Beneficial effects of MG132 on nuclear remodeling, transcript abundance and embryonic development have also been shown for embryos constructed by somatic cell nuclear cloning in mice [22,23], rats [24,25], goats [23] and pigs [7,26,27]. Unlike for addition from 0? h, MG132 added from 16?2 h did not improve oocyte competence by improving nuclear maturation because the percentage of oocytes that were MII at the end of maturation was not affected by MG132 later in maturation. Rather, some of the beneficial effect of MG132 from 16?2 h on the percentage of oocytes that Data are least-squares means 6 SEM of values from six replicates. Values in the same column with different superscript letters are significantly different (P,0.05). e N.S. = non-significant (P.0.10).

Table 5. Effect of treatment with 10 mM MG132 from 0? or 16?2 h of maturation on meiotic maturation at 22 h after initiation of maturation (Experiment 5).Data are least-squares means 6 SEM of values from three replicates. GVBD: germinal vesicle break down; MI: metaphase I; Ana-Telo: anaphase ?telophase; MII: metaphase II. c,d,e Values in the same column with different superscript letters are significantly different (P,0.05 or, for). f N.S. = non-significant (P.0.10).became blastocysts was due to 1) increased cleavage rate through actions not involving fertilization rate and 2) increased competence of the fertilized oocyte to develop to the blastocyst stage. Indeed, the potential of a newly formed embryo to become a blastocyst was improved by addition of MG132 from 16?2 h in two of three experiments evaluated, as indicated by a significant improvement in the percentage of cleaved embryos that became blastocysts. The mechanism by which MG132 late in maturation improves competence of the oocyte to support development is likely to involve arrest of processes mediated by proteasomes that ordinarily compromise the oocyte. One result is likely to be increased transcript abundance for genes required for embryonic development, as shown in the pig oocyte [7]. In the mouse, MG132 improved oocyte competence in aged oocytes but did not affect non-aged oocytes [6]. It might be that MG132 blocked proteasome-mediated degenerative changes in a portion of maturing oocytes of inferior quality caused by prolonged culture during maturation or other reasons. Proteomic analysis was performed to determine possible targets of proteasomal cleavage whose relative expression was altered by MG132 treatment from 16?2 h. Such proteins might be involved in the beneficial effects of MG132 on oocyte competence and may
be important molecules for determining the ability of an oocyte to complete the first cleavage division and support development of the embryo to the blastocyst stage. One limitation to the experimental approach was that less abundant proteins were less likely to be detected by mass spectrometry. Nonetheless, a total of 653 proteins could be analyzed for differences in amount between oocytes treated with vehicle or MG132. Surprisingly, there were a greater number of proteins whose relative expression was decreased by MG132 than there were proteins that were increased. Regulation of intracellular proteins in the presence of MG12 is complex. In HEK293T cells, MG132 can increase ubiquitination of some proteins and decrease ubiquitination of others [28]. Some proteins in the bovine oocyte increase in abundance during oocyte maturation whereas others decline in amount [29]. It is possible that inhibition of the proteasome by MG132 late in maturation protected some proteins from proteolysis, which in turn hastened or exaggerated the maturation-dependent decline in other oocyte proteins. Six of the proteins that were decreased by MG132 (ADSL, AHCY, CDK5, GSTM3, STIP1, and THOP1) and two that were increased by MG132 (CAND1 and GAPDH) are encoded for by transcripts that decrease during nuclear maturation of bovine oocytes [30].

