JB Accepted Manuscript Posted Online 2 March 2015 J. Bacteriol. doi:10.1128/JB.02552-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Novel antibacterial targets and compounds revealed by a high throughput cell

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wall reporter assay

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Asha S. Nayar1, Thomas J. Dougherty1,5, Keith E. Ferguson1, Brett A. Granger2, Lisa

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McWilliams3, Clare Stacey3, Lindsey J. Leach3, Shin-ichiro Narita4, Hajime Tokuda4,

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Alita A. Miller1, Dean G. Brown2 and Sarah M. McLeod1#

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Department of Bioscience and 2Department of Chemistry, Infection Innovative

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Medicines Unit, AstraZeneca R&D Boston, Waltham, Massachusetts, USA

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United Kingdom

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Japan

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School, Boston, Massachusetts, USA

Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire,

Faculty of Nutritional Sciences, University of Morioka, Takizawa 020-0694 Iwate,

Present Address: Department of Microbiology and Immunobiology, Harvard Medical

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Running Head: HTS screen for inhibitors of cell wall biogenesis

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Address correspondence to Sarah M. McLeod, [email protected]

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Abstract

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A high-throughput phenotypic screen based on a Citrobacter freundii AmpC reporter

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expressed in Escherichia coli was executed to discover novel inhibitors of bacterial

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cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here

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we describe the discovery and characterization of sulfonyl piperazine and pyrazole

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compounds, each with novel mechanisms of action. E. coli mutants resistant to

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these compounds display no cross-resistance to antibiotics of other classes.

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Resistance to the sulfonyl piperazine maps to LpxH, which catalyzes the fourth step

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in the synthesis of Lipid A, the outer membrane anchor of lipopolysaccharide (LPS).

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This compound is the first reported inhibitor of LpxH. Resistance to the pyrazole

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compound mapped to mutations in either LolC or LolE, components of the essential

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LolCDE transporter complex, which is required for trafficking of lipoproteins to the

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outer membrane. Biochemical experiments with E. coli spheroplasts show that the

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pyrazole compound is capable of inhibiting the release of lipoproteins from the inner

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membrane. Both of these compounds have significant promise as chemical probes

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to further interrogate the potential of these novel cell wall components for

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antimicrobial therapy.

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Importance

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The prevalence of antibacterial resistance, particularly among Gram-negative

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organisms, signals a need for novel antibacterial agents. A phenotypic screen using

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AmpC as a sensor for compounds that inhibit processes involved in Gram negative

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envelope biogenesis led to the identification of two novel inhibitors with unique

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mechanisms of action targeting Escherichia coli outer membrane biogenesis. One

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compound inhibits the transport system of lipoproteins to the outer membrane, while

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the other compound inhibits synthesis of lipopolysaccharide. These results indicate

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that it is still possible to uncover new compounds with intrinsic antibacterial activity

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that inhibit novel targets related to the cell envelope, suggesting that the Gram-

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negative cell envelope still has untapped potential for therapeutic intervention.

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Introduction The biosynthesis of the bacterial cell wall is a well-established target for

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antibacterial agents such as β-lactams, one of the oldest and clinically most

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prevalent classes of antibiotics. However, with the rise of infections caused by multi-

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drug resistant bacteria, there is a widely recognized need for new antibacterial

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compounds (1-3). The bacterial cell wall is an excellent target for development of

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antibiotics because its synthesis is conserved across bacterial pathogens and absent

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from mammalian cells. This cell wall consists of peptidoglycan, a mesh-like structure

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that plays an essential role in maintaining cell integrity, which is composed of

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repeating β-(1,4) N-acetyl-glucosamine-β-(1,4) N-acetyl muramic acid disaccharide

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strands cross-linked through peptide stems. Whereas several advances have been

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made in reconstituting the biosynthetic steps for peptidoglycan synthesis in vitro for

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chemical interrogation, the later steps in this pathway that utilize membrane-bound

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enzymes make biochemical high-throughput screening challenging (4). Thus,

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phenotypic or cell based screening to find novel inhibitors of cell wall biogenesis is

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an attractive alternative to target based enzymatic screens. Phenotypic screening

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not only overcomes the biochemical hurdles associated with assays needing

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membrane proteins, but also permits interrogation of the entire pathway at once and

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selects for compounds that penetrate into the cell, which has been recognized as a

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significant hurdle in target-based antibacterial drug discovery (5, 6).

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In some Gram-negative bacteria, exposure to β-lactam antibiotics induces the

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expression of the chromosomally-encoded ampC β-lactamase (7, 8). In these

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organisms, AmpC expression is repressed under normal growth conditions by a

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divergently transcribed repressor, AmpR. Treatment with β-lactam antibiotics

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disrupts the balance of peptidoglycan synthesis due to the inhibition of the

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transpeptidase activity of penicillin-binding proteins (PBPs), which are involved in the

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final stage of peptidoglycan synthesis (reviewed in (9)). Continued peptidoglycan

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turnover reactions in the absence of synthesis leads to the accumulation of

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anhydromuramyl peptides, which bind to the AmpR regulator, causing a

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derepression in the expression of the AmpC β-lactamase. Sun et al. have shown

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that inhibitors of other steps of cell wall biogenesis are also capable of inducing

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AmpC β-lactamase production, and that AmpC can be used as a reporter to detect

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cell wall-active compounds (10, 11).

