Discovery of New Inhibitors of Resistant Streptococcus pneumoniae

5926 J. Med. Chem. 2009, 52, 5926–5936 DOI: 10.1021/jm900625q

Discovery of New Inhibitors of Resistant Streptococcus pneumoniae Penicillin Binding Protein (PBP) 2x by Structure-Based Virtual Screening Laurence Miguet,† Astrid Zervosen,‡ Thomas Gerards,‡ Farhan A. Pasha,† Andre Luxen,‡ Martine Disteche-Nguyen,§ and Aline Thomas*,† † Laboratoire de Dynamique Mol eculaire, Institut de Biologie Structurale Jean-Pierre Ebel (CEA/CNRS/UJF), 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France, ‡Cyclotron Research Center, All ee du 6 Ao^ ut, 8, B30, Li ege University, Ulg 4000 Li ege, Belgium, and § Centre d’Ing enierie des Prot eines, Institut de Chimie, B6a, B-4000 Sart Tilman, Li ege, Belgium

Received May 12, 2009

Penicillin binding proteins (PBPs) are involved in the biosynthesis of the peptidoglycan layer constitutive of the bacterial envelope. They have been targeted for more than half a century by extensively derived molecular scaffolds of penicillins and cephalosporins. Streptococcus pneumoniae resists the antibiotic pressure by inducing highly mutated PBPs that can no longer bind the β-lactam containing agents. To find inhibitors of PBP2x from Streptococcus pneumoniae (spPBP2x) with novel chemical scaffold so as to circumvent the resistance problems, a hierarchical virtual screening procedure was performed on the NCI database containing approximately 260000 compounds. The calculations involved ligand-based pharmacophore mapping studies and molecular docking simulations in a homology model of spPBP2x from the highly resistant strain 5204. A total of 160 hits were found, and 55 were available for experimental tests. Three compounds harboring two novel chemical scaffolds were identified as inhibitors of the resistant strain 5204-spPBP2x at the micromolar range.

Introduction a

Penicillin binding proteins (PBPs ) are enzymes involved in the extracellular steps of the biosynthesis of peptidoglycan, which is the main constituent of the bacterial cell wall.1,2 This bacterial envelope is composed of alternating sugar units of N-acetylglucosamine and N-acetylmuramic acid that are cross-linked with species-dependent stem peptides.3 Streptococcus pneumoniae possesses six PBPs, among which five are high molecular mass enzymes: three of them (PBP1a, PBP2a, PBP1b) catalyze both transglycosylation and transpeptidation and belong to class A, while the two others (PBP2b and PBP2x) catalyze transpeptidation only and belong to class B. Essential for the maintenance of the structural integrity of the bacterial cell, PBPs have been targeted by numerous antibiotics of the β-lactam type over the last 70 years.4,5 Streptococcus pneumoniae resists this antibiotic pressure thanks to its mosaic genes that induce highly mutated PBPs that can no longer bind to these antibiotics.6 PBP2x was shown to be the first to be altered in cefotaxime-resistant laboratory mutants7 and also in clinical isolates,8 and both PBP2x and PBP2b were identified as primary resistance determinants.9,10 *To whom correspondence should be addressed. Phone: 33-4-38-7895-72. Fax: 33-4-38-78-54-94. E-mail: [email protected]. a Abbreviations: spPBP2x, penicillin Binding Protein 2x from Streptococcus pneumoniae; R6-PBP2x, 5204-PBP2x, sp328-PBP2x, PBP2x of Streptococcus pneumonia from respectively from the sensitive R6 strain and the resistant 5204 and 328 strains; NAG, N-acetylglucosamine; NAM, N-acetylmuramic acid; ASPRE, active-site penicillin recognizing enzymes; RA, residual activity; BSA, bovine serum albumin; S2d, Nbenzoyl-D-alanyl-thioglycolate; LTV, lactivicin; PLTV, phenoxyacetyllactivicin; CPH, common pharmacophore hypotheses.

pubs.acs.org/jmc

Published on Web 09/11/2009

PBPs belong to the family of active-site penicillin recognizing enzymes (ASPRE). This family also contains β-lactamases that hydrolyze β-lactam rings, and D,D-peptidases that are responsible for cross-linking individual peptidoglycan strands. Even though there are differences in the structural arrangement and characteristics of the protein domains among the ASPRE members, they all share a common penicillin-recognition domain composed of a five-stranded β-sheet framed by two R-helices.11 Moreover, they all possess the ASPRE signature consisting of three motifs bordering the active site: S337XXK, S395XN, and K547XG (according to spPBP2x sequence numbering). The first motif begins with the catalytic serine and is located on the R2 helix, the second motif is located on a loop between the R4 and R5 helices, while the third motif lines the β3 strand. In spPBP2x, the active site forms a rigid network of hydrogen bonds involving S337, K340, S395, N397, K547, S548, and T550. Two aromatic residues W374 and F450 delineate the active site. Until now, 10 crystal structures of spPBP2x have been solved, either free or complexed to β-lactams in acylated forms, as listed in Table 1. To compare the 3D structures of PBP active sites, we will talk throughout this paper of rms deviation between the active sites, calculated on the backbones of the ten residues comprised in the ASPRE signature. The active sites of free or bound R6-PBP2x are very close structurally (rms deviation less than 0.5 A˚) but differ significantly from the sp328-PBP2x active site (rms of 1.2 A˚). This latter structure, refined at 3.2 A˚, is lacking 42 residues in the close proximity of the active site and notably the segment [365-394] which precedes the second conserved motif S395SN located between the R4 and R5 helices.14 Furthermore, its segment 355-364 is R-helix-folded, whereas r 2009 American Chemical Society