Table 6. Effect of treatment with 10 mM MG132 from 0? or 16?2 h of maturation on fertilization rate (Experiment 6).a MG132, 0? h No No Yes Yes Probability of treatment effects MG132, 0? MG132, 16?2 Interaction Data are least-squares means 6 SEM of values from four replicates. N.S. = non-significant (P.0.10). Figure 1. Expression levels and detection of peptide of Cullin-associated NEDD8-dissociated protein1 (CAND1). Panel A: Mean 6 SEM of CAND1 expression for control and MG132-treated oocytes. There was a difference (P = 0.004) between treatments. Panel B: Reporter ion expression for the C peptide fragment of CAND1. 114 and 115 represent two separate biological replicates of control oocytes while 116 and 117 represent two separate biological replicates of MG132-treated oocytes. Panel C: b and y ions and amino acid sequence from one peptide fragment of CAND1. Among the oocyte proteins regulated by the proteasome are proteins involved in RNA processing [2,3] so inhibition of proteasomal activity with MG132 could affect stability and translation of a variety of mRNA. There were 6 annotated proteins identified whose relative expression was increased by MG132 (ACAT1, CAND1, TUBACA1C, P4HB, HYOU1, and GAPDH). The increase in GAPDH may be a direct result of inhibition of the proteasome because intracellular amounts of GAPDH are regulated by ubiquitination [31,32]. Another mechanism may be involved in regulation of CAND1 by MG132. This protein interferes with ubiquitin ligase activity [33]. Perhaps, inhibition of cleavage of ubiquitinated proteins leads to increased synthesis or decreased degradation of CAND1 through feedback mechanisms. Other proteins involved in the ubiquitin pathway were decreased by MG132, notably HSP90B1, THOP1, UBA1, and VCP. None of the 6 annotated proteins increased by MG132 have been identified as a marker of oocyte competence. Nonetheless, an increase in amounts of these proteins could potentially affect oocyte competence. GAPDH, for example, catalyzes an important step in glycolysis. Glycolysis in the bovine oocyte is low and most pyruvate for the oocyte is supplied by the surrounding cumulus cells [34]. There is some evidence, though, that rate of glycolysis in the bovine oocyte is proportional to developmental competence[35]. Another protein increased by MG132 was TUBA1C. Tubulins are important for organelle movement in the oocyte and completion of meiosis [36,37]. Two other upregulated proteins, P4HB and HYOU1, function in protein folding [38,39].

Abstract
Staphylococcus aureus is a major human pathogen and one of the more prominent pathogens causing biofilm related infections in clinic. Antibiotic resistance in S. aureus such as methicillin resistance is approaching an epidemic level. Antibiotic resistance is widespread among major human pathogens and poses a serious problem for public health. Conventional antibiotics are either bacteriostatic or bacteriocidal, leading to strong selection for antibiotic resistant pathogens. An alternative approach of inhibiting pathogen virulence without inhibiting bacterial growth may minimize the selection pressure for resistance. In previous studies, we identified a chemical series of low molecular weight compounds capable of inhibiting group A streptococcus virulence following this alternative anti-microbial approach. In the current study, we demonstrated that two analogs of this class of novel anti-virulence compounds also inhibited virulence gene expression of S. aureus and exhibited an inhibitory effect on S. aureus biofilm formation. This class of anti-virulence compounds could be a starting point for development of novel anti-microbial agents against S. aureus.
Citation: Ma Y, Xu Y, Yestrepsky BD, Sorenson RJ, Chen M, et al. (2012) Novel Inhibitors of Staphylococcus aureus Virulence Gene Expression and Biofilm Formation. PLoS ONE 7(10): e47255. doi:10.1371/journal.pone.0047255 ?University Medicine Berlin, Germany Editor: Stefan Bereswill, Charite Received July 2, 2012; Accepted September 10, 2012; Published October 15, 2012 Copyright: ?2012 Ma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (NIH) Grant P01HL573461 (HS), University of Michigan Life Sciences Institute Innovation Partnership grant (HS and SDL), and NIH Pharmacological Sciences Training Program Grant T32 GM007767 (BDY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: HS, SDL and BDY are co-inventors on a US patent 61/641,590 entitled: Methods and Compositions for treating bacterial infections, filed May 2, 2012. One of the co-authors, MC, is employed by a commercial company (Nanova, Inc.). This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: [email protected] (HS); [email protected] (SDL)

Introduction
Staphylococcus aureus is a major human pathogen that causes skin, soft tissue, respiratory, bone, joint and endovascular infections, including life-threatening cases of bacteremia, endocarditis, sepsis and toxic shock syndrome [1]. Approximately 30% of humans are Staphylococcus aureus carriers without symptoms [2]. S. aureus is also one of the most common pathogens in biofilm related infections of indwelling medical devices which are responsible for billions in healthcare cost each year in the United States [3?]. Bacteria can attach to the surface of biomaterials or tissues and form a multilayered structure consisting of bacterial cells enclosed in an extracellular polymeric matrix [9]. Bacteria in biofilm are particularly resistant to antibiotic treatment [10]. In addition to the difficulty of effectively inhibiting biofilm with conventional antibiotic therapy, treatment is further complicated by the rise of antibiotic resistance among staphylococci. In recent years, methicillin resistance in S. aureus is approaching an epidemic level [2,11?3]. The emergence of antibiotic resistance poses an urgent medical problem worldwide. Current antibiotics target a small set of proteins essential for bacterial survival. As a result, antibiotic resistant strains are subjected to a strong positive selection pressure. Inappropriate and excessive use of antibiotics have contributed to the emergence of pathogens that are highly resistant to most currently available antibiotics [14?6]. The novelapproach of inhibiting pathogen virulence while minimizing the selection pressure for resistance holds great promise as an alternative to traditional antibiotic treatment [17]. The feasibility of such an approach was demonstrated for Vibrio cholerae infections when a novel small molecule was identified that prevented the production of two critical virulence factors, cholera toxin and the toxin coregulated pilus. Administration of this compound in vivo protected infant mice from V. cholerae [18]. In a similar proof-ofconcept (POC) study, a small molecule inhibitor of the membraneembedded sensor histidine kinase QseC was identified. The inhibitor exhibited in vivo protection of mice against infection by Salmonella typhimurium and Francisella tularensis [19]. In a POC study following the same paradigm, we have identified a chemical series of small molecules from a high throughput screen (HTS) that can inhibit expression of the streptokinase (SK) gene in group A streptococcus (GAS) [20]. We previously demonstrated that SK is a key virulence factor for GAS infection [21]. SK activates human plasminogen into an active serine protease that degrades fibrin, a critical component of blood clots and an important line of defense against bacterial pathogens [22,23] Our novel SK gene expression inhibitor also inhibited gene expression of a number of important virulence factors in GAS. The lead compound demonstrated in vivo efficacy at protecting mice against GAS infection, further supporting the feasibility of this novel anti-virulence approach to antibiotic discovery [20].