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In a previous report using a hypersensitive Escherichia coli strain, we

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identified a novel inhibitor of lipoprotein transport to the outer membrane (12). Here,

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we describe the adaptation of the AmpC β-lactamase reporter system (11) for

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phenotypic screening in a high-throughput, 384-well format, that identified another

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novel inhibitor of lipoprotein trafficking as well as a novel inhibitor of

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lipopolysaccharide (LPS) synthesis. Compound 1 targets LpxH, which is involved in

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the biosynthesis of lipid A, the outer membrane anchor of LPS (13) (Figure 1).

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Compound 2 was found to inhibit the function of the LolCDE complex, which is

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required for transport of lipoproteins to the outer membrane (14, 15) (Figure 1).

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Identification of these inhibitors indicates that this screening assay is not restricted to

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identifying compounds that directly impact peptidoglycan synthesis but also has a

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broader scope in finding inhibitors of indirectly-related cell envelope biosynthesis.

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Materials and Methods

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Bacterial Strain Construction

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To engineer the reporter construct, the ampR-ampC locus from a clinical

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isolate of Citrobacter freundii was amplified by PCR using primers

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CfrAmpRAmpCFEcoRI and CfrAmpRAmpCRBsaI (Supplemental Table 1) and the

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Roche High Fidelity Master Mix, according to the manufacturer’s instructions. The

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resulting PCR product was cloned into pCR4 Blunt TOPO vector (Life Technologies,

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WI, USA) to obtain pAN118. The sequence of the ampR-ampC insertion was

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verified by DNA Sanger sequencing using a Life Sciences 3100 series Genetic

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Analyzer. This ampR-ampC region of pAN118 was sub-cloned into the EcoRI site of

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the low-copy vector pWSK129 (16) to create the reporter construct used for

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screening, pAN116. The reporter plasmid pAN116 was then transformed into E. coli

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W3110 ΔampC ΔacrB to create the screening strain ARC4150. The ampC and acrB

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gene deletions were constructed using λ Red-mediated recombination in the E. coli

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strain BW25113 containing plasmid pKD46, as previously described (17). Primers

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used are shown in Supplementary Table 1. Recombinants were selected on Luria-

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Bertani (LB) agar medium containing 25 μg/ml kanamycin and deletions were

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verified by PCR. The ampC deletion was then moved by P1 phage transduction into

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E. coli W3110 (18). The kanamycin resistance gene was excised from the

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chromosome using the FLP recombinase expressed from pCP20 as previously

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described (17). The acrB deletion was subsequently moved by P1 phage

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transduction into the W3110 ΔampC strain and the kanamycin resistance gene was

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removed as described above. The artifact control strain ARC4151 was created by

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transforming the empty vector pWSK129 into W3110 ΔampC ΔacrB.

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To create the LpxH overexpression strain, lpxH from E. coli MG1655

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was amplified by PCR as described above where the upstream primer encoded an

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EcoRI site and the downstream primer encoded a HindIII site (Supplementary Table

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1). The resulting PCR product was digested and cloned into the EcoRI and HindIII

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sites of pPSV35 (19). This plasmid and the empty vector (pPSV35) were then

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transformed by electroporation into E. coli MG1655 ΔtolC, selecting on LB agar

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containing 12 μg/ml of gentamicin.

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High throughput screening

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The screening strain ARC4150 and the artifact strain ARC4151 were

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grown in LB with 25 μg/ml kanamycin at 37 °C to an OD600 of 0.8. The cells were

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mixed with an equal volume of 20% glycerol, divided into aliquots and flash frozen in

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a dry ice and ethanol bath and stored at -80 °C. On each day of screening, cells

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were thawed at room temperature and diluted 1:20 in LB broth supplemented with 25

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μg/ml kanamycin to obtain a final OD600 of 0.02. The cells were grown at 37 °C with

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shaking at 200 rpm to an OD600 of 0.08. 30 μl of cells were then dispensed using a

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multidrop dispenser (Thermo Scientific, Waltham, MA) into each well of 384 well

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plates containing test compounds. For details of how the screening plates were

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prepared, please see Supplementary Methods. The final compound concentration

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was 50 μM with a DMSO concentration of 1.25% (v/v). After the plates were

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incubated at room temperature for 2 h, 10 μl of reaction buffer (20 mM Tris-Cl, pH

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8.0, 20 μg/ml lysozyme and 0.1 mM nitrocefin) was added to each well and

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incubated at room temperature for another hour. The reaction was stopped with a

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final concentration of 10 μg/ml cloxacillin and the plates were read at A490 and OD600

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using an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA). The active

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compounds were re-screened in a 7-point concentration response with a final

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compound concentration range between 200 μM and 3 μM.

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Susceptibility testing Minimum Inhibitory Concentrations (MICs) were determined according to

159 160

the guidelines of the Clinical and Laboratory Standards Institute (20). A preliminary

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toxicity assessment was made by measuring the anti-proliferation activity of

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compounds of interest against Candida albicans, the human lung carcinoma cell line

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A549, and induction of lysis of sheep red blood cells as previously described (21,

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22).

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Inhibition of cellular biosynthetic processes

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Inhibition of cell wall, fatty acid, DNA, RNA and protein biosynthesis was measured

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as previously described in an E. coli W3110 ΔtolC ΔlysA strain (23).

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Microscopy

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Linnaeus Bioscience performed the bacterial cytological profiling as described

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previously (24). E. coli ATCC 25922 ΔtolC were grown at 30 °C with shaking until

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early log phase (OD600 of 0.15 to 0.2). Cells were then mixed with 2x- 10x MIC of

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compound and rolled in test tubes at 30 °C for 120 min. Cells were subsequently

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stained, concentrated by centrifugation and observed by fluorescence microscopy.