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19

it is in a more extended conformation in all other known PBP2x crystal structures. This lack of information and the structural characteristics of the sp328-PBP2x enzyme, along with the fact that high-throughput experiments can be performed on the highly resistant PBP2x from strain 5204 (MIC value for penicillin G of 6 μg/mL19) led us to choose the 5204PBP2x as the target of our lead discovery study. A homology model of this enzyme was built for this purpose. The transpeptidation reaction catalyzed by PBP2x is shown in Scheme 1. The catalytic serine S337 attacks the carbonyl carbon of the D-Ala4 of the “donor” stem peptide, thus a covalent acyl-enzyme complex is formed and the terminal D-Ala5 is released. The carbonyl carbon of the D-Ala4 of the “donor” stem peptide is then attacked by the lateral nitrogen of Lys3 of the second stem peptide, thus forming a covalent bond between both peptides. The β-lactam containing agents behave as mimics of the D-Ala-D-Ala terminus of the “donor” stem peptide. Upon binding to PBP2x, the Oγ atom of the catalytic serine S337 attacks the carbonyl carbon within the Table 1. PBP2x Crystal Structures Known to Datea PBP code resolution (A˚) ligand strain 1PMD 1QME 1QMF 1K25 1PYY 1RP5 2Z2L 2Z2M 2ZC3 2ZC4

3.5 2.4 2.8 3.2 2.4 3.0 2.8 2.6 2.5 2.8

cefuroxime

cefditoren biapenem tebipenem

R6 R6 R6 328 R6 mutant 5259 R6 R6 R6 R6

rmsd (A˚)

refs

nd 0.21 0 1.22 0.42 0.37 0.23 0.12 0.17 0.21

12 13 13 14 15 16 17 17 18 18

a

The RMSD were calculated on the backbone atoms of the ASPRE motif relative to the 1QMF crystal structure. The R6 strain is penicillin sensitive, while the 5259 strain16 and sp328 strain14 are resistant. The 5259 strain shows a MIC for penicillin G of 0.19 μg 3 mL-1,16 while the sp328 strain has a MIC for penicillin of 4 μg 3 mL-1.14 nd: Not determined as only the coordinates of the CR atoms are available.

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chemically reactive β-lactam ring, which results in the opening of the ring and the formation of a long-lived acyl-enzyme complex that leads to cell death.20 How the exact mechanism does work, and notably how the catalytic serine is deprotonated, is still under debate although hypotheses based on molecular dynamics have been proposed.21 The carboxylic group in the R position of the β-lactam ring nitrogen, which mimics that of the substrate D-Ala5, is in appropriate distance to deprotonate the serine as observed in the numerous crystallographic acyl-enzyme complexes. Even though the highly mutated PBPs of resistant strains of Streptococcus pneumoniae can still bind the stem peptides and assume their natural role in the biosynthesis of the peptidoglycan, they cannot recognize β-lactam compounds.6 Research projects on derivatives of β-lactam type agents are still ongoing, as assessed by the tebipenem activity against resistant strain PBP2x, in phase II of clinical trials.22 However, an attempt to drift away from the classical β-lactam scaffold may provide a solution to the cross-resistance problem. Examples have shown up in the literature where some research groups have synthesized new categories of molecules like γ-lactams,23 δ-lactams,24 arylalkylidene derivatives,25 and lactivicin derivatives,26,27 some of them showing activity against resistant-strain PBPs. We present in this paper the use of computational techniques to find compounds possessing new chemical scaffold and targeting the resistant 5204-PBP2x. Valuable leads targeting the D-Ala-D-Ala ligase, involved in the intracellular steps of peptidoglycan biosynthesis, have been found using similar theoretical tools.28,29 We performed a hierarchical virtual screening of the NCI database containing 260071 compounds, using both ligand-based and receptor-based filters. We employed as the first filter a pharmacophore model developed from an in-house database of compounds known to be active against PBP2x. As the second filter, we performed receptor-based molecular docking calculations using FlexX algorithm30 on a homology model of the 5204-PBP2x. The 160 best-ranked compounds resulting from the docking

Scheme 1. Transpeptidation Reaction Catalyzed by PBP2xa

a NAG: N-acetylglucosamine; NAM: N-acetylmuramic acid. Once the catalytic serine S337 is activated, it attacks the carbonyl carbon of the penultimate residue of the stem peptide (D-Ala4) and the D-Ala5 is released. The same carbonyl carbon is then attacked by the lateral nitrogen of the Lys3 of an adjacent stem peptide, resulting in the formation of a “4-3” cross-link.

