activation of inhibitors by sortase triggers irreversible modification

Activation of Inhibitors by Sortase Triggers IrreversibleModification of the Active Site*□S

Received for publication, March 2, 2007, and in revised form, May 24, 2007 Published, JBC Papers in Press, June 1, 2007, DOI 10.1074/jbc.M701857200

Anthony W. Maresso‡, Ruiying Wu§, Justin W. Kern‡, Rongguang Zhang§, Dorota Janik¶, Dominique M. Missiakas‡,Mark-Eugene Duban¶, Andrzej Joachimiak§, and Olaf Schneewind‡1

From the ‡Department of Microbiology, University of Chicago, Chicago, Illinois 60637, the §Midwest Center for Structural Genomicsand Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439, and the ¶Laboratory forthe Design of Bioactive Molecules, Department of Chemistry and Physics, Chicago State University, Chicago, Illinois 60628

Sortasesanchorsurfaceproteins to thecellwallofGram-positivepathogens through recognitionof specificmotif sequences. Loss ofsortase leads to large reductions in virulence, which identifies sor-tase as a target for the development of antibacterials. By screening135,625 small molecules for inhibition, we report here that aryl(�-amino)ethyl ketones inhibit sortase enzymes from staphylo-cocci and bacilli. Inhibition of sortases occurs through an irrevers-ible, covalent modification of their active site cysteine. Sortasesspecifically activate this class of molecules via �-elimination, gen-erating a reactive olefin intermediate that covalently modifies thecysteine thiol.Analysisof the three-dimensional structureofBacil-lusanthracis sortaseBwithandwithout inhibitorprovides insightsinto themechanism of inhibition and reveals binding pockets thatcan be exploited for drug discovery.

The emergence of bacterial strains resistant to antibiotictherapy is a major public health threat (1). Of particular con-cern is Staphylococcus aureus, because this Gram-positivepathogen is the leading cause of infections in the bloodstream,lower respiratory tract, skin, and soft tissue in the United States(2). S. aureus strains exhibiting resistance againstmultiple anti-biotics, such as methicillin-resistant S. aureus, are isolated in30–60% of community and �80% of hospital infections withthis pathogen (3). Vancomycin or other glycopeptides are con-sidered last-resort therapies for methicillin-resistant S. aureus;however, S. aureus strains with intermediate or full resistanceto vancomycin can cause infections for which antimicrobialtreatment may no longer be effective (4).

Surface proteins ofGram-positive bacteria play important rolesduring pathogenesis (5). Sortases anchor these polypeptides to thebacterial cell wall envelope (6). For example, S. aureus sortase Arecognizes proteins destined for the cell surface via an LPXTGmotif in their C-terminal sorting signal (7). Following cleavagebetween the threonine and the glycine residues, an acyl-enzymeintermediate captures cleaved substrate at the active site thiol ofsortase (8). Nucleophilic attack of the amino group of the pepti-doglycan precursor lipid II (C55-PP-MurNAc-(L-Ala-D-iGln-L-Lys(NH2-Gly5)-D-Ala-D-Ala)-GlcNAc) at the thioester intermedi-ate resolves theacyl enzymeand formsanamidebondbetween theC-terminal threonine of surface protein and pentaglycine cross-bridges (9). Lipid II-linked polypeptide is subsequently incorpo-rated into the cell wall envelope of staphylococci (10). The finalproduct of this pathway, protein linked to cell wall pentaglycinecross-bridges, is displayed on the bacterial surface and enablesinteractions between the pathogen and tissues of its host.Surface protein anchoring to the cell wall envelope is thought

to be an essential strategy for bacterial survival during infection,becausemutants lacking genes for one ormore sortase enzymesare attenuated in virulence (11). Inhibition of sortases by smallmolecules may therefore function as a therapeutic strategy forbacterial infections. Previous work described several sortaseinhibitors, includingmethane-thiosulfonates (12), peptide sub-strate-derived affinity labels (13), natural compounds (14–16),vinyl sulfones (17), diarylacrylonitriles (18), bis(indole) alka-loids (19), peptidomimetics (20), isoquinoline alkaloids (16),and threonine analogues (21). However, most of these com-pounds are either of low activity, lack specificity, or displayundesirable structural features that confound therapeutic use.To overcome these obstacles, we have screened a library ofsmall molecules and identified aryl (�-amino)ethyl ketones asmechanism-based inactivators of sortases.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Reagents—S. aureus sortase A (SrtA),2Bacillus anthracis sortase A, SrtB, and SrtC were purified from

* This work was supported by National Institutes of Health Grants AI38897and AI057153 (to O. S.), GM074942 and GM62414 (to A. J.), and GM08043and National Science Foundation Grant 9351490 (to M.-E. D. and ChicagoState University) and by the U. S. Department of Energy, Office of Biologicaland Environmental Research, under contract DE-AC02-06CH11357 (toA. J.). The authors acknowledge membership within and support from theRegion V “Great Lakes” Regional Center of Excellence in Biodefense andEmerging Infectious Diseases Consortium (GLRCE, NIAID-NIH Award1-U54-AI-057153). The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. S1 and Tables S1–S3.

The atomic coordinates and structure factors (code 2OQW and 2OQZ) have beendeposited in the Protein Data Bank, Research Collaboratory for StructuralBioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 To whom correspondence should be addressed: Dept. of Microbiology, Uni-versity of Chicago, 920 E. 58th St., Chicago, IL 60637. Tel.: 773-834-9060;Fax: 773-834-8150; E-mail: [email protected].

