaturated disaccharide 2-acetamido-2-deoxy-3-O-(β-D-gluco-4-enepyranosyluronic acid) D-glucose (Kreil, 1995; Linker and Meyer, 1954). The three-dimensional (3D) structures of S. pneumoniae and S. agalactiae strain 3502 Hyals were elucidated by X-ray analyses in the native as well as in the complex forms with both substrates and products (Jedrzejas and Chantalat, 2000; Li and Jedrzejas, 2001; Li, et al., 2000; Jedrzejas, 2002b). The structures of both enzymes are similar and contain distinct domains with the largest domain being catalytic. The active site of the S. pneumoniae enzyme (SpnHyal), schematically depicted in Figure 1, is located in a long cleft of the catalytic domain that binds the substrate and consists of four main parts: a positive patch, a catalytic triad (His399, Tyr408, and Asn349) responsible for degradation, a hydrophobic/aromatic patch (Trp291, Trp292, and Phe343) responsible for correct positioning of the substrate chains for catalysis, and a negative patch implicated in the release of the product (Glu388, Asp398, and Thr400) (Li et al., 2000). Successive structural (Li et al., 2000; Ponnuraj and Jedrzejas, 2000; Li and Jedrzejas, 2001; Jedrzejas et al., 2002; Mello et al., 2002) and mutagenesis studies (Li et al., 2000; Kelly et al., 2001; Nukui et al., 2003) have enabled the formulation of a firmly supported catalytic mechanism, as reported (Jedrzejas, 2001, 2002a,b). This mechanism is based on acid/base type β-elimination with His399 serving as base and Tyr408 as acid (Figure 1). The Hyal enzymes identified in two S. agalactiae strains, 4755 (SagHyal4755) and 3502 (SagHyal3502), have nearly identical sequences (98% identity) (Ponnuraj and Jedrzejas, 2000) and show 53% identity with the S. pneumoniae enzyme. In contrast to the structure of SpnHyal, a fully active two-domain truncated version (Li et al., 2000), the Fig. 1. Binding of a hyaluronan hexasaccharide in the catalytic cleft of Streptococcus pneumoniae hyaluronan lyase. HA with disaccharide units HA1 (reducing end), HA2 and HA3 (non-reducing end), and the catalytic triad (Tyr408, His399, and Asn349) were extracted from the SpnHyal– hexasaccharide HA complex (Jedrzejas et al., 2002). Oxygen atoms are drawn as black balls and nitrogen as smaller grey balls. The positions of the “patches” forming the binding site with respect to the substrate are indicated. Dotted lines indicate interactions central to the catalytic mechanism according to Li and others (2000). SagHyal3502 structure reveals an additional third domain at the N-terminus, which is presumed to act as a spacer (Li and Jedrzejas, 2001). Bioinformatic studies identified a further domain at the extreme N-terminus in all bacterial Hyal enzymes, including S. pneumoniae and S. agalactiae, that is responsible for substrate binding but not for catalysis (Rigden and Jedrzejas, 2003). Unsaturated disaccharides are the only degradation products of exhaustive digestion of HA by Hyals. These disaccharides might be utilized by bacteria as an additional carbon source (Hynes and Walton, 2000). The degradation of HA in the host ECM decreases the viscoelasticity of the ground substance, which presumably facilitates bacterial spread and their associated toxins into tissues (Hynes and Walton, 2000). Streptococcal Hyal is therefore a significant virulence factor. Human infection by such pathogens is one of the major causes of meningitis, septicemia, as well as many other serious diseases that lead to neonatal mortality (Dillon et al., 1987; Hynes and Walton, 2000). To study the precise role of HA and Hyals in the course of bacterial infection and spread, we undertook the design and development of inhibitors in our laboratories. For bacterial Hyals, L-ascorbic acid (Vc, 1, Figure 2) was described as a competitive inhibitor with an IC50 value of ∼6 mM (Li et al., 2001). Furthermore, various unsaturated fatty acids have been reported to inhibit S. dysgalactiae Hyal, with IC50 values ranging from 1 to 172 μM (Suzuki et al., 2002). The structural basis of the SpnHyal–Vc interaction was elucidated by means of X-ray crystallography (Li et al., 2001). Vc binds in the active site of the enzyme, and among the different interactions, the importance of hydrophobic contacts with Trp292 and Tyr408 was evident. On the basis of the hypothesis that Vc derivatives with increased hydrophobicity would be more potent, L-ascorbic acid-6-hexadecanoate (Vc palmitoate, Vcpal, 2) (Figure 2), a highly effective antioxidant (Klein and Weber, 2001) and Fig. 2. Chemical structures of hyaluronan lyase inhibitors. 