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Resistant mutant selection

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Mutants resistant to compounds 1 and 2 were raised against E. coli MG1655 ΔtolC

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and E. coli ATCC 25922 ΔtolC, respectively. One hundred μl of cells (9.65 x 108

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CFU/ml for E. coli MG1655 ΔtolC and 5.7 x 109 CFU/ml for E. coli ATCC 25922

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ΔtolC) were plated on LB agar containing 8x, 16x, 32x and 64x the MIC of the test

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compound. In addition, 10-fold serial dilutions of the culture were spread on plates

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without antibiotic selection to determine the total number of CFU/ml in the sample. 8

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Plates were incubated at 37 °C for 24 to 48 h. The resistance frequency was

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calculated as the CFU/ml on the compound- containing plates divided by the total

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CFU/ml of the bacterial culture. Resistant colonies were confirmed by plating them

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on LB agar containing compound at 8x - 64x the MIC. Resistant isolates were

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passed onto LB agar without selection three times prior to determining the MICs for

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compound 1 and 2 to ensure that they were stable resistant mutants. Resistant

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mutants were then subjected to whole genome sequencing using an Illumina MiSeq

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V2 instrument to identify mutations as previously described (25).

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Spheroplast release assays

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E. coli MG1655 ΔtolC cells were grown in LB medium at 37 °C to an OD600 of 1.0.

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The cells were converted into spheroplasts as described (26). The Lpp release

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assay was performed as described (12). Briefly, suspensions containing 2 x 108

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spheroplasts were incubated with or without 3.5 μg His-tagged LolA in the presence

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of DMSO or 1.4 μg globomycin or compound 2 at 30 °C for 1 minute. Two hundred

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and fifty μl of LB containing 0.3 M sucrose and 10 μg/ml DNase I was added and

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incubated at 30 °C for 30 min. The spheroplasts were pelleted by centrifugation at

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16,000 x g for 2 min. The supernatant was then diluted 3-fold with 7.15 mM MgCl2

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and was ultracentrifuged at 100,000 x g for 30 min to remove the membranes. The

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supernatants were then analyzed by SDS-PAGE and immunoblotting with anti-Lpp

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and anti-OmpA antibodies.

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Results

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AmpC reporter strain specifically detects inhibitors of cell wall biogenesis

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It was previously shown that the inducible AmpC from C. freundii could be used as a

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sensor for inhibitors of cell wall biosynthesis (11). Here we used a similar inducible

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AmpC from C. freundii introduced into E. coli to develop a higher-throughput 384-

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well format assay. The ampR-ampC region from C. freundii was cloned into a low-

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copy number plasmid and introduced into an E. coli screening strain. A strain of E.

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coli lacking its chromosomal copy of ampC was used to reduce signal background,

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as the endogenous E. coli AmpC system produces a low level of non-inducible

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expression of AmpC (27). Initially we tested a waaP deletion screening strain, which

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impacts permeability via an LPS defect, and found a higher background expression

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of the AmpC reporter. This result was similar to that previously reported by Sun et

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al., in which they also noted an increase in background in their envA (lpxC)

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screening strain (11). Consequently, we decided instead to employ an RND efflux

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pump mutant (ΔacrB) in combination with the ampC chromosomal deletion to

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increase the screen sensitivity, without impacting the expression level of the AmpC

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reporter system (28). Several methods of preparing the cells and various reaction

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buffers were evaluated and compared to those employed by Sun et al. to select

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optimal assay conditions that gave a robust signal and a format amenable to our

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high-throughput robotic screening system. Tris-Cl pH 8 buffer with 20 μg/ml

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lysozyme was found to yield the highest level of ampC induction compared to the

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previously used Z salts (sodium phosphate-based buffer system) (data not shown)

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(11). Several detergents such as CTAB (cetyltrimethylammonium bromide) and

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sodium deoxycholate in the reaction buffer were also tested; however, under these

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conditions some nitrocefin precipitation was observed and no improvement in signal 10

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was seen, so these reagents were abandoned. The optimal conditions for our

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robotic system were found to be those in which the reporter strain was grown to an

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OD600 of 0.08 and incubated with the test compounds for 2 h at room temperature,

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whereupon a detection reagent consisting of Tris-Cl pH 8, lysozyme and the

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colorimetric β-lactam nitrocefin was added (data not shown). Cleavage of nitrocefin

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by the AmpC β-lactamase resulted in a colorimetric change, which was detected at

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A490. Because the amount of nitrocefin cleavage changes over time, a β-lactamase

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inhibitor (cloxacillin) was added after incubation at 25 °C for 1 hour to stabilize the

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signal. The A490 values were normalized relative to the cell density (OD600) to

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account for compounds that caused slower cell growth or cell lysis.