5928 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19

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Figure 1. Alignment of the transpeptidase domain sequences of PBP2x from the sensitive strain R6 and from three resistant strains, 5259, sp-328, and 5204. Blue boxes delineate residues of the active-site pocket, i.e., residues having a heavy atom at less than 10 A˚ from any atom of the cefuroxime ligand (as seen in the 1QMF crystal structure13). The dots mark residues conserved throughout the ASPRE family, and the star indicates the catalytic serine.

calculations were selected. Among them, 55 were available for experimental testing and three showed activities in the micromolar range on the resistant 5204-PBP2x. Results and Discussion Homology Model. Multiple Sequence Alignment. The multiple sequence alignment shows that the 5204-PBP2x sequence shares the closest sequence identity (98%) with the sp328-PBP2x. Whereas the transpeptidase domain (residues 266-616) of the 5204-PBP2x differs by only seven mutations from the sp328-PBP2x, it differs by 41 residues from both the 5259 and the R6 sequences (see Figure 1). The residue substitutions observed in the 5204-PBP2x at positions (T338A, M339F, I371T, R384G, M400T, and N605T) account for nearly all the loss of affinity for β-lactams.31 Within the active site pocket, the sequence of sp5204-PBP2x differs respectively by 4, 5, and 12 residues from the R6, sp328, and 5259 active site sequences. Statistical analyses performed on 89 PBP2x sequences16 have classified these into three families, the R6-like sequences that are susceptible to β-lactams, and two sets of resistant-strain PBP2x, the first one containing a T338A mutation and the second one harboring a Q552E mutation. The sp328 and 5259 strain PBP2x are respectively representative of each of these families. Causal relationship hypotheses were proposed between their resistance and crystal conformation.16 The resistance of sp328-PBP2x is supposed to result from the high flexibility of its active-site attested by the nontraceability of the segment 365-394 which borders the cavity. On the contrary, the 5259-PBP2x structure has a well-defined active-site, and its resistance was attributed to its Q552E mutation located at the entrance of the active site and thus disfavoring the binding of negatively charged β-lactams.16

From both the multiple sequence alignment and the presence of the T338A mutation, the 5204-PBP2x belongs to the same resistant strain family as sp328. This was taken into account during the building phase of the 3D model by assigning some side-chain conformations delineating the active site as they are observed in the sp328-PBP2x crystal structure. Characteristics of the Active Site of PBP2x. To date, despite numerous crystallization efforts,32 no crystal structure of a noncovalently bound compound to PBP2x or of a ligand bound to a resistant strain PBP2x is known. Structural information for modeling the active site of the 5204-PBP2x was thus taken from both the R6-PBP2x that can bind ligands and from the sp328-PBP2x that has the closest sequence. In the R6-spPBP2x crystal structures, the bound ligands are efficiently maintained through a strong network of hydrogen bonds as can be seen in Figure 2. Residues T550, S548, and S395 have strong hydrogen bonds with cefuroxime and with all other bound β-lactams. The hydrophilic neighborhood is highly meshed with interconnecting hydrogen bonds involving residues S395, N397, K340, K547, and S548. The bound penicillins and cephalosporins have their respective 5- and 6-member ring stabilized through a hydrophobic interaction with the conserved W374 residue. Although the active sites of the free R6-spPBP2x are very similar, those of the complexed R6-PBP2x differ (see Figure 3). In the cefuroxime and cefditoren complexes, the Q452 side-chain is rotated toward the outside of the cavity. whereas it is pointing toward the cavity in all other PBP2x structures. The bulky cephalosporins require this rearrangement that involves the twisting of the facing Q552 side-chain,

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Figure 2. Two-dimensional representation of cefuroxime (in purple) as bound in the R6-PBP2x crystal structure (accession PDB code 1QMF) drawn with Ligplot.33 Hydrogen bond distances and hydrophobic interactions are indicated.

Figure 3. Active-site residues after superimposition of the ASPRE signature within the R6 apoenzyme13 (in gray) and the R6-PBP2x bound to cefditoren17 (in orange), cefuroxime13 (in cyan), tebipenem18 (blue), and biapenem18 (in pink). The cefuroxime moiety is atom-type color-coded.

pointing toward the cavity in the free enzymes and completely rotated in the cephalosporins bound enzymes. In the cefuroxime-bound PBP2x, the Q552 side-chain orientation allows the formation of a hydrogen bond with the nearby D567. Another important rearrangement can be seen in the cefditoren complex, where the W374 adopts a different rotamer compared to all PBP2x structures, thus pushing the E378 side-chain inside the cavity. Some other minor structural differences between the R6-enzymes can be observed, attesting to the adaptative plasticity of the active site that allows binding of different ligands. The resistant sp328-PBP2x14 is lacking coordinates for segments 365-394 and 524-535 that border the active site; its segment 355-364 is R-helix-folded, whereas it is in a more extended conformation in all other known PBP2x crystal structures. The conformation of the second conserved motif S395SN, located between the R4 and R5 helices, differs strongly from what can be seen in other PBP2x structures: the essential S395 residue points away from the catalytic cleft, while the N397 points toward the position occupied by the W374. The structural rearrangement of the S395SN motif, which disrupts the local hydrogen bonds network involving residues K340 and S337, was attributed to the presence of two clashing mutations in sp328-PBP2x, respectively S389L and N514H.34