1: Vitamin C, L-ascorbic acid, Vc; 2: L-ascorbic acid-6-hexadecanoate, Vcpal; 3: 1,3diacetylbenzimidazole-2-thione; 4: 1-decyl-2-(4-sulfamoyloxyphenyl)-1Hindol-6-yl sulfamate; and 5: N-substituted benzoxazole-2-thione derivatives. glutathione-S-transferase inhibitor (Mitra et al., 1992), was examined (Botzki et al., 2004). The X-ray structure of the SpnHyal-Vcpal complex [Protein Data Bank (PDB) (Berman et al., 2000) code 1w3y (Botzki et al., 2004)], determined at 1.65 Å resolution, confirmed the hypothesis that additional hydrophobic interactions with Phe343, His399, and Thr400 in the active site lead to increased inhibition. On the basis of these studies of bacterial Hyals inhibition by Vc and Vcpal and the discovery that SagHyal is inhibited by, for example, 1,3-diacetylbenzimidazole-2-thione (3, Figure 2), a new inhibitor was designed, 1-decyl-2-(4-sulfamoyloxyphenyl)-1H-indol-6-yl sulfamate (4, Figure 2) (Walter et al., 2004). In this study, the X-ray structure of a complex of SpnHyal with this new inhibitor, compound 4, is presented and its binding mode compared with that of a hexasaccharide HA substrate and Vcpal. In addition, the newly identified interaction pattern of compound 4 was used to predict N-substituted benzoxazole-2-thiones as additional novel Hyal inhibitors. The initial results on structure-activity relationships (SAR) in this new series support the binding mode derived from the crystallized complexes and provide a basis for further optimization of the molecular structures of these compounds with respect to their selectivity, potency, and pharmacokinetic properties. Results and Discussion Initial design of inhibitors Recent structure-based design approaches using the program LUDI (Böhm, 1992) for docking of molecules from databases into a SagHyal4755 model led to the prediction of numerous potential inhibitors (Botzki et al., 2005). Nineteen compounds were synthesized, from which 13 proved to be active at SagHyal4755 (Botzki, et al., 2005). 1,3-diacetylbenzimidazole-2-thione (3, Figure 2) was the most potent inhibitor (IC50 value of 5 μM at pH 7.4). Another hit from these investigations, indole-2-carboxylic acid, was active only at concentrations above 1 mM. Optimization of this structure resulted in a series of inhibitors based on the 2-phenylindole scaffold (Salmen, 2003; Walter et al., 2004). The hypothesis that hydrophobic chains at the indole nitrogen may mimic the hexadecyl moiety of Vcpal (2, Figure 2) was proposed and later confirmed. Furthermore, the most active compound of the 2-phenylindole series, 1-decyl-2-(4-sulfamoyloxyphenyl)1H-indol-6-yl sulfamate (4, Figure 2), was crystallized in complex with SpnHyal. Structure of the S. pneumoniae Hyal–phenylindole inhibitor complex The overall protein structure of the SpnHyal-phenylindole inhibitor complex (Table I) is similar to that of the twodomain arrangement in the original native apo-form structure of this enzyme (Jedrzejas et al., 1998; Li et al., 2000, Ponnuraj and Jedrzejas, 2000). The crystallized Hyal molecule is composed of two domains, the catalytic domain having an α5/α5 barrel structure (α-domain) and the other Low resolution diffraction limit (Å) High resolution diffraction limit (Å) Completnessa Multiplicitya (%) Sulfate atomsb Non-hydrogen xylitol atoms Non-hydrogen inhibitor atoms Non-hydrogen solvent atoms Number of reflectionsa Mean temperature factor B (Å2) Protein main chain Protein side chain Bond lengths (Å) Bond angles (°) rms deviation from ideal values comprised mainly of an antiparallel, three-layer β-sandwich (β-domain). The catalytic cleft transverses the catalytic αdomain. The active site of the enzyme is located within the barrel domain on one side of the cleft area (Li and Jedrzejas, 2001; Jedrzejas, 2002a,b; Jedrzejas et al., 2002; Mello et al., 2002). The β-domain is located at the C-terminus and does not participate in catalysis. It is presumed to modulate the substrate access to the cleft