2 The abbreviations used are: SrtA, sortase A; SrtB, sortase B; SrtC, sortase C;AAEK, aryl (�-amino)ethyl ketone; DTT, dithiothreitol; SAR, structure-activ-ity relationship; HTS, high-throughput screen; NSRB, National ScreeningLaboratory for the Regional Centers of Excellence in Biodefense andEmerging Infectious Disease; MS, mass spectrometry; MS/MS, tandemmass spectrometry; a-LPETG-d, 2-aminobenzoyl-LPETG-diaminopropionicacid-dinitrophenyl-NH2; MALDI-TOF, matrix-assisted laser desorption ion-ization time-of-flight.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 32, pp. 23129 –23139, August 10, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Escherichia coli strain BL21(DE3) using Ni2�-nitrilotriaceticacid affinity chromatography (8). Reagents were obtained fromSigma-Aldrich unless otherwise noted. Compounds AAEK1(5927860) and AAEK2 (5927943) were purchased from Chem-bridge (San Diego, CA), and their structures were validated by1H NMR and liquid chromatography-MS.Identification of Sortase Inhibitors—The NSRB at Harvard

Medical School provided compound libraries for sortase inhib-itor studies. Purified S. aureus SrtA (2 �M) in black polystyrene384-well plates was incubated with 1 �l of test compound (10ng) inMe2SO for 1 h at 25 °C, followed by addition of 2-amino-benzoyl-LPETG-diaminopropionic acid-dinitrophenyl-NH2(a-LPETG-d) to 2 �M in 50 �l of reaction buffer (5 mM CaCl2,150 mMNaCl2, 50 mM Tris-HCl, pH 7.5) and further incubatedfor 24 h at 37 °C. Fluorescence was measured using an Envisionplate reader (excitation � � 320 nm, emission � � 420 nm, Fig.1A, inset). This assay yielded a Z� of 0.94 (22). Sixteen wells ofpositive control (no compounds added) and sixteen of negativecontrol (no compound or sortase added) wells were performedper plate. FinalMe2SO concentrationswere�2.5% (v/v), a con-centration shown to have no effect on control fluorescence lev-els (data not shown).Data Analysis and Hit Selection—Data were qualified for

analysis by evaluation of within- and across-plate biases andother systematic errors using SpotfireDecision� (SpotFireU.S.,Somerville, MA). Percent activities (%A, the ratio of fluores-cence from a test compound well to the plate positive controlmean, multiplied by 100) and percent inhibition (%I � 100 �%A) were computed. Compounds were rank ordered accordingto %I. 6,154 compounds (4.5% of initial libraries) were activeand displayed %I � 20 (Fig. 1B). Structures of hit compoundswere examined using custom sub-structure search routines inSARNavigator� 1.2 (Tripos, St. Louis, MO) to “filter” reactiveand promiscuous inhibitors (23–26), known mutagens andgenotoxics (27, 28), and molecules lacking good physicochem-ical hit/lead characteristics (23, 26, 29, 30). The alert moleculeswere separated into a salvage set, generating a set of 2,023 com-pounds (1.5% of initial) that were assigned lead-like activities.Analysis of these compounds for structural similarity based oncomputed Tanimoto distances (SARNavigator or Accord� 6.1,Accelrys, SanDiego, CA (31)) was followed by visual inspectionto identify “clean” lead-like clusters and singletons (a molecule/chemotype without a second example) (26, 31). These clusterswere sampled with emphasis on compounds with %I � 50 andpotential for providing SAR in subsequent studies. Analysis ofthese for structural similarity identified �80 clusters with�1150 molecules (median size, 3; mean, 14; and range, 2–63)and �850 putative singletons. Approximately 210 clean com-pounds were selected for secondary assays. The alerts salvageset was combined with the singletons and other weakly activeclean molecules, and this combined set was clustered andexamined visually. Clusters were again sampled on the basis ofactivity, properties, and potential to provide SAR, and the bestrepresentatives and the most active and lead-like singletonswere then added to the 210 to yield a final set of 407 compoundswith potential both as research tools and as therapeutic candi-dates. These 407 compounds were subjected to a secondary

screen using B. anthracis SrtA and papain and data analyzed asdescribed for HTS of S. aureus SrtA.IC50 Determination—S. aureus and B. anthracis sortases (8

�M) were incubated with increasing concentrations of eitherAAEK1 or AAEK2 (0.01–3200 �M) for 1 h at 37 °C, followed byaddition of a-LPETG-d (SrtA), a-KTDNPKTGDEA-d (SrtB),or a-GEKLPNTASNN-d (SrtC) in 300 �l of reaction buffer for15 min at 37 °C, and fluorescence was quantified as describedabove. IC50 values were determined by fitting data to a defaultfour parameter variable slope sigmoidal function in GraphPadPrism� 4.0c using a nonlinear least squares algorithm.Michaelis-Menten Kinetics—S. aureus SrtA (4 �M) was

incubated with or without AAEK1 (50 �M) or AAEK2 (200�M) in the presence of various concentrations of substrate(a-LPETG-d, 0–68 �M) for 15 min at 37 °C in reaction bufferand 300-�l volume, followed by fluorescence measurements.Kinetic constants were determined by Lineweaver-Burke anal-ysis. The amount of cleaved substrate was �5% for all velocitydeterminations. For dialysis experiments, S. aureus SrtA (4�M)was incubated with or without AAEK1 or AAEK2 (0.8 mM) for2 h at 25 °C, followed by dialysis against 1 liter of reaction bufferfor 24 h at 4 °C. Substrate (8 �M a-LPETG-d) was added, andreactions were incubated at 37 °C for 12 h, followed by fluores-cence measurements.MS of Sortase-Inhibitor Complexes—Wild-type or C184A S.