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Antibiotics that inhibit a variety of classes of cellular targets were profiled to

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characterize induction of the AmpC reporter strain (Figure 2). The assay was able to

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detect inhibitors of multiple steps of cell wall biogenesis, such as phosphomycin,

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which inhibits the first committed step of peptidoglycan synthesis performed by

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MurA, as well as cefoxitin, which inhibits one of the last steps in peptidoglycan

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synthesis, the transpeptidation reaction carried out by PBPs. The maximal

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expression of the AmpC reporter (relative to baseline) increased 1.8- and 1.4- fold

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for phosphomycin and cefoxitin, respectively. In addition to inhibition of

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peptidoglycan synthesis, inhibitors of other factors required for outer membrane

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synthesis were also found to induce the AmpC reporter strain. For example, CHIR-

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090, an inhibitor of LpxC (29), which performs the first committed step in the

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biosynthesis of the lipid A component of outer membrane LPS, was also detected by

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the AmpC reporter strain, showing an approximately 1.5-fold induction. Some of the

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compounds displayed concentration curves that had a bell shape due to cell death

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which occurred at concentrations higher than their MIC. Conversely, antibiotics that 11

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inhibit cellular targets that are not involved in cell wall or outer membrane

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biogenesis, such as the translation inhibitors tetracycline and chloramphenicol or the

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gyrase inhibitor ciprofloxacin, did not show induction of the reporter (data not

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shown). These data demonstrated that this reporter strain is specific and suitable for

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detecting molecules that disrupt components of the E. coli envelope.

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High-throughput screening of AmpC reporter assay

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A high-throughput screen to identify compounds that induce the AmpC

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reporter strain was conducted in a 384-well format. Approximately 1.2 million

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compounds from the AstraZeneca collection were screened at a 50 μM test

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concentration. This phenotypic screen resulted in a robust assay with a mean Z’ of

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0.79 ± 0.04 over the 31 screening runs of the entire campaign (30). A chemical

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triage process was applied to remove compounds with undesirable physical-

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chemical properties, commercial antibiotics and known inhibitors of cell wall

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biosynthesis. The remaining compounds were tested in a 7-point concentration

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response assay for determination of potency. As some compounds are colored and

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absorb at 490 nm (the wavelength used to detect the cleaved nitrocefin product), an

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orthogonal artifact assay was used where compounds were tested against the E. coli

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ΔampC ΔacrB strain lacking the C. freundii ampR - ampC reporter construct. To

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correct for artifactual signal at A490, the concentration response data were compared

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to the artifact assay data and normalized by subtraction if an increase in signal was

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detected in the artifact assay, as previously described (31). The resulting hits were

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screened against the yeast strain Candida albicans and an A549 mammalian cell

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line and for lysis of sheep red blood cells to remove promiscuous or broadly toxic

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compounds. 12

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Profile of two compounds from AmpC reporter screen Two compounds were selected for further characterization and to confirm their

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mode of action as inhibitors of cell envelope biogenesis. Compound 1 contains both

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indoline and piperazine scaffolds, has a molecular weight of 453.5 and a measured

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LogD of 2.7 (Figure 1). Compound 2 has a pyrazole core, with a molecular weight of

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345.4 and a measured LogD of 4.3 (Figure 1). Both compounds have high human

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serum protein binding (>99% bound). Compounds 1 and 2 were resynthesized (see

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supplemental materials) and re-tested in the AmpC reporter assay to confirm their

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activity. Compound 1 induced the reporter 0.4-fold and compound 2 induced the

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reporter 0.6-fold over the baseline signal (Figure 2). Both compounds were also

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profiled for antibacterial activity against Gram-negative and Gram-positive species.

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Strong inhibition of growth was observed in an E. coli efflux mutant (ATCC 25922

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ΔtolC) with a MIC of 0.25 μg/ml for compound 1 and 0.125 μg/ml for compound 2

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(Table 1). Compound 2 also had moderate activity against the wild-type E. coli strain

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ATCC 25922 with a MIC of 8 μg/ml and weak activity (MIC of 32 μg/ml) against

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Haemophilus influenzae. Neither compound 1 nor compound 2 were active against

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Staphylococcus aureus or the yeast strain C. albicans and did not cause lysis of

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sheep red blood cells; however, both compounds inhibited the proliferation of the

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human cell line A549 when exposed for 72 h (90% cytotoxic concentration or CC90 of

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23 μM for compound 1 and 84 μM for compound 2) (Table 1 and data not shown).

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In order to confirm the results of the AmpC reporter assay, compounds 1 and

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2 were assayed for inhibition of the incorporation of cellular pathway-specific

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radioactive precursors (23). Both were found to inhibit the incorporation of

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[3H]diaminopimelic acid, which is a component of the E. coli peptidoglycan, indicating 13

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that the activity of these compounds is related to the inhibition of cell wall biogenesis

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(Table 2). Although a decline in [14C]acetic acid incorporation was also observed in

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the presence of compound 1, suggesting a potential inhibition of fatty acid

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biosynthesis, of the 15 independent resistant mutants that were analyzed by whole

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genome sequencing, mutations were found exclusively in the lpxH locus (see below).

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Nonejuie et al. have shown that antibacterial compounds cause distinct

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changes in cellular morphology depending on their mode of action (24). The effects

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of compounds 1 and 2 on the morphology of E. coli ATCC 25922 ΔtolC were

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examined to investigate which cellular pathways they inhibit. The cells were

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exposed to either 2% DMSO (vehicle control), compound 1, compound 2 or

312

meropenem at 2x to 10x MIC for 120 min at 30 °C. The cells were then subjected to

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fluorescence microscopy with staining of membranes by FM4-64, DAPI staining to

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visualize the nucleoid and sytox green to detect membrane permeabilization. All

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three compounds showed distinctly different morphologies, despite the fact that each

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inhibits a component related to cell wall biogenesis (Figure 3). Compound 1 caused

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the cells to elongate relative to the DMSO control as well as faint staining of the

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interior of the cell with sytox green, which indicates loss of membrane integrity.