by covering the cleft area of the α-domain because of its inherently great flexibility and motion (Jedrzejas, 2002; Mello et al., 2002). The full-length enzyme contains additional residues at the N-terminus arranged in two domains (Rigden and Jedrzejas, 2003) that are not catalytically important. The first of these domains is a substrate-binding domain that is thought to act to enhance the Hyal affinity for HA (for full discussion see (Rigden and Jedrzejas, 2003; Rigden et al., 2003). The second, smaller domain acts as a spacer to distance the catalytic domain from the N-terminal substrate-binding domain (Rigden and Jedrzejas, 2003). The inhibitor 4 was found to bind in the cleft present in the α-domain in the area of the catalytic and hydrophobic patch residues (Figure 1). Inhibitor binding was clearly evident from the extra electron density in the active site area, which corresponded to the size and shape of the compound. In general, this new inhibitor is located in the same location in which the HA disaccharide product (HA1 in Figure 1) (Ponnuraj and Jedrzejas, 2000), Vc (Li et al., 2001) as well as Vcpal (Botzki et al., 2004) were found to bind in our earlier studies. The location of this inhibitor in the active site area suggests its competitive nature and its ability to prevent the catalytic function of this enzyme. The binding site of the phenylindole inhibitor Electron density at the catalytic site allowed for the satisfactory modeling of the phenylindole as shown in Figure 3. The inhibitor was located within the electron density with the exception of the sulfamoyl group in 4-position of the phenyl moiety. This presumably reflects this sulfamoyl group solvent exposure and suggests free rotation about the NH2SO2O–phenyl bond. As refinement progressed, it became clear that the 6-sulfamoyloxy group at the indole moiety may adopt two distinct conformations (Figure 3). These alternate binding modes are associated with a small, nearly parallel displacement of the indole ring. The sulfamoyl aValues in brackets are for the highest resolution shell, 2.07–2.00 Å. bEach of the two modeled alternate inhibitor-binding modes comprises 35 atoms. Fig. 3. Stereo view of the SpnHyal cleft with the bound phenylindole inhibitor 4. Density supports the existence of two modes of interaction (modes 1 and 2). The sigmaA-weighted |2Fo-Fc| map electron density is shown at a contouring level of 0.65σ. position in mode 1 is well defined by density. In mode 2, it is not as clearly defined but, if omitted, leads to the presence of significant positive difference electron density. Thus, its inclusion in the model was necessary. It appears that mode 1, shown in more detail in Figure 4, is favored for energetic reasons, but at the resolution available, further occupation refinement is questionable. In the final model, additional difference density was evident at the catalytic site, suggestive of the existence of yet additional modes of inhibitor binding. However, only the conformation shown was well supported by density. Fig. 4. SpnHyal-binding site of the phenylindole inhibitor 4. The orientation shown faces into the substrate-binding cleft with the α-domain toward the top of the page and the β-domain toward the bottom. (A) Detailed representation of the interactions. Carbon atoms are colored as follows—inhibitor white, residues with hydrophobic contacts to the inhibitor orange, other residues green. Red spheres represent bound water molecules and yellow dashed lines indicate hydrogen bonds. (B) Lipophilic potential of the binding site mapped onto a MOLCAD surface. Hydrophobic regions are colored in brown, polar regions in green and green-blue. The 4-sulfamoyloxy substituent is exposed to the solvent. The binding of the phenylindole 4 to SpnHyal involves both hydrophobic interactions of the rings and the aliphatic tail and hydrogen bonds of the 6-sulfamoyloxy group. The indole plane of the inhibitor is in parallel with that of Trp292 (Figures 3 and 4A) in an energetically favorable distance of ca. 3.5 Å. The other side of the indole moiety interacts with the guanidino group of Arg462. Such cation-π interactions frequently contribute to the free energy of protein-ligand binding (Gallivan and Dougherty, 1999). The phenyl