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Building the Model. Docking studies are routinely made on rigid receptors,35 even though attempts to consider the target flexibility in the calculations have recently been published,36-38 as well as attempts to consider multiple targets.39 Modeller40 allows the use of multiple structures as starting templates for building the model, therefore introducing some conformational diversity, which is of major importance because crystallographic data support the internal flexibility of PBP2x. This automatic procedure used in Modeller takes into account all starting structures and for some parts the closest sequence guides the selection of the corresponding structure. The model was built from seven PBP2x crystal structures: four of them from the R6-strain, one from a laboratory mutant, and two from clinically isolated resistant Streptococcus. Some of the starting structures can bind β-lactam ligands (see Table 1), whereas some cannot, such as the sp328-PBP2x whose active site (as defined in the legend of Figure 1) differs by five residues (F339M, A378E, T400M, V572A, and F595Y) from that of 5204-PBP2x. Therefore, the model was refined by integrating specific structural information derived from the available structures. The Q452 side-chain was oriented as in the cefuroxime and cefditoren crystal complexes so that molecules containing bulky substituents could be selected from the docking calculations. The side-chain of residue S395, which has a determinant role in the resistance of sp328,34 was oriented pointing outside the cavity, as observed in the sp328-PBP2x structure only. However, after some cycles of energy minimizations, it returned to a favorable conformation to promote a hydrogen bond with the N397 side-chain as it is in the R6-enzyme complexes. Modeller places the H514 imidazole ring in a similar orientation to that adopted by the N514 side-chain in R6 PBP2x and by the H514 side-chain in 5259-PBP2x. It would clash with the backbone of residue L389 and with the Y392 side-chain (both not visible in the electronic density) if it was modeled as it is in the sp328-PBP2x structure. The energy minimizations showed that the facing mutations S389L and N514H could be accommodated without modification of the R4 helix backbone but with a slight twisting of the H514 imidazole ring. The hydrogen bond between S347 (an alanine in the R6 and 5259 sequences) and T352, observed in the sp328-PBP2x crystal structure, is conserved. The side-chain conformations of W374, N397, and T550 are in an intermediate position compared to those adopted in the R6 and the sp328 enzymes. An unprecedented hydrogen bond links the N397 side-chain with the backbone of W374; this bond might exist in the sp328 structure, as the N397 is pointing toward the W374 side-chain, which is not observed in the X-ray crystal structure. The F339 is pointing toward the neighboring F421, as does the M339 present in all other PBP2x structures. The M339F mutation, present in eight highly resistant strains, was shown to induce a 3-10-fold reduction of the reaction rate of PBP2x with β-lactams.15 The model was further refined by modeling the binding of the naturally occurring inhibitor, lactivicin (LTV) and its derivative, phenoxyacetyl-lactivicin (PLTV), both known to inhibit the 5204 PBP2x.27 These bicyclic γ-lactams have been cocrystallized with spPBP1b,27 whose active site is very close to that of spPBP2x (rms deviation equal to 0.49 A˚). Therefore, there is a strong probability that the interactions formed by the lactivicin molecules within the spPBP1b active


Miguet et al.

Figure 4. The active site of the 5204-PBP2x final model (in purple) superimposed on the crystallographic structures of cefuroximebound R6-PBP2x13 (in cyan) and sp328-PBP2x14 (in orange). The cefuroxime moiety is atom-type color-coded. The R4-R5 loops are represented as ribbons.

site are conserved in the 5204-PBP2x. The lactivicin ligands were manually inserted into the model active site according to their binding pose in the spPBP1b crystal structure, and the active site was energy minimized, as described in the Materials and Methods Section. The final minimized active site (Figure 4) showed rms deviations of 0.61, 1.02, and 0.87 A˚ with respectively the R6-PBP2x, sp328-PBP2x, and spPBP1b. The second conserved segment S395SN showed a rms deviation on all heavy atoms of 1.4 and 0.9 A˚ with respectively the sp328 and cefuroxime-R6 enzymes. The K340 was hydrogen bonded to N397 and S395. Validation of the Model. The backbone conformation of the model was analyzed with the MolProbity41 algorithm: 97.3% of the nonglycine residues lie in the allowed Ramachandran regions. The quality of the model was evaluated by its ability to discriminate known inhibitors from inactive molecules. An in-house database comprising 20 compounds with known activities against sp5204-PBP2x (unpublished results) was built. It contained 10 inactive molecules of various chemical scaffolds and 10 inhibitors, eight of β-lactam type, and the two bicyclic γ-lactams LTV and PLTV. The docking calculations performed with the docking protocol described in the Materials and Methods Section could retrieve all known 5204-PBP2x inhibitors and could discard seven noninhibitors. The best docking solutions of all known inhibitors interacted with active site residues in a consensual binding mode, similar to the covalent adducts observed in PBP2x and PBP1b. The carbonyl groups of the β- and γ-lactam rings interacted with the Oγ S337 and with the nitrogen amide of T550. The docking protocol was also validated as the PLTV predictive binding pose yielded a better FlexX docking score (-24.8 kcal/mol) than LTV (-11.6 kcal/mol), in agreement with experimental data.27

Figure 5. The best CPH superimposed on (a) cefuroxime, (b) lactivicin docked into the 5204-PBP2x active site (N397 residue is not shown for clarity). The hydrogen bond acceptor sites are schematized as blue spheres (A1, A2, A3), and the negative center (N1) is represented by a red sphere.

