Domain requirement of moenomycin binding tobifunctional transglycosylases and developmentof high-throughput discovery of antibioticsTing-Jen Rachel Cheng, Ming-Ta Sung, Hsin-Yu Liao, Yi-Fan Chang, Chia-Wei Chen, Chia-Ying Huang, Lien-Yang Chou,Yen-Da Wu, Yin-Hsuan Chen, Yih-Shyun E. Cheng, Chi-Huey Wong*, Che Ma*, and Wei-Chieh Cheng*

Genomics Research Center, Academia Sinica, Sec. 2, 128 Academia Road, Nangang, Taipei 115, Taiwan

Contributed by Chi-Huey Wong, November 19, 2007 (sent for review October 2, 2007)

Moenomycin inhibits bacterial growth by blocking the transglyco-sylase activity of class A penicillin-binding proteins (PBPs), whichare key enzymes in bacterial cell wall synthesis. We compared thebinding affinities of moenomycin A with various truncated PBPs byusing surface plasmon resonance analysis and found that thetransmembrane domain is important for moenomycin binding.Full-length class A PBPs from 16 bacterial species were produced,and their binding activities showed a correlation with the antimi-crobial activity of moenomycin against Enterococcus faecalis andStaphylococcus aureus. On the basis of these findings, a fluores-cence anisotropy-based high-throughput assay was developed andused successfully for identification of transglycosylase inhibitors.

penicillin-binding proteins � transglycosylase inhibitors �fluorescence anisotropy

Many common bacterial pathogens, such as Staphylococcusaureus, Streptococcus pneumoniae, and Enterococcus fae-

calis, have become multidrug-resistant and have emerged as apublic health concern, creating an urgent need for new antibiotics.

The bacterial cell wall, or its peptidoglycan synthesis pathway,has been targeted for the development of antibacterial agents(1). The synthesis of peptidoglycan consists of several steps,including the formation of lipid I and lipid II, followed by thefinal transglycosylation and transpeptidation of lipid II to formpeptidoglycan (1, 2). Many current antibiotics are �-lactamderivatives that target transpeptidation. To our knowledge, nomedicines have yet been developed to inhibit the transglycosy-lation process. The only known potent inhibitors for transgly-cosylase (TG) are moenomycin complexes (flavomycin), includ-ing moenomycin A (Moe A) (Fig. 1A, compound 1), A12, C1, C3,and C4 (3, 4). Among these, Moe A is the most abundant agentin its family (3, 4). The unique antibacterial properties of MoeA have prompted chemists to synthesize moenomycin fragmentsand derivatives (5–7) in an attempt to develop new antibiotics.Recently, the total synthesis of Moe A (8), and of its biosynthesispathway (9), has been reported. However, due to poor bioavail-ability, f lavomycin is currently used only as a growth promoterin animal feeds (10).

The characterization of class A penicillin-binding proteins(PBPs) and the identification of TG inhibitors require functionalPBP and lipid II as the substrate for the enzyme. However, thelimited availability of lipid II has hampered the development ofeffective enzymatic assays for identification of inhibitors. As aresult, the majority of the screening methods that are used tosearch for TG inhibitors, including the low-throughput methodsthat use surface plasmon resonance (SPR) (12) or radioactiveassays (13–16), rely mainly on moenomycin (11). Developmentof a TG activity assay that is amenable to high-throughputscreening (HTS) is thus desirable for inhibitor identification.

In this study, we compared the binding of moenomycin tovarious truncated PBPs and concluded that the transmembrane(TM) domain is critical for moenomycin binding. Consequently,

the full-length PBP was used to develop a high-throughput assay.We devised a general method for expressing and purifying classA PBPs from 16 bacterial species and characterized their moeno-mycin binding activity. A fluorescence anisotropy (FA)-basedassay was developed by using the PBP that had higher bindingaffinity and was successfully used for the HTS of TG inhibitors.

Author contributions: T.-J.R.C., C.-H.W., C.M., and W.-C.C. designed research; T.-J.R.C.,M.-T.S., H.-Y.L., C.-W.C., C.-Y.H., L.-Y.C., Y.-D.W., and C.M. performed research; Y.-F.C.,Y.-H.C., Y.-S.E.C., and W.-C.C. contributed new reagents/analytic tools; T.-J.R.C., M.-T.S.,H.-Y.L., and C.-W.C. analyzed data; and T.-J.R.C., C.-H.W., C.M., and W.-C.C. wrote thepaper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

*To whom correspondence may be addressed. E-mail: chwong@gate.sinica.edu.tw,cma@gate.sinica.edu.tw, or wcheng@gate.sinica.edu.tw.