moiety of the inhibitor is sandwiched between Trp291 and Asn580. The interaction with Trp291 involves an approximately perpendicular ring arrangement (Figure 3). The aliphatic tail fits into a surface crevice lined mainly by hydrophobic residues—Met579, Trp291, and Phe343—and also by Arg336, Glu388, and His399. The lipophilic potential mapped on the surface of the binding site depicts a continuous hydrophobic region extending from Trp292 outward to Phe343, which aligns with the indole ring and nearly the complete decyl chain (Figure 4B). The 6-sulfamoyloxy group forms a network of hydrogen bonds with protein and water. The interactions associated with binding mode 1 (Figure 4A) seem more favorable, with charge-assisted bidentate H bonds between Arg466 and two sulfamoyl oxygen atoms. Both the oxygen atoms and nitrogen atoms are additionally linked to the protein by through–water interactions. In binding mode 2, there is only a single direct H bond of a sulfamoyl oxygen with the indole NH of Trp292. In this case, a water molecule mediates additional interactions with the carboxylate of Asp352. The binding site of the phenylindole inhibitor overlaps with that of the HA substrate (see also Figure 1). Figure 5A presents an alignment of the structure of functional SpnHyal-hexasaccharide HA complex (Jedrzejas et al., 2002) (substrate-based hexasaccharide bound to SpnHyal, PDB code 1loh) with the SpnHyal-inhibitor complex (root mean square [rms] of the backbone atoms 0.37 Å). The amino acids of the catalytic triad, Asn349, His399, and Tyr408, are involved in interactions with the phenylindole. A water-mediated hydrogen bond (Figure 4A) links the amide NH2 of Asn349 and the oxygen in 6-position of the indole in binding mode 1, the imidazole ring of His399 contributes to the fit of the decyl chain, and the phenyl moiety of Tyr408 is approached by the NH2 function of the 6-sulfamoyloxy group. Also, the hydrophobic patch (Trp291, Trp292, and Phe343) that participates in the precise positioning of the substrate belongs to the binding site of the inhibitor. Comparing the binding modes of the HA substrate and the inhibitor, it is obvious that the indole moiety of compound 4 is essentially coplanar with HA and is located in the HA-binding region around the scissile 1,4-glycosidic bond. The Nacetyl group of HA is directly related to the 6-sulfamoyloxy substituent of compound 4. The N-acetyl-D-glucosamine unit of HA approximately corresponds to the decyl chain. Comparison of the binding modes of the phenylindole and Vcpal Apart from the alkyl chains, the phenylindole 4 and Vcpal are prima facie structurally dissimilar (Figure 2). Nevertheless, their bound conformations and the binding modes are similar to some extent and are therefore compared in some detail. X-ray crystallography of the co-crystallized SpnHyal– Vcpal complex resulted in the PDB structure 1w3y (Botzki et al., 2004). Figure 5B presents a superposition of 1w3y onto the complex of SpnHyal with compound 4 (rms of the backbone atoms 0.07 Å). The vitamin C moiety, as illustrated by the structure of SpnHyal–Vcpal complex, binds in a ring-opened form, and it occupies the same region as intact Vc (Li et al., 2001). This Vcpal ring opening also occurs during the oxidation of Vc into L-dehydro-ascorbic acid which is rapidly degraded under physiological conditions (pH 7.4) via L-diketo-gulonate to yield L-erythrulose and oxalate (Simpson and Ortwerth, 2000). The L-ascorbic acid moiety of Vcpal binds within the catalytic site overlapping with the indole ring of 4. Most protein–Vcpal interactions are hydrophobic. The hydrophobic face of the Vc portion is stacked with the indole ring of Trp292. This structural arrangement helps the Vc moiety of Vcpal, like Vc itself, to bind in the narrowest part of the binding site comprising the amino acids Trp292, Arg462, and Arg466 (Li et al., 2001). The Vcpal palmitoyl moiety fits approximately in the same hydrophobic surface crevice as the decyl chain of the phenylindole. The binding of the proximal aliphatic tails of Vcpal and the phenylindole is generally similar, starting from overlapping positions of the palmitoyl C1 and the indole N1 atoms (distance ∼1 Å). A significant difference is evident at the distal ends of the tail