The Virtual Screening Process. The Pharmacophore Model. An initial pharmacophore model was built using the 33 active molecules of the training set (Table 1, Supporting Information). A maximum of four chemical features were allowed so that nine common pharmacophore hypotheses (CPH) could be generated, all based on three hydrogen bond acceptor sites and one negative center (AAAN). The best hypothesis was selected by survival, vector, and volume scores (respectively equal to 2.615, 0.886, and 0.269). It retrieved 30 active molecules from the in-house database and discarded all five inactive molecules. The outlying compounds (Loracarbef,42 a bridged-lactam23 and N-benzoyl43,44 D-alanyl-thioglycolate ) exert a maximum of three pharmacophoric features. The best CPH, which is composed of four features, namely three hydrogen bond acceptor sites and one negative center, is shown in Figure 5. The negative center (N1) and the hydrogen bond acceptor (A2) represent respectively the carboxylate moiety and the carbonyl carbon of the β-lactam compounds, both directly involved in the enzymatic mechanism. The best CPH was refined using an external set of three PBP2x inhibitors as 4-quinolone derivatives,45 thus chemically different from the training set molecules. These molecules match well to three features out of the four of the best CPH, with a

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tolerance of 2 A˚. Thus we screened the NCI database using the best CPH found here-above with an additional optional criterium indicating that at least three features out of the four should be present in the selected hits. Among the 260071 compounds within the NCI database, a total of 6151 were retrieved using this pharmacophore model and were subjected to further screening by molecular docking. The geometric and score details of the pharmacophore model are available in the Supporting Information. The Docking Calculations. Prior to the docking calculations, the multisearch option of the SYBYL interface46 generated 36211 initial molecular conformations from the 6151 compounds selected by the pharmacophore mapping step. These were subjected to the FlexX-Pharm47 docking procedure. A scoring filter was set arbitrarily to -25 kcal/mol so as to retain 25% of the docked solutions. Those compounds satisfying the two receptor-based pharmacophoric restraints, thus having hydrogen bond acceptors facing the Oγ of S337 and the backbone nitrogen of T550, were retained. The best conformer of each compound in terms of FlexX score was visually inspected, and those having favorable interactions with residues known to contribute

Figure 6. Dose-response curve from inhibition of the 5204-PBP2x by compound 2. An IC50 value of 71 μM ((4%) was calculated from this curve.

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significantly to the stabilization of β-lactam compounds, and particularly S548, S395, and K547, were selected. Interestingly, this screening procedure could retrieve solutions containing a peptidomimetic end, mimicking the natural substrate of PBPs. Some β-lactam containing solutions could also be selected, thus validating the overall procedure. These compounds were not retained for experimental tests to avoid cross-resistance problems. Finally, 160 molecules were proposed for experimental validation. Experimental Results and Structural Analysis. Among these predicted inhibitors, 55 were available for experimental tests and 43 could be tested due to good properties such as high solubility and no interference with experimental assays (absorbance 1000 336 ((15%) 255 ((25%)

219 ((5%) 71 ((4%) 72 ((11%)

no inhibition 259 ((1%) 88 ((15%)

a

Calculated logP48 and IC50 values. % Standard error of estimate.


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Figure 7. Best CPH superimposed on compounds 1, 2, and 3 that have respectively their carbons colored in cyan, gray, and green. Table 3. Pharmacophore Details for the Three Hits compd