This article contains supporting information online at www.pnas.org/cgi/content/full/0710868105/DC1.

© 2008 by The National Academy of Sciences of the USA

cytoplasm

OOAcHN

HO

L-AlaD-GluL-LysD-AlaD-Ala

O

O

HO

HOHO

AcHNO

O

O

OAcHN

HO

P-O O P

O

-O O

OL-AlaD-GluL-LysD-AlaD-Ala

O

O

HO

OHOAcHN

O O

O

OAcHN

HO

P-O O

PO

-O O

OL-AlaD-GluL-LysD-AlaD-Ala

O

O

HO

HOHOAcHN

O

per iplasmLipid II

OHO

OH

HO

OOHO

AcHN

O

OHO

NHAc

O

OHOO

O

H 2N

OHO

HOHO

HOO

CONH 2

OP

O

O

OH

HO

COOH

OC

NH

O OH

KD (µM)

0.44 + 0.04

2.36 + 0.24

0.64 + 0.10

9.4 + 0.1

> 103

Penicillin Binding Proteins(Bi-functional Transglycosylase)

TG + TP

TM + TG

TG

TP

TM Transglycosylase Transpeptidase

1 844

Transglycosylase Transpeptidase88

Transglycosylase

1 409

Transglycosylase195

Transpeptidase736

409

444

TM

A

B

Moenomycin A (1)

844

Transglycosylase

Fig. 1. Inhibition of TG by moenomycin and binding affinities of truncatedPBP variants. (A) Moe A (1) inhibits the transglycosylation step in bacterial cellwall synthesis. (B) Schematic representation of PBP variants used for moeno-mycin binding studies. The full-length PBP (TM � TG � TP) contains amino acidresidues 1–844 of the E. coli PBP1b P02919. TG � TP (residues 88–844), TM �TG (residues 1–409), TG (residues 195–409), and TP (residues 444–736) aretruncated variants with that portion of the full-length PBP1b. The moenomy-cin binding constant (KD) for these variants was determined by using SPR.

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ResultsDomain Requirement of Moenomycin Binding to Class A PBPs (Bifunc-tional TGs). Transglycosylation is mainly catalyzed by bifunctionalclass A PBPs (17). PBPs are the major enzymes responsible forlast-step cell wall formation by polymerization of N-acetylglucosamine-N-acetylmuramyl pentapeptide, and cross-linking between pentapeptides, and thus have been consideredas one of the important targets for antibiotics discovery. Thesemembrane-anchored enzymes consist of three distinct proteindomains—TM, TG, and transpeptidase (TP)—from theiramino-to-carboxyl termini. The recent description of x-ray crys-tal structures of the PBP2 extracellular domain from S. aureus(18) and the TG domain of PBP1a from Aquifex aeolicus (19)have provided invaluable structural insights into the plausiblemechanism of cell wall peptidoglycan polymerization. Lacking,however, is information regarding the role of the TM domain ofPBPs in catalysis, although it has been speculated to interact withthe lipid moiety of moenomycin or lipid II (20, 21). To addressthis question, protein constructs containing different domains ofPBP1b from Escherichia coli were expressed and purified withsuitable detergents [see supporting information (SI) Materialsand Methods and SI Fig. 7 ] and used for analysis of moenomycinbinding by using SPR. Five E. coli PBP1b variants were expressedand purified: (i) the full-length protein containing the TM, TG,and TP domains (TM � TG � TP), (ii) TG � TP, (iii) TM �TG, (iv) TG alone, and (v) TP alone (Fig. 1B). After immobi-lization of the target proteins on the sensor chip, differentconcentrations of moenomycin were passed through the surface,and the binding affinities were determined. As shown in Fig. 1B,the binding affinity of moenomycin to PBP without the TMdomain (TG � TP) was �5-fold lower than with the full-lengthPBP1b. On the other hand, the binding affinity of TM � TG issimilar to that of the full-length protein. These results indicatethat the TM domain plays an important role in moenomycinbinding.