aureus SrtA (10 �M) was incubated with or without AAEK1(800 �M) at 25 °C for 1 h in reaction buffer. Trypsin (5 �g) wasadded, and sortase inhibitor complexes were digested at 37 °Cfor 12 h. Reaction aliquots (500 �l) were subjected to reversed-phase high-performance liquid chromatography on C18 col-umn (2 � 250 mm, C18). Cleaved peptides were eluted with alinear gradient of acetonitrile in 0.1% trifluoroacetic acid (100min, 0.5 ml min�1 fractions collected). Peak fractions (Abs.215)were dried, dissolved in 15 �l of 30% acetonitrile, 0.1% triflu-oroacetic acid mixed with saturated �-cyano-4-hydroxycin-namic acid suspensions, spotted onto sample plates, and air-dried. For MALDI-TOF experiments, samples were ionizedwith anN2UV laser in a Reflectron time-of-flight spectrometer(Applied Biosystems) in reflectron mode. Two hundred lasershots were conducted at an accelerating voltage of 25,000V andlaser intensity of 2,075 (repetition rate, 3 Hz). The instrumentwas calibrated using bovine serum albumin as an internalstandard control, and scans were processed using BiosystemsVoyager 6004 software. Peptides with m/z 1555.74 and m/z1693.78 were subjected to tandem mass spectrometry.Synthesis of Olefin Intermediates—Inhibition intermediate

12, an �,�-unsaturated olefin, was prepared in two steps fromcommercial thiophene-2-carbaldehyde by procedures summa-rized here. The comparable 1-(thiophen-3-yl) products wereprepared from their corresponding aryl aldehydes. Chromato-graphically pure products were isolated by Still-type isocratic60-Å SiO2 adsorption chromatography at medium pressure(0.7 MPa, Biotage SP1TM system, KP-SILTM, 40–63 �M), using3–15% linear gradients of EtOAc in hexanes with 2- to 4-col-umn volume initial and final isocratic steps. Subsequent spec-troscopic data, pulsed Fourier transform 1H and 13C NMR,electrospray ionization-MS, and Fourier transform IR, wereconsistent with the structures reported and literature data

Aryl (�-Amino)ethyl Ketone Inhibitors of Sortase

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available. Purified products were stored under argon at �20 °Cin the dark until use. rac-1-(Thiophen-2-yl)prop-2-en-1-ol (11)(32) was prepared from vacuum-distilled thiophene-2-carbal-dehyde by treatment with 1.10–1.20 equivalents of vinylmag-nesium chloride in anhydrous Fluka tetrahydrofuran at15–20 °C for 3–4 h (reaction 0.7 M in aldehyde, scale 3.4–15mmol) (33). After weakly acidic aqueous workup, tert-butyl-methyl ether extraction, and standard drying, chromatographicisolation gave the indicated pure product (yields, 74–92%).1-(Thiophen-2-yl)prop-2-en-1-one (12) (34) was preparedfrom purified 11 or directly from the crude propenol extract of11 after drying. The extract was treated with 1.50 equivalent ofthe oxidant N-methylmorpholine N-oxide and 0.15 equivalentof the Fluka catalyst tetra-N-propylammoniumperruthenate inanhydrous Fluka dichloromethane at 0–5 °C for 15–17 h (reac-tion 0.10 M in alcohol, scale 0.6–1.8 mmol) (35). After catalystadsorption/filtration (ICN GmbH SiliTech SiO2, 32–63�M/Celite 545�), filtrate evaporation, and extraction of the res-idue with hexanes, chromatographic isolation gave the indi-cated pure product (yields: 59% on 63% conversion from pure11, 24% direct from extract). Compounds were diluted to 0.57–0.58 M in 50% aqueous Me2SO containing sortase reactionbuffer (see above). Compounds were further diluted to assayconcentrations of 5 or 50 �M, and assays were performed asdescribed for the DTT experiments.Effect of DTT on AAEK1- and AAEK2-mediated Inhibition of

Sortase—Reactions included 4 �M S. aureus SrtA, 8 �Ma-LPETG-d, inhibitor (50 �M AAEK1 or 200 �M AAEK2), andincreasing concentrations of DTT (2–500 �M) and were incu-bated at 37 °C for 1 h, followed by fluorescence measurements.Crystallography—Protein expression and purification was