SU5416 is a Ligand of the AHR
To confirm that this molecule is a direct ligand of the AHR and not working through some other agonist, we performed competitive binding assays of the AHR using a radioligand. Photoaffinity experiments incubating 125IBr2N3DpD with the hepatic cytosolic fraction from C57BL/6J mice (AHRb isoform) were conducted as described in the Methods [21]. Increasing concentrations of SU5416, TCDD, BNF, and 1,2-Benzanthracene (a ligand of low receptor affinity) were added. As shown in figure 2C, SU5416 competitively displaced the radiolabel with efficacy similar to TCDD.
Figure 2. Induction of DRE-mediated transcription by SU5416 is AHR dependent. A. The AHR-mutant C35 cell line was transfected with the AHRb, lacZ gene and a 36DRE-Luc construct. Controls were transfected with the empty pSPORT vector plus the reporter constructs. After 24 h, the cells were treated with 3 mM SU5416 or 0.3% (v/v) DMSO, then incubated for 18 more h. Induction of AHR activity was determined by normalizing the luciferase activity to b-galactosidase activity. White bars: Empty vector. Grey bars: AHR. Error bars: SD; (n = 3). B. Induction of DRE-mediated transcription by SU5416 is ARNT dependent. The ARNT-deficient C4 cell line was transfected with the human ARNT or the pSPORT parent vector. These cells were also cotransfected, treated and assayed as in A. White bars: Empty vector. Grey Bars: ARNT. Error bars: SD; (n = 3). C. SU5416 is a ligand of the AHR. The hepatic cytosolic fraction from C57BL/6J mice was incubated with 1 nM of the radioligand 125BR2N3DpD, in the presence of increasing concentrations of competitor, SU5416, TCDD, BNF or 1,2-Benzanthracene. Ordinate: Specifically bound radioligand in the presence of competitor divided by specifically bound radioligand in the absence of competitor. Abscissa: The concentration of competing ligand, represented as log of molar concentration. Each data point represents the average of two determinations. Competitive binding to the C57BL/6J
cytosol produced the IC50 values of SU5416 = 2.1 nM, TCDD = 1.5 nM, BNF = 2.8 nM, and 1,2-Benzanthracene = 13.7 nM.

In utero Exposure to SU5416 Stimulates Closure of DV
We have previously shown that genetically altered mice that express only 10% of the AHR display a patent ductus venosus (DV) in the liver in nearly all cases [22]. We additionally identified that in utero activation of the receptor in the hypomorphs with TCDD successfully closed the DV [5]. To test the role of SU5416 as an in vivo ligand and its potential effect on embryology and vascular development, we performed timed matings of female AHRfxneo/+ mice to male AHRfxneo/fxneo mice. The pregnant dams were treated at embryonic day E18.5 with a single dose of SU5416 at 110 mg/kg, or an equivalent volume of the vehicle, corn oil. At 4 weeks of age, the pups were sacrificed, and DV status was examined by hepatic perfusion with trypan blue. As seen in Table 1, only 1 of 25 AHRfxneo/fxneo pups treated with corn oil possessed a closed DV. In the experimental group, 13 of 22 animals of this phenotype exposed to SU5416 had a closed DV.activity indicating loss of binding to the DRE, which is clearly in contrast to the long duration DRE-binding seen with TCDD. Of note, when we did titrate SU5416 doses as high as 10 mM, we did observe as much as 20% of TCDD response (1 nM) as far out as 96 hours (data not shown). This SU5416 data is similar to the known plasma half-life of 30 minutes, although VEGF-receptor inhibitor effects have been shown to last as much as 72 hours in culture [23]. We further analyzed whether the AHR antagonist CH223191 could inhibit the ability of SU5416 to activate the DRE in 101L-hepatoma cells. It has previously been shown that this antagonist inhibits TCDD but not some of the other ligands of the AHR including some polycyclic aromatic hydrocarbons. We first performed a titration of the AHR antagonist in culture with either 1 nM TCDD or 100 nM SU5416. As can be seen in figure S2B, the effects of TCDD are inhibited whereas minimal inhibition is shown for SU5416. In figure S2C, we show a titration of SU5416 with only a small amount of inhibition of activity by the antagonist (10 mM).