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Compound 2 also caused cell elongation, but in contrast to compound 1, the cells

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were quite swollen and the nucleoids appear to be less condensed than seen with

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the vehicle control. Some of the cells were also very brightly stained with sytox

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green, which indicates loss of membrane integrity. Both of these morphologies are

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quite different from meropenem, which targets PBPs (reviewed in (32)). In the

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presence of meropenem, the majority of cells were elongated with distinct bulges in

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the middle, and the nucleoids were de-condensed compared to the vehicle control,

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as previously described (24).

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Mode of Action of Compound 1 To further define the cellular target of compound 1, resistant mutants were

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generated using an E. coli MG1655 ΔtolC strain. The frequency of resistance

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ranged from 2.1 x 10-8 at 8 x MIC to 4.7 x 10-8 at 64 x MIC of compound 1. Fifteen

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stable resistant mutants were subjected to whole genome sequencing using an

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Illumina platform. All fifteen mutants were found to have single amino acid changes

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in LpxH. LpxH is essential for cell viability and catalyzes the fourth step in the

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biosynthesis of lipid A, the outer membrane anchor of LPS (13, 33, 34). These

336

mutations mapped to four different residues in LpxH: G48, L84, F141 and R149.

337

The MIC of compound 1 for each of these four mutants increased by more than 512-

338

fold compared to the parent strain (Table 3, left panel). The susceptibility of these

339

LpxH mutants did not change for control antibiotics such as levofloxacin, meropenem

340

and tetracycline, indicating that these mechanisms of resistance are specific to

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compound 1. Interestingly, these LpxH mutants also showed no change in

342

susceptibility to the LpxC inhibitor PF1090, which targets an earlier step in the same

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pathway as LpxH (35). These results strongly suggest that this compound inhibits

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bacterial cell growth through inhibition of LpxH activity.

345

To expand on these results, high copy suppression with LpxH was used to

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confirm it as the cellular target of compound 1. lpxH from E. coli was cloned onto a

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plasmid under the control of an IPTG-inducible promoter (pPSV35) (19) and

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transformed into E. coli MG1655 ΔtolC. In the presence of 50 μM IPTG, the MIC of

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compound 1 against a strain overexpressing LpxH increased to greater than 128

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μg/ml while other antibacterial compounds that do not inhibit LpxH showed no

351

change in MIC (Table 3, right panel). These data are consistent with compound 1 15

352

having a mode of action through inhibition of LpxH, as the susceptibility to this

353

inhibitor decreases upon increased copy number of LpxH in the cell.

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Mode of Action of Compound 2

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25922 ΔtolC (a pathogenic strain of E. coli with an engineered deletion of the efflux

359

pump TolC) to define its mode of action. The frequency of resistance was

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determined to be 7.3 x 10-7 at 32x MIC. At concentrations lower than 32x MIC,

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confluent growth was observed. Whole genome sequencing was performed on ten

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stable resistant mutants using an Illumina platform. Each isolate carried a mutation

363

in a single gene locus. Mutations were mapped to either lolC, lolE, the predicted

364

promoter region of lpp or a locus that is predicted to encode two overlapping,

365

divergent genes annotated as Z2510 and Z2511, as further defined below.

366

Mutants resistant to the pyrazole compound 2 were raised in E. coli ATCC

The Lol mutations corresponded to either a single amino acid change, G254V,

367

in LolC, or three different amino acid changes in LolE: G195S, P365C, and D367Y.

368

LolC and LolE are both essential for cell viability and are members of an ABC

369

transporter complex, LolCDE, which is responsible for releasing lipoproteins from the

370

inner membrane for transport to the outer membrane (14, 36). All of these

371

mutations in either LolC or LolE caused very large increases in the MIC of compound

372

2 (>1024-fold), but not to other classes of antibiotics (Table 4). Interestingly, a

373

mutation in the predicted promoter region of lpp was also isolated. This mutation

374

caused a 32-fold increase in the MIC of compound 2. Lpp is one of the most

375

abundant E. coli outer membrane lipoproteins where it interacts both covalently and

376

non-covalently with peptidoglycan to stabilize the cell surface structure (37, 38).

377

When lipoprotein trafficking to the outer membrane is disrupted, Lpp accumulates in

16

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the inner membrane where it binds covalently to peptidoglycan and is lethal for the

379

cell (39, 40). It has been shown that mutations in Lpp can cause resistance to

380

globomycin or myxovirescin, antibiotics that inhibit the signal peptidase II protein

381

(LspA), which cleaves the signal peptide from lipoproteins (41-43). This is a step in

382

lipoprotein maturation that occurs prior to LolCDE releasing lipoproteins for transport

383

to the outer membrane. In order to confirm that this lpp promoter mutation

384

(presumably due to decreased lpp transcription) is responsible for resistance to

385

compound 2, an E. coli strain carrying a deletion in lpp was tested for susceptibility to

386

compound 2. The E. coli MG1655 ΔtolC Δlpp strain (12) showed a 64-fold increase

387

in the MIC of compound 2 relative to the E. coli MG1655 ΔtolC parental strain (data

388

not shown), indicating that a loss of Lpp in the cell leads to compound 2 resistance.

389

There was no change in susceptibility of these lpp mutants to compounds that do not

390

inhibit lipoprotein transport to the outer membrane, as expected (Table 4). We have

391

previously identified a structurally distinct inhibitor of the LolCDE complex (12)

392

whose mutations conferring resistance mapped to LolC N265K and LolE L371P.