carried out as described (36). The construct did not include 36N-terminal amino acids (signal peptide) and encoded a 242-amino acid SrtB polypeptide with a 24-residue His tag at its Nterminus. A 2mMprotein stock solution in 10mMTris-HCl, pH7.4, 20 mM NaCl, and 1 mM DTT was used for crystallization.Selenomethionine-labeled SrtB was prepared using the methi-onine biosynthesis inhibition method (37). Inhibitors werediluted to 10 and 50mM in crystallization buffer for AAEK1 andAAEK2, respectively. The SrtB/AAEK adduct structures wereobtained using vapor diffusion at 25 °C and crystallized underdifferent conditions as compared with apo-protein. The SrtB/AAEK1 adduct crystallizes in a different space group (see Table3). The structures of SrtB/AAEK1 and SrtB/AAEK2 adductswere determined by single-wavelength anomalous dispersionphasing using HKL3000 (38) and selenomethionine-labeledenzyme. The structures were auto traced usingARP/wARP andrefined with REFMAC against the averaged peak data (39). Theinitial models of SrtB/AAEK1 and SrtB/AAEK2 were adjustedmanually using COOT and refined to the final crystallographicR of 18.7% and Rfree of 25.9% for SrtB/AAEK1, and to the finalcrystallographic R of 18.6% and Rfree of 22.6% for SrtB/AAEK2,both with zero � cutoff (see Table 3). The stereochemistry ofthe structures was examined with PROCHECK and the Ram-achandran plot. The final model of SrtB/AAEK1 does not havesufficient density for nine N-terminal residues (64–65 and85–191) and four internal residues (238–241). The final modelof SrtB/AAEK2 does not have sufficient density for four inter-

nal residues (236–240). Atomic coordinates have been depos-ited in the Protein Data Bank.

RESULTS

Screening for Sortase Inhibitors—A biochemical assay thatmonitors S. aureus sortase A cleavage of the fluorescence reso-nance energy transfer substrate (a-LPETG-d) between thethreonine and glycine residues was optimized for HTS (8).Translation of this assay into a 384-well format yielded a Z� of0.94, which is well above the value suggested for successful HTSassays (Fig. 1A) (22). Using the NSRB library, 135,625 smallmoleculeswere screened in duplicate for inhibition of SrtA (Fig.1A, inset). Of the compounds assayed, 6,154 displayed percentinhibitions (%I) �20% (Fig. 1B). Of these, �two-thirds wereplaced in a salvage set for being reactive, genotoxic, promiscu-ous (frequent hits in unrelated assays), or lacking in drug-likeproperties (see “Experimental Procedures”) (23–30). Theremaining lead-like activities were clustered on the basis ofstructure to identify common structural cores (chemotypes)and selected based on SARs. After combining these with themost active compounds from the salvage set, a total of 407compounds was attained (Fig. 1B).To examine specificity of inhibition, the 407 compounds

were screened for inhibition of B. anthracis SrtA and papain, aeukaryotic protease with an active site thiol (40). Many of themost potent and specific sortase inhibitors belonged to five che-motypes (Table 1): Class I, aryl (�-amino)ethyl ketones; Class II,N-aryl maleamides and aryl fumaramides, and related com-pounds; Class III,N,4-diaryl-2-aminothiazoles; Class IV, 3-het-eroatom-substituted (N-alkyl/aryl)pyrrolidine-2,5-diones; andClass V, variously substituted maleimide-furan Diels-Alderproducts. Of these, the aryl (�-amino)ethyl ketones (AAEK)were the most active inhibitors of sortases from staphylococciand bacilli (Table 1). These compounds areMannich bases (41),with a propiophenone (or related heteroaromatic) core, bearinga�-arylamino or�-dialkylamino substituent. Two compounds,AAEK1 and AAEK2, were selected for further characterizationbecause of their substantial inhibition of sortase, limited inhi-bition of papain, and presentation of heavy atoms to assist ineventual x-ray structure determination (Table 2).Inhibition of Sortases by AAEKs—Inhibitory concentration

(IC50) values of AAEK1 and AAEK2 for sortases from B.anthracis and S. aureus were determined. AAEK1 and AAEK2were �10- and 3-fold more potent inhibitors of B. anthracisSrtA than of S. aureus SrtA (Fig. 2, A and B). Inhibition of B.anthracis SrtAwas severalfold greater than inhibition of SrtB orSrtC (Fig. 2, B–D). As different sortase enzymes recognizeunique substrates, it seems plausible that observed differencesin IC50 values may be due to differences in active site configu-ration for members of this enzyme family. Nevertheless, thedata suggest that AAEK1 and AAEK2 function as inhibitors ofall four sortases tested.Incubation of S. aureus SrtA in the presence of inhibitor

(AAEK1 or AAEK2) with increasing concentrations of peptidesubstrate caused no alterations in Km but decreased Vmax withan apparent non-competitive profile (supplemental Fig. S1A).Lineweaver-Burke transformation of the data corroborated thisnotion, revealingVmax values of 88.8 7.8 (AAEK1) and 58.0




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3.6 (AAEK2) compared with 139.0 5.4 observed in theabsence of inhibitor (supplemental Table S1). Following a 2-hincubation of SrtAwith AAEK1 or AAEK2, reactions were sub-jected to dialysis and then assayed for activity. Dialysis failed torestore activity, consistent with the notion that these com-pounds cause irreversible inhibition of sortases even in theabsence of substrate (supplemental Fig. S1B). If so, AAEK-me-

diated inhibition likely precedes enzyme nucleophilic attack atthe scissile peptide bond.Inhibition by AAEKsOccurs at the Active Site Thiol of Sortase—