chains. The shorter chain length in compound 4 (10 carbons, as opposed to the 16 of Vcpal) enables the distal methylene groups to fit into a surface pocket of the enzyme, in contrast to the solvent exposure of the end of the palmitoyl moiety. This fit comes along with closer binding of the terminal chain of the phenylindole to the rings of Tyr408 and His399. The 4-sulfamoyloxyphenyl moiety points to a surface region of SpnHyal that is not occupied by Vcpal. Inhibitory activity on Hyals The inhibitory effects of Vc (1), Vcpal (2), and the phenylindole inhibitor 4 were measured at pH 5.0 utilizing two Hyal enzymes, SagHyal from strain 4755 and SpnHyal. Concentrations for 50% inhibition (IC50) are summarzied in Table II. Vc was only weakly active against both enzymes with IC50 values of 6 mM for SagHyal and 32 mM for SpnHyal under our reaction conditions, respectively. On the contrary, Vcpal inhibited SagHyal with an IC50 of 4 μM, and the inhibition of SpnHyal was about 25-fold lower (Botzki et al., 2004). The phenylindole compound, 4, inhibits SagHyal with IC50 of 11 μM. Its IC50 could not be determined against SpnHyal because the phenylindole has limited solubility under our assay conditions. Following the trend of SagHyal “selectivity” observed on Vc and Vcpal, an IC50 value of compound 4 against SpnHyal was estimated to be between 200 and 300 μM. It is interesting to consider the possible reasons for the markedly lower inhibition of SpnHyal compared with SagHyal. The overall sequence identity of the crystallized Hyals SpnHyal (Li et al., 2000) and SagHyal from strain 3502 (Li and Jedrzejas, 2001) is only 53%, but the amino acids of the inhibitor-binding sites are essentially identical. The structures of the α- and the following β-domains of both enzymes are similar and have rms deviations of 1.54 and 1.18 Å, respectively. The flexibility behavior of both enzymes was analyzed by molecular dynamics (Jedrzejas et al., 2002, Mello et al., 2002), indicating similar major modes of motion, (1) rotation/twisting of the whole αdomain relative to the top half of the β-domain and (2) opening/closing of the substrate-binding cleft (Jedrzejas et al., 2002; Mello et al., 2002). Nevertheless, there may be differences in the free energy of the conformational changes leading to different substrate kinetics (apparent Km, Vmax) and/or inhibitor affinity. However, because Vmax does not differ significantly between the two enzymes and because a reduction of the HA concentration did not significantly lower IC50 values, the weaker SpnHyal activity of all Vcc Vcpalc R: (CH2)2Ph R: (CH2)3Ph ND, not determined. The assays were performed at pH 5.0 as described in the text. aSagHyal from strain 4755. bThe standard error of the mean IC50 (SEM) of four experiments was in all cases lower than 10%. cThe data of Vc and Vcpal are from Botzki and others (2004). dNot soluble under assay conditions in concentrations >50–100 μM. compounds should be rather attributed to the inhibitors themselves, for example, to slower association or higher dissociation rates because of the specific topology around the binding cleft or the flexibility of the enzyme–inhibitor complex. From Figure 5A, it becomes obvious that the inhibitors occupy only about half of the hexasaccharidebinding site, especially the product exit position where HA1 is located. The inner positive patch that interacts with HA3 disaccharide of the HA substrate (Figure 1) does not contribute to inhibitor binding. Motion mode 1 (see above) likely triggers the one-disaccharide unit shift of HA via specific conformational changes of the positive patch relative to residues from the active site and the negative patch (Jedrzejas et al., 2002; Mello et al., 2002). Thus, the flexibility of the enzyme–substrate and the enzyme–inhibitor complexes may differ. Structure-based design of benzoxazole-2-thiones as Hyal inhibitors The knowledge of the binding modes of Vcpal and the phenylindole 4 within the active site of SpnHyal provides several options for the design of more potent inhibitors. The combination of a small hydrophilic molecule like Vc or 6-sulfamoyloxyindole with a larger lipophilic residue like the hexadecanoic or the decyl moiety appears to be an appropriate strategy. Mainly