matched sites

align score

vector score

volume score

fitness

1 2 3

A1 A2 - N1 - A2 A3 N1 - A2 A3 N1

0.995341 1.140240 0.749978

0.857189 0.89953 0.80792

0.321549 0.308869 0.263158

1.349288 1.258199 1.446096

The compounds 1, 2, and 3 were active in the presence of 0.01% Triton X-100. The inhibition of 5204-PBP2x was not time-dependent; inhibition activity with compounds 1, 2, and 3 was observed without preincubation time. This indicated that these compounds were not promiscuous inhibitors.49,50 Kinetic studies were performed to determine the enzyme inhibition model that describes the interaction between the 5204-PBP2x and the inhibitors. This can be done by measuring the initial rates of S2d hydrolysis near the Km value and up to 10 times the Km value in the presence of different inhibitor concentrations.51 In the presence of the highest concentration of S2d that can be studied by spectrometry (i.e., 7 mM), a linear relationship between the initial rates and the substrate concentration was still observed. This indicated that it is only possible to determine the kcat/Km value of S2d (value equal to 350 ( 50 M-1 s-1) as described by others52 but not to study the initial rates of S2d hydrolysis up to 10 times the Km value. Therefore, it was not possible to determine the inhibition model of 5204-PBP2x by kinetic studies. The HPLC analyses proved that the purity of compounds 1 and 2 was higher than 95% and that of compound 3 was 85%. The three hits superimposed on the best CPH are represented in Figure 7, and their pharmacophore scores and fitness data are detailed in Table 3. They all possess a negative center and two hydrogen bond acceptor sites. Compounds 2 and 3 contain the A2, A3, and N1 features, typical of β-lactams. Compound 1 possesses feature A1 instead of A3, like the Loracarbef42 inhibitor. The lowest-energy conformers of the three hits docked in the 5204-PBP2x model are represented in Figure 8a,b,c. The three hits possess a common sulfonate center, which mimics the carboxylate moiety of the known β-lactam inhibitors. The transpeptidation reaction catalyzed by PBP2x involves as the first step the deprotonation of the catalytic S337, which can then perform the nucleophilic attack of the

Figure 8. Lowest energy conformers of respectively compounds 1, 2, and 3 docked in the active site of the 5204-PBP2x model. The T550 and W374 residues are respectively located above and under the ligands.

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carbonyl carbon of the D-Ala4 or of the β-lactam ring. The sulfonate group common to the three hits is ideally positioned to deprotonate the catalytic S337. It is in strong hydrogen bonds with the serine residues S395 and S548. All three hits possess a phenyl ring leaned back on the W374 side-chain. Compounds 2 and 3 both harbor three phenyl groups, so that a local hydrophobic core forms with the adjacent W374 and F450 residues. This hydrophobic core is also present in the cefuroxime and cefditoren R6-PBP2x complexes with respectively a furan and a thiazole ring. It is interesting to note that compounds 2 and 3 would clash with the Q452 side-chain positioned as it is in the biapenem and tebipenem R6-PBP2x crystal complexes. Compounds 2 and 3 possess an internal hydrogen bond between the sulfonate group and the amide nitrogen located in the ortho position. They are hydrogen bonded to residues T550, S548, S395, N397, and S337. The N397 side-chain conformation allows a hydrogen bond with the oxygen atom linking the two phenyl rings. Conclusion We have conducted a hierarchical virtual screening of the NCI database comprising 260071 compounds to identify novel leads of a resistant-strain spPBP2x. This enzyme is involved in the biosynthesis of pneumococcal bacterial membrane and is a primary resistance determinant.9,10 A qualitative pharmacophore model was developed from an in-house database containing molecules known to be active and inactive on PBP2x. Compounds matching this pharmacophore were retrieved from the NCI database and docked in a homology model of the highly resistant strain 5204-PBP2x. Among the highest-ranked solutions, some molecules contained chemical scaffolds known to be recognized by the enzyme such as a peptide group or a β-lactam ring, thus validating the homology model as well as the docking protocol. To avoid the cross-resistance problems, the β-lactam containing molecules were not retained for experimental testing. Among the 43 biochemically tested molecules, three hits with novel chemical scaffold could be identified: two ortho-phenoxyl diphenylurea derivatives and one aminothiadiazole derivative. They all showed higher activity on the resistant strain 5204-PBP2x than on the sensitive strain enzyme. The diphenylurea core compounds have IC50 values close to 70 μM against 5204-PBP2x, while the aminothiadiazole compound has an IC50 value close to 220 μM. These molecules act as noncovalent binders, which is to our knowledge unprecedented for this target. They offer promising starting points for chemical optimization. Finally, it is important to note that PBP2x inhibitors can generally target other members of the ASPRE family. Penicillin G for example is known to be recognized by the staphylococcal β-lactamase, Streptomyces R61 D,D-peptidase, and many PBPs from Escherichia coli and Staphylococcus aureus. We thus expect that compounds presented in this study might target other members from the ASPRE family. Detailed activity profile of these compounds and their optimization derivatives are underway. Materials and Methods All calculations were performed on four Intel-based Linux workstations. Ligands Preparation. In-House Database. The pharmacophore model was developed from an in-house database totaling 41 molecules. Among these, 36 were assessed inhibitors of