Correlation of PBP Binding Activities and MIC of Moenomycin. Be-cause the TM domain contributes significantly to moenomycin

binding, we set out to prepare full-length PBPs from 16 bacteria(10 Gram-negative and 6 Gram-positive) (Table 1) for moeno-mycin binding studies. The genes of class A PBPs were identifiedby using the National Center for Biotechnology Informationdatabase (www.ncbi.nlm.nih.gov/BLAST/), and all were con-firmed to have TG and TP motifs (22, 23) (see SI Fig. 6). Thetarget genes were amplified from respective genomic DNA fromeach individual bacterial species and cloned into expressionvectors for recombinant protein production using E. coli as host.The enzymatic activities of the purified proteins were confirmedby lipid II polymerization (data not shown) and moenomycinbinding. As shown in Table 1, varied steady-state affinity (KD)values were found among PBPs from different species. None-theless, all of the measured KD values fell into the range of 10�7

M, close to the reported inhibition concentration for the trans-glycosylation process (12). Interestingly, among the Gram-positive bacteria we tested, the KD values of PBP from E. faecalisand S. aureus correlate with the MIC values, suggesting that themoenomycin binding site of PBP may be a good target fordevelopment of antibiotics against these species.

Development of an FA-Based Assay for Identification of TG Inhibitors.Based on the conclusion that the full-length proteins (TM � TG �TP) should be used for moenomycin binding studies, we designedan FA-based assay to monitor the binding affinities of smallmolecules toward TG. Previous studies on the complex crystalstructure (18) and the structure–activity relationship (6) betweenmoenomycin and TG suggested that modification of compound 1to compound 2 (Fig. 2A) will not dramatically reduce binding andantibacterial activities. Furthermore, the amine moiety in 2 can beconjugated with any fluorophore and used as a probe for bindingstudies (24, 25). Indeed, compound 2 was linked with fluorescein byusing 6-carboxyfluorescein N-hydroxysuccinimide ester to preparethe fluorescent probe 3 (F-Moe, Fig. 2B). To investigate whetherthe fluorophore or the structural modification interferes with thebinding between the targeted protein and the small molecule, thePBP binding affinities for Moe A and the fluorescent probe werecompared by using SPR. The determined steady-state affinity (KD)

Table 1. Correlation of antimicrobial activity and PBP binding affinity of moenomycin for 16 bacterial strains

Species MIC, �M*

Reported values†

Gene sequence ID‡ SPR� KD, nM§ FA� KD, nM¶MIC, �M Citation

Gram-negativeBordetella pertussis 0.011–0.021 — PBP1a (NP�882163) 1,270 594 � 230Citrobacter freudii — 352 28 PBP1b (CAA90232) 728 —Escherichia coli 80 55–110 28 PBP1b (NP�414691) 440 54 � 17Haemophilus influenzae — — PBP1b (AAX88775) 966 174 � 13Helicobacter pylori — 1.3 29 PBP1a (NP�207392) 334 25 � 14Klebsiella pneumoniae 80 13.75–27.5 28 PBP1b (NTUH-2044) 819 78 � 24Neisseria gonorrhoeae — 1.69 28 PBP1 (YP�207272) 450 47 � 26Pseudomonas aeruginosa 40 55–110 28 PBP1b (YP�793163) 866 42 � 29Salmonella enterica 160 13.75–110 28 PBP1b (YP�149541) 561 138 � 113Shigella flexneri 40 27.5–110 28 PBP2 (YP688236) 290 —

Gram-positiveBacillus subtilis 0.3–0.6 0.07–0.43 30 PBP1a/1b (NP�390113) 1,690 197 � 80Clostridium difficile �160 220–440 30 PBP (CAJ67615) 582 37 � 33Enterococcous faecalis 0.075–0.3 0.1 31 PBP2a (NP�814430) 619 276 � 14Enterococcous faecium — �40.5 10 PBP1 (EAN08787) 94 56 � 11Staphylococcus aureus 0.075 0.02–0.12 10 PBP2 (NP�371974) 393 30 � 29Streptococcus pneumoniae 0.625 — PBP1b (NP�359500) 900 38 � 24

*MIC of moenomycin against different bacterial species.†MIC values of moenomycin from the literature.‡Sequence ID of class A PBP genes from individual bacterial strains. Homologs of class A PBPs were identified by using BLAST against the NCBI database.§KD of PBP homologs and moenomycin using SPR. Average values are shown.¶KD of PBP homologs and moenomycin using our FA-based assay.