hydrophilic, but more potent, inhibitors than Vc can serve as optimal anchors in the center of the binding site. Such compounds have been suggested by structure-based ligand design methods (Botzki et al., 2005). The most active inhibitor proposed by the program LUDI (Böhm, 1992) was 1,3-diacetylbenzimidazole-2-thione (3, Figure 2). Replacement of one of the N-acetyl fragments by oxygen has led to the benzoxazole-2-thione scaffold, which may be suggested to overlay with the indole moiety of compound 4. If this is the case, alkanoyl chains at the nitrogen should point into the same direction like the aliphatic tail of 4. Indeed, benzoxazolethione derivatives 5 (Figure 2) with different Nalkanoyl substituents were found to inhibit SagHyal from strain 4755. Comparison of their putative binding mode with that of the phenylindole 4, Vcpal, and the hexasaccharide substrate was used to optimize the benzoxazole-2thione lead. Docking of the benzoxazole-2-thione scaffold into the SpnHyal-phenylindole 4 complex in close superposition with the indole moiety of compound 4 suggests additional interactions (Figure 5C). The benzoxazole oxygen forms a H bond with the amide NH2 group of Asn290, and the thione sulfur is 2.65 Å distant from the indole NH of Trp291. The oxygen of the N-alkanoyl substituents interacts with the guanidine moiety of Arg462, with the OH group of Tyr408 and possibly also with a protonated Nτ imidazole atom of His399. If one projects the benzoxazolethione scaffold in this position into the SpnHyal-hexasaccharide complex, conclusions about optimal substituents can be derived. As shown in Figure 5C, 3-phenyl substituted Npropionyl derivatives are promising because the region at the 1,3-glycosidic linkage within HA1 corresponds to the two methylene groups and because the N-acetylglucosamine residue of HA1 overlaps with the terminal phenyl ring. Results on SagHyal strain 4755 inhibition indeed show that the 3-phenylpropionyl derivative is more potent than its acetyl and butyryl homologs (Table II). The position of the 3-phenylpropionyl moiety also corresponds to the methylene groups 2–5 and 2–6 of the alkyl chains of the phenylindole 4 and Vcpal, respectively. On the basis of these comparisons, further derivatives with the common scaffold 5 should share the following structural elements: a benzoxazole moiety as anchor in the upper part of the binding site, a propionic acid as spacer and a variety of ring-based 3-substituents such as morpholine, piperazine, cyclohexyl, phenyl, and 4-carboxyphenyl occupying the space of the N-acetylglucosamine residue. The synthesis of the representatives of the new compounds is currently ongoing. Conclusions Our continuing search for new and novel inhibitors of streptococcal Hyal recently led to the structure-based design, synthesis, and analyses of Vcpal (compound 2), 1,3-diacetylbenzimidazole-2-thione (compound 3), and the most recent 1-decyl-2-(4-sulfamoyloxyphenyl)-1Hindol-6-yl sulfamate (compound 4). The last compound, 4, with an IC50 value of 11 μM against SagHyal from strain 4755 is one of the most potent inhibitors of this enzyme known to date. The X-ray structure of the SpnHyal– phenylindole 4 complex demonstrated this compound binding in the active site of the enzyme and rationalized its competitive nature. Interactions with catalytic Asn349 and the amino acid residues of the catalytically essential hydrophobic patch were the basis for the inhibitory activity. On the basis of the binding modes of the phenylindole 4, a new compound, N-(3-phenylpropionyl)-benzoxazole-2thione, was predicted and confirmed as yet another strong Hyal inhibitor (IC50 value of 15 μM at SagHyal). All Hyal inhibitors described are being further investigated for their antibacterial properties in vivo. The inhibition of the spread of pathogenic Streptococci, especially in the early stage of their infections, will prevent the onset of bacterial disease, namely bacteremia and meningitis. The prospective importance of Hyal as target for the adjuvant therapy of such diseases prompts our further search for new inhibitors. In this respect, the binding modes of the phenylindole and the benzoxazole-2-thione lead structures provide the necessary information for further structure-based inhibitor