393

Compound 2 was found to be resistant to these mutants as well. The MIC of

394

compound 2 increased 64-fold when tested against each of these mutants (data not

395

shown).

396

In addition to mutations in loci related to outer membrane lipoproteins, five

397

different mutations (4 single amino acid changes and one frame shift mutation) in

398

Z2510 were also isolated. Z2510 is a putative AcrR family transcriptional repressor

399

of unknown function that is encoded by some pathogenic strains of E. coli.

400

Additionally, there is a putative open reading frame annotated as Z2511 that is

401

divergent and partially overlapping Z2510. Therefore, these mutations would also

402

affect Z2511. Z2510 and Z2511 are positioned near an operon predicted to encode 17

403

an efflux system suggesting they may be involved in regulating this process. These

404

mutations caused a 32- to 64-fold increase in MIC to compound 2 (Table 4). The

405

MIC of levofloxacin was also increased 2- to 4-fold in these strains, which suggests

406

that these mutations cause a non-target related efflux-mediated change in

407

susceptibility to some antibiotics. Also, E. coli strains that lack Z2510 and Z2511,

408

such as some E. coli K-12 strains, remain susceptible to compound 2 (data not

409

shown), suggesting that these resistant mutants are not indicative of the mode of

410

inhibition by compound 2.

411 412

Taken together, these data suggest that compound 2 inhibits bacterial growth

413

by targeting LolCDE to block lipoprotein transport to the outer membrane. To

414

determine whether compound 2 prevents trafficking of lipoproteins from the inner

415

membrane to the outer membrane, we measured Lpp release from spheroplasts to

416

purified LolA in the presence of compound 2. As a control, globomycin was also

417

tested as it inhibits a previous step in lipoprotein processing. Spheroplasts were

418

prepared from E. coli and incubated with purified His-tagged LolA. The spheroplasts

419

were then removed by centrifugation and the supernatant was analyzed by SDS-

420

PAGE and Western blot with anti-Lpp antibody. The amount of Lpp released from

421

the spheroplasts indicates lipoprotein releasing activity. The blot was also probed for

422

OmpA, another outer membrane protein not dependent on LolCDE for transport to

423

the outer membrane. As shown in Figure 4, the appearance of Lpp in the

424

supernatant was dependent on the presence of both spheroplasts and purified LolA.

425

Both globomycin and compound 2 decreased the amount of Lpp that was released

426

from the spheroplast. There was a slight decrease in the amount of OmpA released

427

from the spheroplast seen in the presence of compound 2 and globomycin indicating

18

428

that these compounds may also have some effects on protein synthesis or

429

translocation across the inner membrane by the Sec machinery. To examine the

430

effect of the LolC and LolE mutations that confer resistance to compound 2,

431

spheroplasts were prepared from the E. coli ΔtolC LolE L371P and LolC N265K

432

resistant mutants. Compound 2 was not able to inhibit release of Lpp from these

433

mutant spheroplasts at the same concentration used to inhibit release from

434

spheroplasts from the susceptible parent strain (Figure 4). These data are

435

consistent with compound 2 inhibiting E. coli growth through the LolCDE complex.

436

Globomycin, which does not target LolCDE, is still capable of inhibiting Lpp release

437

from the mutant E. coli spheroplasts, as expected (41).

438 439

19

440 441

Discussion An assay using an inducible AmpC reporter system as a sensor for molecules

442

that inhibit processes related to E. coli cell wall biogenesis was first developed and

443

validated for high-throughput screening. In conjunction with the cell wall phenotypic

444

reporter, the strain also had a deletion of an RND efflux pump to improve detection

445

of weak inducers of the reporter. The system exhibited a surprisingly broad

446

response to a range of disturbances in the cell envelope assembly process,

447

identifying novel inhibitors of both outer membrane lipoprotein transport and LPS

448

biosynthesis. Therefore, this assay is capable of identifying inhibitors of a number of

449

different targets in a single screening campaign, many of the targets, such as the Lol

450

transport system not being amenable for screening as an enzyme assay in a cell-

451

free system. The results of this AmpC reporter screen also illustrate that disruption

452

of systems involved in envelope assembly impacts peptidoglycan biosynthesis and

453

turnover processes, as has been demonstrated recently by a role for lipoproteins in

454

the functioning of key cell wall (PBP) synthetic enzymes (44).

455

One of the inhibitors identified in this reporter screen was the sulfonyl

456

piperazine compound 1 with antibacterial activity against an efflux mutant of E. coli.

457

Mutants resistant to this compound mapped to LpxH, which suggests the mode of

458

action of this compound is to inhibit the production of LPS. Over-expression of LpxH

459

in an efflux mutant of E. coli abolished sensitivity to compound 1, further implicating

460

LpxH as the target of this compound. Numerous inhibitors of LpxC, which performs

461

the first committed step in this pathway have been reported (29, 35, 45); however,

462

none of these compounds have advanced beyond Phase I clinical testing. To our

463

knowledge compound 1 is the first reported inhibitor of LpxH. LpxH is essential for

464

growth in E. coli and present in most γ –proteobacteria; however, it has never been

20

465

exploited as a target for therapeutic intervention (13, 33, 34). Due to its lipophillicity

466

and high protein binding in serum, compound 1 lacks the physical properties

467

necessary for dosing in mammalian systems. The narrow antibacterial spectrum of

468

this compound also limits its clinical use. It is possible that compound 1 does not

469

permeate the outer membrane of some of the Gram negative organisms of high

470

unmet medical need, such as P. aeruginosa, which are well known for their

471

impermeable outer membranes and active efflux systems. Additionally the high rate

472

of spontaneous resistance to compound 1 indicates that targeting this protein alone

473

may not lead to a single agent therapy, but an LpxH inhibitor may need to be

474

combined with another agent targeting a different step. In that regard, compound 1

475

may have promise as a chemical probe of LpxH function and further assessment of

476

the potential of LpxH as a target for antibacterial therapy.