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values are very similar (4.4 � 10�7 for Moe A vs. 5.2 � 10�7 M forF-Moe) (Fig. 2 C and D), suggesting that F-Moe is a validfluorescent probe that is useful for the FA-based binding assay.

The anisotropy of F-Moe increased significantly during incu-bation with E. coli PBP1b, presumably because of the formationof an F-Moe–PBP1b complex (Fig. 3A). In contrast, the anisot-ropy of F-Moe was unchanged when incubated with BSA, up to100 �M (data not shown). The protein concentrations were

further titrated down to establish the dose-dependency of theanisotropy changes. The deduced dissociation constant (KD) forF-Moe was in the nanomolar range (54 � 17 nM) for E. coliPBP1b (Fig. 3A). However, the signal of the FA assay using theE. coli PBP1b is only medium, with an anisotropy of 0.12. Wethus screened different class A PBPs to improve the assay, i.e.,to obtain higher anisotropy. Of all homologs tested, the FA assayusing Helicobacter pylori PBP1a produced the best signal-to-noise ratio with KD of 25 � 14 nM and with anisotropy reaching0.2 upon binding to F-Moe (Fig. 3B). To develop an assay forinhibitor screening, F-Moe was preincubated with H. pyloriPBP1a, and unlabeled Moe A was then added at variousconcentrations. A decrease in anisotropy was observed as theconcentration of Moe A increased, and from this competitionanalysis the inhibition constant (KI) and IC50 values weredetermined as 0.47 � 0.10 �M and 0.36 �M, respectively (Fig.3C). This result validates the FA assay, which can be used toscreen for inhibitors that compete with moenomycin for bindingto PBP.

HTS for TG Inhibitors. The FA assay was used to screen a collectionof 57,000 small molecules, along with Moe A derivatives (Fig. 4),

O HO

OH

HO

O O HO Ac HN

O

O HO

NH Ac

O

O HO O

O H 2 N

O HO

HO HO HO

O

CO NH 2

O P O

O

OH

H O

CO OH

N

NN

O

O OH

NO 2

O NH

NH 2

O HO

OH

HO

O O HO Ac HN

O

O HO

NH Ac

O

O HO O

O H 2 N

O HO

HO HO HO

O

CO NH 2

O P

O

O

OH

H O

CO OH

N

NN

O

O OH

NO 2

O H N

O

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O

O

OH N H

O

F-Moe (3 )

2

A

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0 50 10 0 15 0 20 0 25 0 30 0

0

5

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Ti me (s ec )

Re s

p on

s e (

R U

)

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Ti me (s ec )

Res

pon

se (

R U

)

0 200 40 0 60 0 800 1000 5

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[M o enom yci n A] (n M)

Re s

pons

e (R

U)

0 20 0 40 0 0 20 0 40 0 60 0 80 0 1000 5

10

15

20

25

[F -M oe] (n M)

Re s

pons

e ( R

U)

C

D

Fig. 2. Design of an FA assay for TG. (A and B) Chemical structures ofmodified Moe A (2) and fluorescein-labeled moenomycin, F-Moe (3). (C and D)Results of SPR analysis of the binding activity of Moe A (C) and F-Moe (D) to E.coli PBP1b. Shown are responses for moenomycin binding to immobilized E.coli PBP1b. The data were analyzed by using steady-state affinity and fitted toa 1:1 interaction model (Insets). The KD values deduced from the intercepts ofthe x axis and the dotted lines are 440 and 520 nM for Moe A and F-Moe,respectively.

0.0 0.5 1.0 1.5 2.0 2.5

0.025

0.050

0.075

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KD = 54 + 17 nM

E. coli PBP1b (μM)

Flu

ores

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e A

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y (A

)

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cent

age

chan

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inF

luor

esce

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sotr

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(%)

A

B

C

-8 -7 -6 -5 -4

Fig. 3. Development of a high-throughput FA assay for TG. (A) Concentra-tion-dependent changes in FA were observed when E. coli PBP1b bound toF-Moe. (B) Improved FA assay with H. pylori PBP1a. The concentration-dependent FA changes were performed similarly to A but using the H. pyloriPBP1a. The maximum anisotropy value was 0.2. (C) Displacement of thePBP1a-bound F-Moe complex by unlabeled moenomycin at various concen-trations. The changes in FA are defined as [(Aobs � Amin)/(Amax � Amin) � 100%].The KD and IC50 values were calculated as described in SI Materials andMethods.