design to optimize potency, selectivity, and pharmacokinetic properties. Materials and methods Materials Hyaluronan HA from Streptococcus zooepidemicus was purchased from Aqua Biochem (Dessau, Germany). Bovine serum albumin (BSA) was obtained from Serva (Heidelberg, Germany). All other chemicals were of analytical grade and were obtained either from Merck (Darmstadt, Germany), from Fisher Scientific (Pittsburgh, PA), or Sigma Chemical (St. Louis, MO). Enzymes and inhibitor preparations Stabilized SagHyal, that is, 200,000 IU of the lyophilized enzyme (0.572 mg from strain 4755, plus 2.2 mg BSA and 37 mg Tris–HCl per vial according to the information from the supplier) was kindly provided by id-Pharma (Jena, Germany). S. pneumoniae Hyal (Jedrzejas et al., 1998; Jedrzejas, 2000, 2001; Li et al., 2000) was produced as previously described (Jedrzejas, et al., 1998). The enzyme was concentrated to 5 mg/mL in 10 mM Tris-HCl buffer, 150 mM NaCl, 1 mM dithiothreitol (DTT) (pH 7.4) using centrifugal spin devices with a 50-kDa molecular weight cutoff (Millipore, Billerica, MA). This preparation was used for inhibition studies and for the production of crystals of complex with inhibitor. Enzyme concentration was determined photometrically at 280 nm using a calculated molar absorption coefficient of 127,090 L mol−1 cm−1 (Jedrzejas et al., 1998) according to Pace and others (1995). L-ascorbic acid-6-hexadecanoate was obtained as described (Botzki, et al., 2004). 1-decyl-2-(4-sulfamoyloxyphenyl)-1H-indol6-yl sulfamate (4) (Walter et al., 2004) and the benzoxazole2-thiones 5a–5c were synthesized in our laboratory as described (Braun, 2005). Determination of inhibitory activity on Hyals The inhibitory effects of the compounds on the Hyals from S. pneumoniae and S. agalactiae strain 4755 were measured using a turbidimetric assay (Di Ferrante, 1956). Enzyme activity was quantified by determining the turbidity caused by the residual high molecular weight substrate (molecular weight >6–8 kDa) precipitated with cetyltrimethyl ammonium bromide (CTAB). The incubation mixture contained 120 μL of citrate-phosphate buffer (solution A: 0.1 M Na2HPO4/0.1 M NaCl, solution B: 0.1 M citric acid/0.1 M NaCl, and solutions A and B were mixed in appropriate proportions to reach pH 5.0), 30 μl of BSA solution (0.2 mg/mL in water), 30 μL of HA substrate solution (2 mg/mL in water), 50 μL of H2O, 10 μL of dimethyl sulfoxide (DMSO), and 30 μL of enzyme solution. The assays were normalized by using equiactive concentrations of 2.9 ng of Hyal from S. agalactiae strain 4755 or 1.7 ng of Hyal from S. pneumoniae in 30 μL of BSA solution. To determine the enzyme activities in the presence of the test compounds, instead of 10 μL of DMSO, 10 μL of varying concentrations of inhibitors were used. The final DMSO concentration was 3.8% (v/v). After incubation of the assay mixture for 30 min at 37°C, 720 μL of a 2.5% (m/v) cetyltrimethylammonium bromide solution (2.5 g of CTAB dissolved in 100 mL of 0.5 M sodium hydroxide solution, pH 12.5) was added to precipitate the residual high molecular weight substrate and to stop the enzyme reaction. This mixture was again incubated at 25°C for 20 min, and the turbidity of each sample was determined at 600 nm with an Uvikon 930 UV spectrophotometer (Kontron, Eching, Germany). Experiments were performed in quadruplicate. The turbidity of the sample without inhibitor was taken as reference for 100% enzyme activity, whereas the turbidity of the sample without enzyme (30 μL BSA) was taken as reference for 0% enzyme activity. The activities were plotted against the logarithm of the inhibitor concentration, and IC50 values were calculated by curve fitting of experimental data with Sigma Plot 8.0 (SPSS, Chicago, IL). Molecular models of S. pneumoniae Hyal in complex with ligands Crystallization of the complex The hanging drop vapor diffusion method (McPherson, 1999) using Linbro culture plates (Hampton Research, Aliso Viecho, CA) at room temperature was used to grow the crystals of the enzyme–inhibitor complex. Equal volumes of protein, reservoir solution (1 μL each), and various amounts of inhibitor solutions (0.1, 0.5, and 1.0 μL) were mixed and equilibrated against 1 mL of the reservoir solution which contained 60– 