477

Another inhibitor identified in the AmpC reporter screen was the pyrazole

478

compound 2 with antibacterial activity primarily against E. coli. Mutants at relatively

479

high frequencies resistant to compound 2 mapped primarily to single amino acid

480

changes in the LolC and LolE proteins, which form a complex with two copies of the

481

LolD ATPase protein to form an inner membrane ABC transporter. This LolCDE

482

complex is essential for cell viability and is responsible for releasing lipoproteins from

483

the inner membrane to LolA, a periplasmic molecular chaperone, for transport to the

484

outer membrane component, LolB (14, 36). Compound 2 was found to directly

485

inhibit release of lipoproteins from spheroplasts to purified LolA, which confirms the

486

genetic evidence found by mapping resistance mutations. These data suggest the

487

mode of action of compound 2 is to inhibit lipoprotein transport to the outer

488

membrane. In addition mutations in the lpp locus, which encodes one of the major

489

outer membrane lipoproteins in the cell, also conferred resistance to compound 2,

21

490

which further suggests that compound 2 inhibits lipoprotein trafficking to the outer

491

membrane, as mis-localization of Lpp to the inner membrane is lethal to E. coli (39,

492

40). Thus, mutations that decrease the amount of Lpp in the cell should alleviate

493

some of the toxicity that occurs in the presence of the inhibitor. Resistance

494

mutations were also found in an uncharacterized putative transcriptional repressor;

495

however, how these mutations lead to resistance to compound 2 is currently not

496

known. These mutations do not cause as large a shift in MIC versus compound 2 as

497

those isolated in LolC or LolE and this gene is not found in many susceptible strains

498

of E. coli. It is therefore likely these mutations correspond to a modest resistance

499

mechanism for compound 2.

500

Inhibitors of outer membrane lipoprotein trafficking have been previously

501

reported (12); however, to date none of these molecules have been used

502

therapeutically. Compound 2 lacks the physical properties and antibacterial

503

spectrum required for clinical use, but can be employed as a tool to aid in exploration

504

of lipoprotein transport to the outer membrane, such as the physiological impact of

505

disruption on outer membrane integrity. Compound 2 also displayed relatively high

506

resistance rates and large shifts in MIC with single step mutations. These properties

507

are similar to what was reported with the structurally distinct pyridine imidazole

508

series that targets the LolCDE complex (12). Whether this observed high resistance

509

emergence is a property of the target or these particular compounds still remains to

510

be determined. Unfortunately, crystal structures are not available for either of these

511

targets, so the binding modes of both compounds to their targets are unknown at

512

present. Thus the possibility of modifying the compounds to pick up additional

513

binding interactions and strengthen compound targeting must await structural

514

information.

22

515

One challenge in antibiotic discovery is that it has become evident that the

516

physicochemical properties of the chemical libraries used for screening are not

517

favorable for finding new antibiotics (46, 47). In addition, it has been argued that

518

because of pre-existing mutations in bacterial populations, single gene targets are

519

susceptible to single-step, high-level resistance, and will need to be paired with

520

agents that target other functions (6, 48). The results in the two targets presented

521

here support these ideas, as well as the recently published results with mutations

522

developing in the clinic to the Anacor/GSK oxaborole compound which targets the

523

leucyl t-RNA synthetase editing site (49). Multiple therapeutic compound strategies

524

for single gene targets are employed as a matter of course in tuberculosis therapy

525

(50). Inasmuch as inhibition of either the LpxH or LolCDE targets would impact the

526

cell envelope integrity, these compounds could act to potentiate a partner antibiotic’s

527

efficacy by improved influx through the outer membrane.

528 529

The rise of antibacterial resistance, particularly among Gram-negative

530

pathogens, signals a growing need for novel antibacterial compounds. The results

531

described above demonstrate that the AmpC reporter screen, combined with

532

improved sensitivity due to efflux pump inactivation, is capable of uncovering a

533

broader range of new molecules with intrinsic antibacterial activity that inhibit novel,

534

cell envelope-related targets. The present screen results also suggest that

535

interfering with the biogenesis of the Gram-negative cell envelope still has

536

significant, untapped potential for therapeutic intervention, possibly by exploiting

537

these novel targets to resensitize cells to current antibiotics by disruption of outer

538

membrane assembly.

539

23

540 541 542

Acknowledgments We would like to thank the AstraZeneca Infection Bioscience Department for

543

MIC testing, James Whiteaker and Robert McLaughlin for whole genome

544

sequencing, Amy Kutschke for helping with assays to measure inhibition of

545

macromolecular synthesis pathways and Helen Plant for help with high-throughput

546

screening. We also thank Boudewijn de Jonge and Kirsty Rich for input and editing

547

of the manuscript. We also wish to thank Joe Pogliano and employees of Linnaeus

548

Bioscience for their bacterial cytological profiling services and helpful discussions.