S. aureus sortase A was incubated with or without AAEK1.Enzyme preparations were subjected to tryptic digestion, pep-tides separated by reversed-phase high-performance liquidchromatography, and fractions yielding absorbance at 215 nmwere analyzed by mass spectrometry. A compound with m/z1555.74 eluted in fraction 50, and its mass measurement was inagreement with the predicted m/z 1555.73 of the tryptic pep-tide QLTLITCDDYNEK, encompassing the active site residueCys-184 (Fig. 3A). MS/MS of m/z 1555.74 confirmed the pep-tide sequence QLTLITCDDYNEK (supplemental Table S2).MS failed to identify a compound with m/z 1555.74 in sortasepreparations that had been incubated with AAEK1 (Fig. 3B). Incontrast, m/z of 1693.78 was present in sortase preparations(fraction 54) that had been treated with AAEK1 but not in sor-tase preparations without inhibitor (Fig. 3,C andD). MS/MS ofm/z 1693.78 confirmed the peptide sequence QLTLITXD-DYNEK (supplemental Table S2), where X represents Cys-184,which has been chemically modified by the inhibitor and is inagreementwith the addition of a thienylpropanonemoiety (Fig.3C, inset, supplemental Table S3). If so, the amine moiety of

FIGURE 1. HTS of sortase and cheminformatics. A, a fluorescence resonanceenergy transfer-based assay using a-LPETG-d as sortase substrate in a 384-well plate format was used in this HTS (Z�-factor of 0.94). The inset demon-strates the assay design used to identify sortase inhibitors. B, compoundsdisplaying inhibition �20% of mean positive control (6,154) were computa-tionally filtered to remove reactive inhibitors, promiscuous inhibitors, etc.(see “Experimental Procedures”). Filtered molecules were placed in a Salvageset, and remaining Lead-like actives were clustered and sampled based onlead-likeness and potential for later SAR analysis, with emphasis on the mostactive compounds. Approximately 210 “clean” compounds were selectedfrom the Lead-like Clusters. To allow for discovery of chemical tools as well(54), these were combined with representatives from the Salvage set (aftertheir re-clustering) to yield a final set of 407 compounds to take into thesecondary screen for specificity.

TABLE 1Representative classes of sortase inhibitors revealed by HTSShown are the core structures of five chemotypes of sortase inhibitors with a clustersize �6 that demonstrate limited inhibition of papain. The range of percent inhibi-tions observed for the class and the median (brackets) are shown for sortase A (S.aureus and B. anthracis) and papain (C. papaya). R, R�, and R indicate a hydrogenatom or an alkyl-type substituent (alkyl, methylene, or methine); Ar indicates acarbocyclic aromatic or heteroaromatic, including complex arrays; X indicates aheteroatom functional group such asOR, -NHR, etc.; and a dashed line indicates thepresence/absence of a ring unsaturation.




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AAEK1 must first be eliminated in order for the compound toreact with thiol in the active site of sortase. The modificationdid not occur when AAEK1 was incubated with a mutantenzyme carrying an alanine substitution at the active site resi-due (C184A). Thus, modification of sortase with AAEK1requires an active site Cys-184 (Fig. 3E).X-ray Structure Determination of the Sortase-AAEK Adducts—

To gain insights into themechanism of inhibition by the AAEKclass, we solved the three-dimensional structure of B. anthracissortase BwithAAEK1 andAAEK2 by x-ray crystallography andsingle-wavelength anomalous dispersion. Electron densitieswere refined to 2.1Å (AAEK1,R� 18.7%,Rfree� 25.9%) and 1.6Å (AAEK2, R � 18.6%, Rfree � 22.6%) resolution, respectively(Table 3). The use of experimental phases was important tocorrectly trace structural changes in the active site. For AAEK1,clear electron density corresponding to the thienylpropanoneadduct was observed near Cys-233 (the functional equivalent ofCys-184 in S. aureus SrtA), consistent with covalent modifica-

tion determined by MS (Fig. 4C). For the SrtB-AAEK2 adduct,�50% of Cys-233 was modified, most likely due to lower solu-bility of the compound under crystallization conditions. Thisfortuitous result allowed us to directly compare bound andunbound structures to determine what changes occur uponinactivation by the AAEK class. BothAAEK1 andAAEK2mod-ify sortase in a similar fashion, i.e. the �-carbon of the inhibitoris covalently linked to Cys-233 with the aryl group interactingwith a critical tyrosine (Tyr-138) (Fig. 4A). Cys-233, Asp-234,and His-140, which make up the catalytic triad, undergo sub-stantial rearrangements upon reaction with the AAEKs. Themost important change is Cys-233, which undergoes a rotationof �180 degrees to accommodate the ligand. In addition, Arg-243 swings away from the active site and is in excellent positionin the ligand-free form to stabilize an oxyanion intermediate ofthe inhibitor, a step that would be required prior to eliminationof amine from the AAEK (Fig. 4B). Further, the SrtB-AAEKadduct revealed two binding pockets, one cationic (above) andone anionic (below) the AAEK aryl group, which may beexploited for the engineering ofmore specific and potent inhib-itors (Fig. 5, A and B).Sortase Converts AAEKs to Reactive Olefin Intermediates—

We hypothesized that the �-carbon of AAEK1 or AAEK2 maybe linked via Michael-type addition by the sortase thiol to anolefin intermediate generated by amine elimination (42, 43). Ifso, addition of exogenous thiol would be expected to “capture”the olefin intermediate, thereby preventing the modification of

FIGURE 2. AAEK1 and -2 inhibit different sortases from bacilli and staph-ylococci. S. aureus SrtA (A) and B. anthracis SrtA (B), SrtB (C), or SrtC (D) wereincubated with increasing concentrations of AAEK1 (F) or AAEK2 (E) fol-lowed by the addition of substrate (a-LPETG-d for SrtA, a-KTDNPKTGDEA-dfor SrtB, or a-GEKLPNTASNN-d for SrtC) and fluorescence measurements. IC50is the concentration of compound that inhibits 50% of sortase activity and isdisplayed in micromolar in the upper right corner. Data were fit using an non-linear least squares method in GraphPad Prism�, and represent the mean ofthree independent determinations.