Cheng et al. PNAS � January 15, 2008 � vol. 105 � no. 2 � 433

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at 50 �M. High controls are assays with 1 �M moenomycin, andlow controls are assays with 2.5% DMSO. Other proteins, suchas BSA, were included as a control to confirm that the anisotropyincrease is not the result of nonspecific binding. The Z� value, astatistical parameter ranging from 0 to 1 used to evaluate therobustness of HTS (26), was determined as 0.895 from �100independent experiments. Eleven possible hits that showed atleast 75% inhibition in the screening were selected for additional

studies involving antimicrobial assays and IC50 determinationsfor PBP binding (Fig. 5A). Among these hits, two moenomycinanalogues (2 and 4) and three small molecules (compounds 6–8)were confirmed to have both antibacterial and TG bindinginhibition activities (Table 2).

DiscussionWe have shown that the TM domain is important for Moe Abinding. A general method was thus devised for preparing thefull-length class A PBPs from various bacterial strains. We foundthat the binding affinity of Moe A to PBP correlated with itsantibiotic activity for E. faecalis and S. aureus. By using thefull-length PBP1b, an FA-based HTS assay was developed foridentification of TG inhibitors as antibacterial agents.

We were concerned about the nonspecific binding of the C25lipid of Moe A. By using a TP variant as a control, it wasconfirmed that the determined binding affinities of moenomycinto the PBP1b variants are significant and specific. Bindingstudies using truncated PBP1b variants further showed that thebinding affinity of the TM � TG variant is similar to that of thefull-length protein. The role of the TM domain in Moe A bindingwas revealed in the observation that the binding affinity of theTG � TP variant decreased 5-fold compared with that offull-length PBP1b (Fig. 1B). These studies suggest that oneshould use the full-length PBP, or the truncated PBP containingthe TM and TG domains, for moenomycin binding studies andinhibitor discovery and that the TM domain may be importantfor the structure of the enzyme.

In the course of our study, several moenomycin analogues (2,4, and 5) were prepared to evaluate the reliability of theFA-based assay. As shown in Table 2, the differential bindingaffinities (IC50 values) of these moenomycin analogues by theFA-based assay are comparable with published results (12, 27).The modification of Moe A to 2 resulted in a slight decrease inTG binding affinities. Dimerization of 2 by means of an eight-carbon spacer produced 4, with a 2-fold increase in both PBPbinding and antimicrobial potency, although 4 is still less potentthan Moe A (1). More significantly, compound 5, without theC25 lipid moiety, could barely displace the F-Moe probe 3,indicating that the hydrophobic part of moenomycin is crucial forits activity. It is noteworthy that the order of inhibition potencyis in agreement with the MIC values (Table 2).

O HO

OH

HO

O O HO

Ac HN

O

O HO

NH Ac

O

O HO O

O H 2 N

O HO

HO HO HO

O

CO NH 2

O P

O

O

OH

H O

CO OH

N

N N

O

O OH

NO 2

O H N

N H

O

O OH

HO

OH

O O OH

NH Ac

O

O OH

Ac HN

O

O OH O

O NH 2

O OH

OH OH OH

O

H 2 NO C

O P

O

O

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H O

CO OH

N

N

N

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O 2 N

O N H

H N

O

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OH

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O O HO Ac HN

O

O HO

NH Ac

O

O HO O

O H 2 N

O HO

HO HO HO

O

CO NH 2

O P

O

O

OH

H O

CO OH O

N

NN

O

O OH

NO 2

O NH

NH 2

4

5

Fig. 4. Chemical structures of moenomycin derivatives (4 and 5).

F-Moe

PBP

Compounds

+

+

+

Flu

ores

cenc

e A

niso

trop

y (%

)

100

75

50

25

0

A

IC Determinationand

Antimicrobial Activity

50

B

6 7 8

NN

N

N

NHSO

N+O O

-OOO

BrOH

O

Fig. 5. Screening for small molecules as TG inhibitors using the class A PBPs. (A) HTS for TG inhibitors using the FA assay. Protein structure graphics were createdfrom Protein Data Bank (www.pdb.org) ID code 2OLV (12). (B) Chemical structures of the HTS hits (compounds 6–8).