SU5416-induced Upregulation of CYP1A1 is Similar in Murine AHRb and AHRd Splenocytes
As the above in vitro experiments were performed in cell lines, we next utilized AHRb (C57BL/6J) and AHRd congenic mice (on a C57BL/6J background). Spleens from these mice were harvested and suspended in culture media, and exposed to titrating doses of TCDD and SU5416. These data are presented in figure 4, where the graphs show normalized data from 0 to 100% response. Normalized data was chosen to allow comparison of CYP1A1 upregulation to its maximum in AHRb versus AHRd mice. After 4 hours of culture, TCDD induced CYP1A1 more rapidly and to a higher degree in wild-type than AHRd splenocytes, with an EC50 of 0.461 nM in wild-type and 1.894 nM in AHRd animals. Figure 4B shows that SU5416 induced CYP1a1 similarly in AHRb and AHRd mice, with an EC50 of 0.682 nM in wild-type and 0.730 nM in AHRd mice. Figures S3A and B show the total fold change seen by qPCR analysis of splenocytes after exposure to TCDD and SU5416, to allow an assessment of the potency of AHR activation of these two ligands with CYP1A1 induction as the readout. As can be seen in the figure, TCDD elicits more CYP1A1 in AHRb compared to AHRd mice, whereas SU5416 leads to the same or more CYP1A1 in AHRd mice. By this readout, TCDD and SU5416 have similar potency in AHRd cells, and TCDD is a stronger ligand in AHRb cells.

SU5416 Upregulates CYP1A1 and CYP1B1
The above data clearly shows that SU5416 is a ligand of the AHR. We now focused our attention on the strong response of SU5416 to the AHRd polymorphism in the screening assay, and compared the activity of this ligand in the high and low affinity polymorphisms. We utilized the wild type rat hepatoma cell line, 5L, which harbors the high affinity AHR isoform, and our newly created AHRd-15 cell line. As seen in figure 3A, we first performed a titration with TCDD and measured EROD activity. As expected, the activity of TCDD was shifted by 1.5 orders of magnitude to the left for the AHRb isoform. In contrast, when SU5416 was tested in vitro, the two curves virtually overlapped (figure 3B), showing equal potency for cytochrome P450 induction using the two cell lines. We also tested BNF, which as expected, showed a strong response with the 5L cell line and no response with the AHRd-15 cell line (figure 3C). As these experiments were done in cell lines, and in addition the AHRd-15 line combines a rat cell line with a transfected murine AHR, we further tested the ability of SU5416 to activate the AHR in vivo. Six-week old C57BL/6J mice (AHRb) and DBA/2J (AHRd) were orally administered 30, 80, or 120 mg of SU5416 per kg of body weight.

Phosphorylations were also attempted with di-tert-butyl or dicyanoethyl phosphoramidites to produce di-tert-butyl or dicyanoethyl instead of dibenzyl phosphate. Neither of these phosphates was stable on silica gel, and b-elimination products were obtained after chromatography. TFA deprotection of crude di-tert-butyl phosphate, and NH4OH deprotection of crude dicyanoethyl phosphate both gave b-elimination products as well. Thus, the dibenzylphosphate was chosen to carry through to the final products 1 and rac-2. Hydrogenation of the crude dibenzyl phosphate (1S,3R,4R)13 went very slowly, giving a complex crude mixture. Thus, (1S,3R,4R)-13 was purified by reverse-phase semi-preparative high performance liquid chromatography (HPLC). With pure dibenzyl phosphate, hydrogenation at atmospheric pressure worked very well, and gave a very clean final product 1, similar to our experience with a-ketoamides [14].X-ray crystallography
During the synthesis of the inhibitors, Michael addition of tristhiomethyl methide to an a,b-unsaturated ketone 8 produced three stereoisomers of 9, which could not be readily separated (Figure 3). Two diastereomers of a subsequent synthetic intermediate, (1S,3R,4R)-11 and rac-11, were separated by chromatography. Each diastereomer was crystallized, and the relative stereochemistry was determined.