TABLE 2The structures of ten representatives from the AAEK chemotype classThe mean percent inhibitions of sortase A (S. aureus and B. anthracis) and papain(C. papaya) are presented.




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sortase (Fig. 6A). To test this hypothesis, AAEK-mediated inhi-bition of sortase was examined in the presence of DTT. Enzymekinetic analysis andmass spectrometry revealed that DTT doesnot react with AAEK1 or AAEK2 in the absence of sortase (datanot shown). However, in the presence of sortase, increasingamounts of DTT prevented inactivation by AAEK1 or AAEK2and, at 250 �M DTT, AAEK2-mediated inactivation was com-pletely abolished (Fig. 6B). Addition of DTT alone (withoutinhibitor) did not stimulate sortase activity and could notrestore the activity of enzymes whose active site was alreadymodified (Fig. 6B). Taken together, these observations suggest

FIGURE 3. AAEK1generates a covalent adduct with Cys-184 of S. aureusSrtA. S. aureus wild-type or mutant SrtA (C184A) were incubated with (B, C,and E) or without (A and D) AAEK1, followed by digestion with trypsin. Reac-tion products were separated by reversed-phase high-performance liquidchromatography and collected fractions subjected to MALDI-TOF MS. Com-pounds with m/z 1693.78 (fraction 54) and m/z 1555.74 (fraction 50) corre-spond to tryptic peptides encompassing the modified and unmodified activesite Cys-184 of sortase, respectively.

TABLE 3SrtB-AAEK adductsSummary of crystal, data collection, and crystallographic statistics for complexeswith AAEK1 and AAEK2.




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that DTT interferes with modification of the active site by theAAEK inhibitors.We sought to test whether the proposed olefin of AAEK1

can indeed modify sortase and synthesized the predictedintermediate (Fig. 6C, 12). Incubation of sortase with 12caused concentration-dependent and irreversible enzyme inhi-bitionwith aKi of 69�M (21�M), similar toKi values observedfor its parent AAEK1 compound. No sortase inhibition was

FIGURE 4. Three-dimensional structure of the active site of sortase B mod-ified by AAEK1. A, thienylpropanone adduct to sortase Cys-233 (blue), theproduct of the reaction of sortase B with AAEK1, is compared with the sortase

B structure at 1.6 Å (orange). The adduct occupies the position of three watermolecules (orange spheres) and is stacking against Tyr-138. B, the catalytictriad of sortase B (His140, Asp-234, and Cys-233) and Arg-243 are in closeproximity to the inhibitor adduct and undergo substantial structural shifts.The SrtB (green) and SrtB-AAEK adduct (blue) are superimposed. The figurewas generated using PyMOLTM. C, thienylpropanone modification of theactive site of sortase. Electron density map of the B. anthracis sortase B-AAEK1adduct demonstrating Cys-233, Tyr-138, and the thienylpropanone. Green,sulfur; red, oxygen. The figure was generated using COOT.

FIGURE 5. Surface charge rendering of the sortase-AAEK adduct struc-tures. AAEK1-SrtB (A) and AAEK2-SrtB (B) adducts revealed modification ofthe active site thiol of sortase. Electrostatic potential (red for negative andblue for positive) of the active site was generated using GRASP. An ionicpocket is located above and below the aryl group of each adduct. Ligandsatoms are color-coded as follows: yellow, sulfur; red, oxygen; green, chloride;and light blue, carbon.




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observed with a control compound 11, the corresponding alco-hol derivative of the olefin intermediate (Fig. 6C). Together,these data corroborate the hypothesis that sortase inhibition byAAEKs proceeds via a reactive olefin intermediate that forms acovalent adduct at the enzyme active site thiol.

DISCUSSION

The emergence of S. aureus strains with broad antibioticresistance represents a rapidly growing challenge for both hos-pital- and community-acquired infections. Antibiotic resist-

ance has also been observed in other Gram-positive bacteria,including an agent of bioterrorism, B. anthracis (44). Wide-spread use of antibiotics for human therapeutic or food indus-trial purposes is thought to be the primary selectionmechanismfor the emergence of drug resistance strains (45). Staphylococ-cal resistance mechanisms have been reported for all knownantibiotics, which necessitates identification of new therapeu-tic targets and development of new drugs that can be used forthe treatment of bacterial infections (46). Sortases, a family oftranspeptidases that immobilizes polypeptides in the envelopeof Gram-positive bacteria, recognize surface protein substratesand attach their C-terminal end to an amino group acceptor oflipid II, which is subsequently incorporated into the cell wall(47). Sortases are required for the pathogenesis of many differ-ent bacterial infections and their selective inhibition may be oftherapeutic value.Several recent studies sought to identify inhibitors of sorta-