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Although moenomycin and its analogues are potent againstGram-positive bacteria, their poor pharmacokinetic propertiesdemand unique approaches for antibiotic development. Anefficient and economical TG assay in an HTS format mayfacilitate the identification of new TG inhibitors. Using ourFA-based assay, a hit rate of 0.02% was achieved with a Z� valueof 0.895, suggesting that this assay could be used as a robustprimary screen to quickly identify potential hits from largecompound libraries. The selected hits would be further screenedby using antibacterial assay and lipid II polymerizing activityanalysis, to identify leads. As with all f luorescence-based assays,our FA-based assay cannot be used to screen fluorescent com-pounds for TG inhibitors. Nonetheless, the FA-based PBPbinding assay has been demonstrated to be a powerful HTS assayfor the discovery of new antibacterial agents.

Materials and MethodsSPR Analysis. Purified PBP and its variants were immobilized onto CM3 sensorchips (GE Healthcare) to the level of �1,500–2,000 relative units via aminecoupling. The chips were then passed over with different concentrations ofMoe A (0–2,000 nM). Immobilization and data collection were performedwith BIAcore T100 (GE Healthcare) at 25°C.

FA Measurements. FA measurements were carried out in triplicate in 384-wellplates by using laser fluorimetry (IsoCyte; Blueshift Biotech). Various buffers,salts, pH values, and divalent cations (Ca2�, Mg2�, Co��) were optimized forFA measurements. KD and KI determinations were carried out in100 mM NaCl,10 mM Tris, pH 8.0 (see SI Materials and Methods for details).

HTS for TG Inhibitors. The FA assay was used to screen 50,000 purchased smallmolecules (ChemBridge) and 7,000 from our proprietary collections. Thecompounds were transferred to 96-well plates (Freedom Evo; Tecan Schweiz)and then to 384-well plates, using a multidispenser (Labcyte) to prepare thecompound plates for screening. The H. pylori PBP1a (10 �g/ml) in 100 nMF-Moe, 10 mM Tris, 100 mM NaCl, pH 8.0, at a final volume of 40 �l was addedto 384-well plates (Freedom Evo 150; Tecan). One microliter of 2 mM stocksolution of compound was added to wells by using a multidispenser (Labcyte).The last two columns of every plate were controls containing 10 �M moeno-mycin and 2.5% DMSO, respectively. After a 30-min incubation, changes in FAwere determined with Isocyte (Blueshift Biotech). Hits that showed �75%reduction compared with the control anisotropy values were selected forfurther confirmation.

Determination of MIC. The MIC of tested compounds was determined inaccordance with the National Committee for Clinical Laboratory standard. Theexperiments were conducted in 96-well microtiter plates using 2-fold dilutionsin Muller–Hilton broth with (S. pneumoniae) or without (Bacillus subtilis, E.faecalis, S. aureus) blood. Exponentially growing cells at 5 � 105 cells permilliliter were incubated with test compounds at various concentrations. After18- to 24-h incubation at 37°C, MIC was determined as the minimal concen-tration of the compound that prevents bacterial growth.

ACKNOWLEDGMENTS. We thank Drs. Evan F. Cromwell and Steve Miller ofBlueshift Biotechnologies, Inc., for help with FA detection and Dr. AndrijBuchynskyy for chemistry discussions. This work was supported in part byNational Health Research Institute, Taiwan, Grant NHRI-EX95-9515SC (to C.M.)and National Science Council, Taiwan, Grant NSC95-2113-M-001-019-MY2 (toW.-C.C.).

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Table 2. Inhibition of TG activity and the antibacterial determinations of selected hits

Compound* IC50, �M†

MIC, �M‡

B. subtilis(ATCC23857)

E. faecalis(ATCC29212)

S. aureus(ATCC29213)

S. pneumoniae(ATCC49619)

1 0.36 0.33 0.04 �0.01 0.332 2.10 2.50 10.0 1.25 20.04 0.92 1.25 5.0 0.625 20.05 125.00 — — — —6 34.00 0.25 0.25 1.0 4.07 3.70 0.25 1.0 4.0 —8 9.30 4.0 �4.0 �4.0 �4.0

*Compounds 2, 4, and 5 are moenomycin derivatives. Compounds 6–8 were HTS hits.†IC50 values were determined by using the fluorescence anisotropy assay shown in Fig. 5A.‡The MICs of moenomycin against different bacterial species were determined as described in Materials andMethods. ATCC, American Type Culture Collection.

Cheng et al. PNAS � January 15, 2008 � vol. 105 � no. 2 � 435

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