65% saturated ammonium sulfate, 0.2 M NaCl, 2% dioxane, and 0.1 M sodium citrate buffer (pH 6.0) as reported for the native enzyme crystallization (Jedrzejas et al., 1998). X-ray diffraction The crystals of the enzyme–inhibitor complex were cryoprotected using 30% xylitol (w/v), 3.5 M ammonium sulfate, and 0.1 M sodium citrate buffer (pH 6.0) as reported for the native crystals (Li et al., 2000) and frozen in liquid nitrogen. Standard fiber loops (Hampton Research) of a suitable size were used to pick up and mount the frozen crystals under nitrogen flow at –180°C. The X-ray diffraction for the inhibitor complex data was collected using rotation (oscillation) photography and a Quantum 4u CCD detector. The crystallographic setup of beamline 5.0.1 of the Berkeley Center for Structural Biology, Advanced Light Source, Lawrence Berkeley National Laboratory was used. The collected data were analyzed, indexed, integrated, and scaled using the HKL2000 software package (Otwinowski and Minor, 1997). The resultant crystals were isomorphous to the native S. pneumoniae Hyal crystals (Jedrzejas, 1998). Structure solution and refinement The structure was solved by rigid body refinement in Crystallography and NMR System (CNS) (Brunger et al., 1998) using the crystal structure of S. pneumoniae Hyal in complex with Vcpal (Botzki et al., 2004) as a search model. The iterative approach proceeded with alternating cycles of computational refinement with CNS (Brunger et al., 1998) and manual rebuilding using O (Jones, et al., 1991). No intensity or sigma-based cut-offs were applied to the data. SigmaA-weighted map coefficients (Read, 1986) were used throughout. The Rfree value (Brunger, 1992), calculated for a test set of 5% of reflections, was used to monitor the progress of refinement. Difference density at the active site was apparent even in initial maps. The aliphatic portion of the inhibitor was relatively easily placed, but different orientations of the remainder of the inhibitor were required. The possibilities were assessed using the quality of electron density maps, B-factors, and the Rfree value as criteria. Sulfate and xylitol molecules, deriving from the crystallization and cryo-cooling solutions, respectively, were modeled into suitably shaped regions of electron density. Final statistics for the model are summarized in Table I. Programs of the CCP4 package (Collaborative Computational Project, Number 4, 1994) were used for manipulations and structural superpositions made with LSQMAN (Kleywegt, 1999). PYMOL was used to generate illustrations (DeLano, 2003). Modeling approaches were performed using the BIOPOLYMER module of the software package SYBYL 6.9 (Tripos, St. Louis, MO). Energy minimizations were based on the AMBER all-atom force field (Cornell et al., 1995) with AMBER all-atom charges (distance-dependent dielectric constant of 4) up to an rms gradient of 0.1 kcal/mole/Å (Powell conjugate gradient). Surface areas and lipophilic potentials (protein variant with the new Crippen parameter table [Heiden et al., 1993; Ghose et al., 1998]) of the model were calculated and visualized by the program MOLCAD (Brickmann et al., Tech. University Darmstadt, Germany) contained within SYBYL. The diffraction data were collected at the Berkeley Center for Structural Biology, Advanced Light Source, Lawrence Berkeley National Laboratory using beamline 5.0.1. The coordinates and structural factors of the SpnHyal–inhibitor complex have been deposited to the PDB with accession numbers 2BRP and PDB2BRP for the coordinates and structure factors, respectively. This study was supported in part by NIH grant AI44079 (M.J.J.), by the Graduate College 760 of the Deutsche Forschungsgemeinschaft, and by the Studienstiftung des Deutschen Volkes (A.B.). Conflict of interest statement None declared. 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Daniel J. Rigden, Alexander Botzki, Masatoshi Nukui, R. Brandon Mewbourne, Ejvis Lamani, Stephan Braun, Erwin von Angerer, Günther Bernhardt, Stefan Dove, Armin Buschauer, Mark J. Jedrzejas. Design of new benzoxazole-2-thione-derived inhibitors of Streptococcus pneumoniae hyaluronan lyase: structure of a complex with a 2-phenylindole, Glycobiology, 2006, 757-765, DOI: 10.1093/glycob/cwj116