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spPBP2x as known from crystallographic studies and/or biochemical or microbiological data. All the active molecules were β-lactams except two γ-lactams,23,27 three quinolone derivatives,45 and a thioester analog of the stem peptide.51 A bridged γlactam23 active on the structurally and functionally related streptococcal spPBP1b was also considered as active to provide some molecular diversity. Indeed, the active sites of spPBP1b27 and R6-spPBP2x13 are very similar (rms deviation of 0.49 A˚) and their ASPRE signatures share 70% of sequence identity. Five inactive molecules on spPBP2x were included in the database: a peptide compound mimicking the natural substrate53 and derivatives of arylalkylidene rhodanine and arylalkylidene iminothiazolidin-4-ones.51 The three quinolone derivatives45 active on spPBP2x were considered separately in an “external set” used for the refinement phase of the pharmacophore model. The in-house database is fully detailed in the Supporting Information. The low-energy conformers of the compounds were generated using the OPLS-2005 force field54 and the hybrid Monte Carlo multiple minima/low mode search (MCMM/LMod) as implemented in the LigPrep55 module of the Schro¨dinger Suite (Schro¨dinger LLC, New York). Relevant ionization states were obtained using the Hammett and Taft methodology implemented in the Epik56 module. NCI Database. The 2003 release of the NCI database, containing 260071 compounds, was screened. The compounds were available from the ZINC version 6 Web site48 in mol2 format, annotated with calculated logP and in multiple tautomeric forms when necessary. Relevant protonation states and atomic charges were calculated as performed for the in-house database. The Pharmacophore Model. A qualitative pharmacophore model was derived from the training set of 33 active molecules using the PHASE module57 of the Schro¨dinger Suite (Schro¨dinger LLC, New York). Common pharmacophore features hypotheses (qualitative pharmacophore models) were developed by comparing a set of conformational models with a number of 3D configurations of chemical features shared among the training set molecules. One hundred conformers were generated for each compound of the training set using the MCMM/LMod method with OPLS-2005 force field54 with a 10 kcal/mol cutoff value of energy difference and a distancedependent dielectric function. Nine common feature hypotheses were generated using a tree-based partitioning algorithm with maximum tree depth of 4. The allowed tolerance on the intersite distances was set to 1 A˚. The features like hydrogen bond acceptor (A), hydrogen bond donor (D), hydrophobic group (H), negatively charged group (N), positively charged group (P), and aromatic ring (R) were considered. Receptor Preparation. A homology model of the PBP2x from the highly resistant strain 5204 was built using the comparative modeling tool Modeller,40 which finds the most probable structure for a sequence given its alignment with related crystallographic structures called templates. The model is obtained by the optimal satisfaction of spatial restraints derived from the alignment and expressed as probability density functions for the features restrained. The optimization procedure is a variable target function method that applies a conjugate gradient algorithm to position all non-hydrogen atoms. The seven PBP2x crystal structures that were available at the beginning of this study (with respective accession PDB codes 1QMF, 1QME, 2Z2L, 2Z2M, 1PYY, 1RP5, and 1K25) were used as templates for building the homology model, and their sequences were aligned using the program ClustalW.58 Among the 10 models generated, the one showing the lowest energy and restraint violations was selected for further refinement using energy minimization with the SYBYL7.3 program.46 The protonation states were assigned from previous pKa calculations.21 The molecular system was described with the Amber force field,59 and Kollman charges60 were assigned to all atoms. A distance-dependent dielectric function was chosen for the


electrostatic interactions, and a nonbonded cutoff of 8 A˚ was applied. The model was energy-minimized using steepest descent algorithm until a gradient of 0.01 kcal/mol 3 A˚ was reached. The quality of the model was assessed with Molprobity.41 Docking Procedure. The incremental construction algorithm FlexX30 in the SYBYL7.346 program was used for all docking studies, with the FlexX-Pharm47 option, which allows selection of binding poses meeting specified pharmacophoric criteria. During the docking process, FlexX performs a complete systematic search for the conformation, orientation, and position of a compound in the defined binding site. The active site was defined as a sphere of 8 A˚ radius centered on the catalytic S337 residue. The hydrogen bond donors located at the Oγ of the catalytic S337 and at the backbone nitrogen of T550 were introduced as receptor-based pharmacophoric constraints. These atoms are known to be essential to ligand binding in PBP2x; indeed, the distance between the carbonyl oxygen of the β-lactam ring and the amide nitrogen of T550 is less than 2.9 A˚ in the cefditoren, cefuroxime, tebipenem, and biapenem crystal complexes. To be selected in the filtering process, ligands should possess hydrogen bond acceptor atoms facing these two donor sites. The flexibility of the ligands was taken into account by combining two conformational search algorithms: prior to the docking procedure, the multisearch option of the SYBYL interface46 was used to generate six conformers for each compound, and for each of these conformers, 30 conformers were generated with the FlexX algorithm so that a maximum of 180 conformers was built per ligand. The FlexX score value, as an estimation of the free energy of binding of the lowest energy conformation, was used to rank compounds. Biological Evaluation. PBP2x-5204, PBP2x-R6, and R6PBP1b from Streptococcus pneumoniae were prepared as previously published.31 Standard protocols were used for preparing fluorescein-labeled ampicillin61 and S2d.43,44 Screening Experiments with R6-PBP2x and 5204-PBP2x. High throughput screening is widely used in drug discovery. To study a maximum number of compounds with different PBPs in a short-time assay, conditions were developed that allow the use of 96-well microtiter plates for the assays. A preincubation of the compounds with the targets, lasting 1 h in the case of the sensitive R6-PBP2x and 4 h in the case of the resistant 5204-PBP2x was performed to allow the detection of slow binding inhibitors. The residual activity (RA) of PBPs was then determined by observing the hydrolysis of the thioester S2d catalyzed by the noninhibited enzyme. Assays were done under the following conditions: compounds were dissolved in DMF (concentration: 100 mM); screening experiments were done with final concentrations of 1 mM or 100 μM, then diluted with 10 mM sodium phosphate buffer (pH 7.0). The final concentration of DMF in the assays was 1%. In the preincubation step, 0.09 μM R6-PBP2x was incubated with the compounds in 10 mM sodium phosphate buffer (pH 7.0) and 0.01 mg/mL BSA for 60 min at 25 °C, while 0.6 μM 5204-PBP2x was incubated with the potential inhibitors in 10 mM sodium phosphate buffer (pH 7.0), 70 mM D-alanine, and 0.01 mg/mL BSA for 4 h at 25 °C (volume 127 μL). The RA were determined by measuring the initial rate of hydrolysis of 1 mM S2d in the presence of 0.5 mM 4,40 -dithiodipyridine or 1 mM DTNB (volume: 150 μL). The increase of absorbance at 324 nm (4,40 -dithiodipyridine: ε[Δε] = 20000 M-1 s-1) or at 412 nm (DTNB: ε[Δε] = 13600 M-1 s-1) were observed for some minutes with a microtiter plate reader PowerWave (Bio-Tek Instruments). The rate of spontaneous hydrolysis of S2d in the presence of the inhibitors was also determined in the absence of the enzymes. All assays were done three times, and the determination of RA in the absence of inhibitors was repeated six times on each plate. Screening Experiments with R6-PBP1b. Unfortunately, S2d is a very poor substrate of PBP1b and it is not possible to perform high throughput screening with this enzyme. The use of