Figure 4. X-ray crystal structures of intermediates (1S,3R,4R)-11 and rac-11 are shown above as displacement ellipsoid drawings (50%). The positional disorder of the benzyl group in rac11 is shown as lighter lines. Hydrogen atoms are omitted for clarity. Structural depiction of the stereochemistries of (1S,3R,4R)-11 and rac11 are shown below each crystal structure. ?after geometry optimization was 3.16 A; with the trans-pyrrolidine torsion angle fixed during geometry optimization, the distance was ?3.67 A (Figure 6).Discussion Stereochemical results of inhibitor synthesisFigure 3. Cyclohexyl ketone inhibitor 1 was synthesized by the method shown. Thermodynamic control in the Michael addition resulted in the anti-Ser-trans-cyclohexyl stereoisomer of 9 as the major product (Figure 4). The chiral center adjacent to the Ser carbonyl was easily epimerized due to the electron-withdrawing effects of both the a-amide and a-ketone, resulting in an enantiomeric mixture of a second diastereomer, rac-9. Because the unnatural D-Thr-the original Ser configuration intact (Figure 4). The minor isomer, rac-11, proved to be a racemic mixture. The absolute configurations were assigned as (1R,3R,4R)-11 and (1S,3S,4S)-11, in which the stereocenter of the Ser analogue was partially epimerized to the syn-Ser-trans-cyclohexyl configuration (Figure 4).Pin1 PPIase Enzyme Assays
The a-chymotrypsin protease-coupled assay was used to evaluate inhibition of Pin1 by compounds 1 and rac-2 with the same substrate concentration as described previously [10,14]. The IC50 values of the two diastereomers were determined to be 260630 mM for 1, and 6168 mM for rac-2. Preincubation with Pin1 for 15 minutes did not result in improved inhibition.

Molecular modeling
Each of the three cyclohexyl ketone inhibitors was docked flexibly, with geometry minimization, into the Pin1 active site. The resulting docked stereoisomers, (1S,3R,4R)-1, (1R,3R,4R)-2, and (1S,3S,4S)-2, are shown in Figure 5. The total energies, Cys113?S–C = O ketone distances, and angles are reported in Table 1. Figure 5. Models of cyclohexyl ketone inhibitors were docked with dynamic minimization. (A) (1S,3R,4R)-1 in orange, (B) (1R,3R,4R)-2 in blue, (C) (1S,3S,4S)-2 in green, and (D) superposition of all atoms of 1 and rac-2. Models were based on PDB 2Q5A [32], and minimized using Sybyl 8.1.1 [42]. Images were prepared using MacPyMol [44]. Figure 6. Pin1 is proposed to stretch the prolyl ring by binding phosphate and C-terminal residues tightly, creating a transpyrrolidine conformation of the substrate and forcing pyramidalization of the prolyl nitrogen in the twisted-amide mechanism. Distance measurements are from calculated structures of AcroH in the ground state and the trans-pyrrolidine transition state.
containing inhibitors were more potent than the L-Thr in work by Zhang et al [32], both diastereomers 1 and rac-2 were tested for Pin1 inhibition. Inhibitor 1, corresponding to the native L-Ser-LPro stereochemistry of Pin1 substrates, had an IC50 value of 260 mM, while rac-2, an enantiomeric mixture of D-Ser-L-Pro and L-Ser-D-Pro analogues, had an IC50 value of 61 mM. Preincubation did not result in improved inhibition, suggesting that they are not slow-binding inhibitors. We obtained a crystal structure of the similarly substituted, reduced amide inhibitor 4 bound in the Pin1 active site, suggesting that the ketones also bind in the active site [27].

conformation, and the trans-pyrrolidine AcroH conformation ?was 0.51 A (Figure 6). This effect of stretching the ring conformation may provide insight into the mechanism of Pin1. In either of the proposed mechanisms: (1) nucleophilic-addition [26], or (2) twisted-amide [25], the nitrogen of the prolyl ring must become pyramidalized and deconjugated from the carbonyl in the transition state [22,24,25]. If binding of substrate to the catalytic site forces the Pro ring into a trans-pyrrolidine conformation, the nitrogen lone pair and the carbonyl p-bond would no longer be conjugated (Figure 6).