ses. Oh and colleagues (14) evaluated medicinal plant extractsfor sortase inhibition; the best results were obtained with Coc-culus trilobus extracts. A lipid glucose conjugate from Fritil-laria verticillata as well as isoquinoline alkaloids from Coptischinensis are other inhibitor candidates (15). Isosteres of thescissile bond, i.e. threonine-glycine for sortaseA, achieved inhi-bition when the LPXT peptides were decorated with either dia-zoketone or chloromethylketone (13). These peptide-derivedinhibitors display favorable Ki values, however their rates ofinactivation are slow. Vinyl sulfones also react with thiols, butthe corresponding peptide vinyl sulfones present even slowerrates of sortase inactivation than diazoketone or chloromethyl-ketone derivatives (17). Finally, phosphorous isosteres of pep-tides are effective transition state analogs and inhibitors of zincproteases. Peptide mimics carrying replacement of the scissilepeptide bond indeed inhibited sortase (20). As with all otherpeptide-derived compounds, the further development of thesetypes of inhibitors toward a therapeutic use is obstructed bytheir chemical features, including high molecular weight andundesirable pharmacological properties. Finally, random irre-versible inhibition of thiol groups and of sortases can beachieved with small molecules such as methane-thiosulfonates(12, 17). Because such compounds lack specificity, their chem-ical properties preclude drug development.Previous studies on small molecule inhibitors of sortases

have been limited to the screening of 1000 compounds, whichidentified diarylacrylonitriles as potential inhibitors (18). In thisstudy, compounds with IC50 values between 10 and 1000 �Mwere reported, and one compound functioned as competitiveinhibitor with very favorable Ki value (18). Diarylacrylonitrileshave been proposed to bind to the active site of sortase,although this hypothesis has not yet been supported by exper-imental evidence (18).Here we screened a library of 135,625 small molecules for

inhibition of S. aureus SrtA with a fluorescence resonanceenergy transfer assay that measures enzymatic cleavage of thefluorogenic substrate a-LPETG-d between its threonine andglycine residues. By combining a cheminformatic approachwith secondary specificity assays, we identified several newclasses of sortase inhibitors, each one a distinct series with acommon structural core. Class I, AAEKs, display drug-like

FIGURE 6. An olefin intermediate modifies the active site thiol followingsortase activation of AAEK1. A, proposed structure of the olefin intermedi-ate and its reaction with thiol. B, reactions were performed in the presence orabsence of DTT and the degree of peptide cleavage assayed 1 h later, asdescribed for supplemental Fig. S1. The symbols (� and �) represent thepresence or absence of the indicated reagents, whereas the numbers refer tothe concentration of DTT. The far right reaction “1hr” refers to the addition ofDTT to the sample 1 h after the reaction with inhibitor was started. C, reactionswere performed as in B, except that the concentration of AAEK1 and theputative elimination intermediates 11 and 12 were assayed at 5 and 50 �M.The mean S.D. of three independent experiments are presented in B and C.Ar, thiophen-2-yl.




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properties, high relative levels of inhibition of sortase, as well asgood specificity. To investigate the mechanism of inhibition bythis class, we focused on compounds AAEK1 and AAEK2 dueto their high selectivity for inhibition of sortase and presence ofheavy atoms for x-ray structural studies. Kinetic studies sug-gested non-competitive inhibition with substrate and dialysis/re-assay hinted at an irreversible modification. MS confirmedthat the active site thiol of sortase was covalently modified witha derivative of AAEK1, implying the parent compound hadundergone a change during reaction with sortase.Comparison of the crystal structures of B. anthracis sortase

B-AAEK1 and sortase B-AAEK2 adductswith the 1.6-Å ligand-free enzyme revealed some remarkable structural changes.Although a majority of the enzyme structure is virtually identi-cal, there are some conformational changes that may be attrib-uted to adduct formation. Remarkably, side chains of all resi-dues in or near the active site that are part of the sortase barrel(loop between�2 and 3, strands�4 and�7) show virtually iden-tical conformation in ligand-free enzyme and in the complexwith inhibitors (Asn-102, Leu-106, Phe-121, Asp-123, Arg-125,Tyr-138, and His-140 of the catalytic triad). In contrast, theregion of the active site contributed by residues on the loopbetween �5 and �6 (Phe-189, Tyr-191, Tyr-235, and Arg-243)showmuchhighermobility and quite different conformation inligand-free and ligand-bound structures.In the ligand-free sortase, the region following theAsp-234 is

disordered. However, upon AAEK1 adduct formation thisregion becomes ordered and can be traced up to Leu-237 andfor AAEK2 up to Tyr-235. Interestingly, the region near theactive site between Thr-186 and Tyr-191 becomes more disor-dered upon adduct formation. The electron density for AAEK1is very well defined, and electron density for AAEK2 is goodwith the aromatic ring and the chlorine atoms well defined(data not shown), although it shows lower occupancy. Bothinhibitors react in a very similar manner with sortase B. In bothcases the �-carbon is covalently linked to Cys-233 (in agree-ment with MS data from sortase A) and the aromatic moietyinteracts with Tyr-138 and Asn-102. Both adducts are fullyaccessible to the solvent. As mentioned above, although themajority of protein amino acid side chains are in very similarconformations in both structures, there are some remarkabledifferences in the residues that make up the sortase catalytictriad. The most important is a change in the conformation ofCys-233, which, to accommodate the inhibitor, must rotate�180°. Adduct formation swings Arg-243 away from the activesite.However, in ligand-free sortase, its guanidiniumgroup is inexcellent position to stabilize an oxyanion intermediate of theinhibitor and thus may contribute to AAEK activation.The most significant structural finding is that the AAEK

adducts are situated in a crevice between two pockets, eachopposite in charge character. The anionic pocket proximal tothe carbonyl oxygensmay bewhat draws these compounds intothe active site through interaction with the electropositiveammoniummoiety. Further, the aryl rings are adequately posi-tioned such that substituent changes could present the appro-priate opposite charges to foster interactions that would addpotency and selectivity. This principal is partially demonstratedby the sortase-AAEK2 adduct, where the presence of two chlo-

rine atoms shifts the aryl group closer to the cationic pocket. Apreliminary investigation using para-substituted AAEKs sug-gests there is a correlation between inhibition of sortase andincreasing anionic character of the substituent, perhaps reflect-ing interactions with the cationic pocket.3