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fluorescein labeled ampicillin requires the use of SDS-PAGE for the separation of the protein and does not allow high throughput assays. Inhibition studies with PBP1b were only performed for those compounds showing good inhibition of spPBP2x. PBP1b (0.7 μM) was incubated with the potential inhibitors in 10 mM sodium phosphate (pH 7.0) for 60 min at 30 °C. Active PBP was then counter-labeled with 10 μM fluorescein-labeled ampicillin for 20 min. The reaction was stopped and analyzed by SDS-PAGE, followed by fluorescence visualization using a Molecular Imager FX (Bio-Rad) and the program Quantity One (Bio-Rad). Background fluorescence was subtracted. Screening of “False-Positives”. A testing compound was considered as an inhibitor if RA was smaller than 80%. In this case, to detect false positives, assays in the presence of 0.01% Triton-X-100 were done. As described in the literature, promiscuous inhibitors (false positives) are slow binding, noncompetitive inhibitors. To avoid a detailed kinetic research,51 it is possible to identify such compounds by performing assays in the presence of Triton-X-100,49,50 which eliminates inhibition by promiscuous compounds. Experiments were done under the same conditions as described above for the different PBPs in the presence of 0.01% Triton-X-100 v/v. Experiments were also performed without preincubation with Triton-X-100. Determination of IC50 Values. IC50 values were determined for RA values smaller than 50%. (RA = 50%, IC50 = c; c: concentration of compound in the assay; RA > 50%, IC50 > c). Experiments were done under the same conditions as described above for the different PBPs in the presence of decreasing concentrations of inhibitors. IC50 values were determined from a plot of the residual activities against inhibitor concentrations and 0.01% Triton X-100. Fit was determined using the software Sigma Plot (Systat software) and equation 1 as already published27 Y ¼ y0 þ ða bÞ=ðb þ xÞ

ð1Þ

Purity of the Hits. The purity of the compounds of the NCI database that were found as hits from the virtual screening procedure was controlled by HPLC. The analytical method was HPLC of reverse-phase (XTerra RP18 (150 mm 4.6 mm, 3.5 μm), Waters, Milford, Ireland) with an HPLC-chain consisting of a pump (Waters 600), a UV detector (PDA Waters 996), a Waters temperature control module, and an auto sampler (Waters 717plus). Wavelengths between 198 and 400 nm were recorded. The peak area of the compounds was calculated using Empower software (Waters, Zellik, Belgium). The following protocol was used: solvent A: water (Millipore quality) containing 0.1% trifluoroacteic acid and solvent B: acetonitrile (HPLC-Gradient grade, Fisher Scientific, Leicestershire, UK). Flow: 0.7 mL/min; gradient: 0-2 min 100% A; 2-32 min: 0-100% B; 32-36 min: 100% B; 37-56 min: 100% A. The injection volume was 20 μL.

Acknowledgment. We are indebted to Patricia Amara, Laurent David, and Jean-Marie Frere for critical reading of the manuscript. We thank Otto Dideberg for very enthusiastic and helpful discussions at the beginning of the work. We thank Andre Zapun for the gifts of R6-PBP2x and 5204PBP2x, and we thank Andrea Dessen and Alexandre Dos Santos Martins for the gift of PBP1b. We thank the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, for supplying the 160 compounds. This work was supported by the European Commission Sixth Framework Program grant LSMH-CT-EUR-INTAFAR 2004-512138. Supporting Information Available: The in-house database, the best pharmacophore hypothesis for the training set and external

Article

set molecules, as well as the mass spectrometry analyses of the three hits. This material is available free of charge via the Internet at http://pubs.acs.org.

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Discovery of New Inhibitors of Resistant Streptococcus pneumoniae

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