549 550

Supplementary information is available

551

24

552

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27

711 712 713 714 715 716 717

Figure Legends Figure 1: Chemical structures of compounds 1 and 2 Figure 2: AmpC induction by cell wall inhibitors

718

Induction of the C. freundii ampC reporter plasmid in the presence of cell wall active

719

compounds. Expression of ampC is detected upon cleavage of the colorimetric β-

720

lactam analog, nitrocefin, at A490. Induction is calculated as the A490 /OD600 ratio to

721

correct for cell density. The LpxC inhibitor is CHIR-90 (29). The effects of

722

compounds 1 and 2 on the reporter are also shown.

723 724

Figure 3: Morphology and staining of E. coli ΔtolC in the presence of inhibitor

725

compounds

726

E. coli ATCC25922 ΔtolC was treated with compounds as indicated for 120 min and

727

stained with FM4-64 (red), DAPI (blue) and sytox green (green) as previously

728

described (24). An overlay of FM4-64 and DAPI is also shown. Scale bars are 1

729

μm.

730 731

Figure 4: Compound 2 inhibits Lpp release from spheroplasts.

732

Spheroplasts were prepared from E. coli MG1655 ΔtolC (Parent), LolE (L371P) or

733

LolC (N265K). Spheroplasts were incubated with purified LolA in the presence of

734

DMSO (vehicle control), globomycin or compound 2. The amount of Lpp released to

735

LolA was detected by SDS-PAGE and subsequent immunoblotting with anti-Lpp

736

antibodies (upper panel). OmpA, whose release from the spheroplast is

737

independent of LolA was also detected with an anti-OmpA antibody (lower panel).

738 28

Table 1: Antibacterial activity of compounds 1 and 2 Bacterial Species Escherichia coli

Strain ATCC 25922

Description Wild type

Compound 1 MIC (μg/ml) >64

Compound 2 MIC (μg/ml) 8

Escherichia coli

ATCC 25922 ΔtolC

Efflux mutant

0.25

0.125

Haemophilus influenzae

ATCC 49247

Wild type

>64

32

Pseudomonas aeruginosa

PAO1

Wild type

>64

>64

Staphylococcus aureus

ATCC 29213

Wild type

>64

>64

Candida albicans

ATCC 90028

Counter screen

>64

>64

29

Table 2: Inhibition of E. coli macromolecular synthetic pathways Incorporation IC50 (μg/ml)1 Compound

Target

Protein [14C]-Leucine

Cell wall [3H] DAP2

Fatty Acid [14C] acetic acid

RNA [3H] uridine

DNA [3H] thymidine

Erythromycin

Protein Synthesis

30

>256

>256

>256

>256

Ampicillin

Cell Wall Synthesis

>256

16

>256

>256

>256

Triclosan

Fatty Acid Synthesis

>256

1.5

0.0156

2

2

Rifampicin

Transcription

64

>256

>256

64

>256

Ciprofloxacin

DNA Replication

30

>256

>256

1

0.02

CCCP

Membrane Potential

50

0.15

0.07

0.25

0.3

Compound 1

Cell Wall Synthesis

>256

20

0.43

>256

>256

>64 Compound 2 Cell Wall Synthesis >64 164 Incorporation of radiolabeled precursors was measured in E. coli ΔtolC ΔlysA 2 DAP= diaminopimelic acid 3 IC50 value is based on maximum inhibition of less than 40% 4 IC50 value is based on maximum inhibition of less than 42% due to the compound solubility

>64

>64

1

30

Table 3: Susceptibility of compound 1 to resistant isolates (left panel) and LpxH overexpressing strains (right panel) MIC (μg/ml) LpxH

LpxH

LpxH

LpxH

pPSV352

pPSV35

pPSV35-LpxH3

pPSV35-LpxH

Parent1

(G48D)

(L84R)

(F141L)

(R149H)

0 IPTG

50 μM IPTG

0 IPTG

50 μM IPTG

Compound 1

0.25

>128

>128

>128

>128

0.125

0.125

0.125

>128

Levofloxacin

0.008

0.008

0.008

0.008

0.004

0.004

0.004

0.004

0.004

Compound

Meropenem

0.016

0.031

0.016

0.016

0.008

0.031

0.031

0.031

0.031

PF1090

0.008

0.008

0.008

0.008

0.004

0.004

0.004

0.004

0.004

0.5

0.5

0.5

0.5

0.5

0.125

0.25

0.0625

0.125

Tetracycline 1

E. coli MG1655 ΔtolC E. coli MG1655 ΔtolC plus empty vector pPSV35 control E. coli MG1655 ΔtolC pPSV35 expressing LpxH under an IPTG-inducible promoter

2 3

31

Table 4: Relative sensitivity of compound 2-resistant isolates

Compound

Parent1

LolC

LolE

LolE

(G254V)

(G195S)

(P365C)

MIC (μg/ml) LolE Plpp2 (D367Y)

(A49G)

Z25103

Z2510

Z2510

Z2510

(G28R)

(A32P)

(G46D)

(G119fs4)

Compound 2

0.125

>128

>128

>128

>128

4

4

8

8

4

Levofloxacin

0.004

0.004

0.004

0.004

0.004

0.004

0.016

0.016

0.016

0.008

Meropenem

0.016

0.016

0.016

0.016

0.016

0.016

0.016

0.016

0.016

0.016

Tetracycline

0.5

0.5

0.5

0.5

0.5

0.5

1

1

1

1

1

E. coli ATCC 25922 ΔtolC 2 promoter of lpp 3 Z2510 is a putative transcriptional repressor from the AcrR family 4 fs=frame shift mutation

32