MS and x-ray crystallographic studies of sortase-AAEKadducts revealed a thienylpropanone covalently tethered viaits �-position to the active site cysteine (Fig. 4). The adductdiffers from the parent compound by the absence of a di-methylamine moiety. Elimination of this group from anAAEK would generate an aryl vinyl ketone, an electrophilicolefin that would be expected to react with an available thiol(41, 48). Indeed, excess thiol DTT prevented inactivation ofsortase by the AAEKs, and the putative olefin eliminationproduct, i.e. intermediate 12, was also a covalent inactivator,thereby corroborating the hypothesis. Thus, a general modelfor sortase inhibition by the AAEK class can be proposed(Fig. 7). The aqueous milieu, the surface accessibility of theactive site, and the orientation of the adducts in it suggest aninitial interaction between the ammonium form of the AAEKsand regions of negative electrostatic potential on the sortases(Fig. 5, pocket 2). This attraction of the AAEKs to the sortaseactive site could be followed by further stabilizing interactionswith the active site tyrosine and other residues. The path toalkylation of the cysteine by the AAEKs most likely then pro-ceeds via an elimination-addition mechanism (41, 48–50).Following deprotonation of AAEKs by an active site base,

enolate 13 is formed. This enolate could be stabilized by thesame active site features (the guanidinium of the conservedarginine, shown here with 13) that stabilize the oxyanion that

3 A. W. Maresso and O. Schneewind, unpublished observation.

FIGURE 7. Model for the mechanism of inhibition of sortase by AAEKs.Deprotonation of the � carbon is conjectured to occur via a base in the activesite of sortase. This generates enolate 13, which may be stabilized in a mannersimilar to the oxyanion intermediate of sortase during catalysis (e.g. by theguanidinium of a conserved active site arginine). This intermediate eliminatesan amine, here dimethylamine, from the �-position to generate 14. The elec-trophilic nature of 14 allows it to serve as an acceptor in a Michael-type con-jugate addition by the thiol of the active site cysteine. This resulting enolatemight also be stabilized by the guanidinium moiety; subsequent protonationby enzyme or medium would then generate the stable AAEK thioetheradduct observed.




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forms during sortase cleavage of substrate (Fig. 7) (45). Follow-ing �-elimination (133 14), the vinyl group of the aryl vinylketone is a suitable electrophile for the sulfur nucleophile of theactive site thiol (here presented as the thiolate anion) (41, 48).The Michael-type conjugate addition of the thiol to the vinylgroup then renders sortase inactive.The model presented herein suggests the AAEK compounds

are mechanism-based inhibitors, which are a class of inactiva-tors with examples among commercial drugs, and so have cleartherapeutic potential (51). Although there are no AAEKsamong these, previous work revealed that AAEKs displaydiverse in vivo properties, including anti-inflammation and glu-tathione reduction in mammalian organisms (52). Of note,AAEK2 has been demonstrated to possess antimicrobial activ-ity against Gram-positive but not against Gram-negative bac-teria (53). We have observed similar effects for AAEK1 and -2against both S. aureus and B. anthracis.3 Whether this antimi-crobial property of AAEKs relates specifically to the inhibitionof sortases has not yet been determined, although this hypoth-esis is being explored. Evaluation of the in vivo inhibition ofsortases in S. aureus and B. anthracis requires development ofassays with improved sensitivity and specificity over those thatare currently available. Preliminary results on the toxicity ofAAEKs for mammalian cells suggest that AAEK1 and AAEK2display 70- and 40-fold higher growth inhibitory activity towardmicrobial cells.3 Collectively, these results suggest that AAEKcompounds are suitable for further development. We seek toeventually interrogate such lead compounds for their therapeu-tic potential as sortase inhibitors in animalmodels of anthrax orstaphylococcal disease.

Acknowledgments—We acknowledge Sue Chiang and other membersof theNSRB for technical assistance duringHTS.We thankD. J. Burnsand Y. C. Martin (Abbott GPD) for cheminformatic advice and JohnWilmes (Reed College) for programming support. M.-E. D. wishes tothankV.H. Rawal (TheUniversity of Chicago (UC)) for sponsorship asa UC Visiting Fellow.

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SchneewindDominique M. Missiakas, Mark-Eugene Duban, Andrzej Joachimiak and Olaf

Anthony W. Maresso, Ruiying Wu, Justin W. Kern, Rongguang Zhang, Dorota Janik,Site

Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active

doi: 10.1074/jbc.M701857200 originally published online June 1, 20072007, 282:23129-23139.J. Biol. Chem.

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activation of inhibitors by sortase triggers irreversible modification

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