Application of -Lactamase Enzyme Complementation to the High-Throughput Screening of Toll-Like Receptor Signaling Inhibitors

Abstract

We describe a successful application of -lactamase fragment complementation to high-throughput screening (HTS) for Tolllike receptor 4 (TLR4) signaling inhibitors. We developed a stable cell line, HeLa/CL3-4, expressing MyD88/Bla(a) and TLR4/Bla(b), in which the two -lactamase fragments complement with each other by virtue of spontaneous MyD88-TLR4 binding via their Toll/IL-1R (TIR) domains. Inhibition of the MyD88-TLR4 binding leads to the disruption of the enzyme complementation and a loss of the lactamase activity. We used a 384-well plate format to screen 16,000 compounds using this assay and obtained 45 primary hits. After rescreening these 45 hits and eliminating compounds that directly inhibited -lactamase, we had five candidate inhibitors. We show that these five act as inhibitors of TLR4-MyD88 binding and are variously effective at inhibiting lipopolysaccharide-stimulated cytokine release from RAW264.7 cells. One compound is effective near 100 nM. None of the compounds showed any cytotoxicity at 20 M. In the innate immune system, Toll-like receptors (TLRs) function indispensably as sensors of microbial molecules. Upon recognition of microbial components, TLRs quickly engage with their adaptors and initiate innate immune signals by sequentially recruiting downstream signaling mediators. The TLR-mediated innate immune signals generally provide immediate host protection and stimulate the host’s adaptive immune response (Akira et al., 2001; Medzhitov, 2001; Hoebe et al., 2004). Since the first mammalian Toll homolog was discovered (Medzhitov et al., 1997), at least 11 mammalian TLRs have been identified (Ulevitch, 2004). All TLRs initiate cellular signaling through their cytoplasmic Toll/interleukin-1 receptor (TIR) domain, which triggers signaling pathways by TIR-TIR homophilic interaction with TIR domain containing TLR adaptors. Myeloid differentiation primary response protein 88 (MyD88) is the most common TLR adaptor (Kawai et al., 1999). Others include MAL/TIRAP, TRAM and TRIF, but all TLRs use MyD88, with the exception of TLR3, which uses only TRIF (Akira et al., 2006). Despite the fact that TLR-mediated innate immune signals are required to combat invading pathogens, when signaling is not well regulated, uncontrolled activation can result in the disruption of host homeostasis leading to long-term inflammation and septic shock. Although there are naturally occurring negative regulators of TLR signaling that function as part of cell homeostasis (Kobayashi and Flavell, 2004; Ulevitch, 2004; Han and Ulevitch, 2005; Miggin and O’Neill, 2006), these are not adequate in all situations. A number of authors have commented on the need for pharmacological means to regulate TLR signaling, and some efforts are underway (Cristofaro and Opal, 2006; Földes, 2006; Leaver et al., 2007). We have exploited a new approach to screen libraries for TLR signaling inhibitors. Based on our previous study of in vivo detection of TLR-TLR and TLR-adaptor association using -lactamase enzyme fragment complementation (Lee This work was supported by National Institutes of Health grants GM066119 (to P.S.T.), MH081265 (to P.S.T.), and MH074404 (to H.R.). This is publication 18911-IMM from the Department of Immunology, The Scripps Research Institute. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.038349. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: TLR, Toll-like receptor; HTS, high-throughput screening; DMSO, dimethyl sulfoxide; LPS, lipopolysaccharide; TIR domain, Toll/interleukin-1 receptor homology domain; ELISA, enzyme-linked immunosorbent assay; HEK, human embryonic kidney; DMEM, Dulbecco’s modified Eagle’s medium; IL, interleukin; pIpC, polyinosinic-polycytidylic acid; TNF, tumor necrosis factor ; NFB, nuclear factor B; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; HA, hemagglutinin; FCS, fetal calf serum; AM, acetoxymethyl ester; TIRAP, toll-interleukin 1 receptor domain containing adaptor protein; S/B, signal-to-background ratio; MALP-2, macrophage-activating lipopeptide 2 kDa; CpG, cytosine phosphate guanine; TLR4CD, Toll-like receptor 4 transmembrane-cytoplasmic domain; Bla, -lactamase. 0026-895X/07/7204-868–875$20.00 MOLECULAR PHARMACOLOGY Vol. 72, No. 4 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 38349/3253327 Mol Pharmacol 72:868–875, 2007 Printed in U.S.A. 868 http://molpharm.aspetjournals.org/content/suppl/2007/07/09/mol.107.038349.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from et al., 2004), we have developed an application of cell-based -lactamase enzyme complementation to the high-throughput screening of small molecules. We found that highthroughput screening of 16,000 compounds resulted in five inhibitors of TLR4-MyD88 interaction and that they specifically inhibit the MyD88-dependent TLR signaling pathway. Materials and Methods Reagents and Antibodies. Potassium clavulanate as a lactamase inhibitor was from Sigma-Aldrich (St. Louis, MO), and the lactamase substrate, CCF2/AM, was from Invitrogen (Carlsbad, CA). The 16,000 target compounds used for screening were from Maybridge (Cornwall, England). For the experiments in Figs. 4 through 9, they were prepared at 10 mM in DMSO. Lipopolysaccharide (LPS) from Escherichia coli (O111:B4) was purchased from List Biological Laboratories (Campbell, CA), and polyinosinic-polycytidylic acid (pIpC) was from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK). Anti-FLAG M2-agarose and antibody were obtained from Sigma-Aldrich, and anti-HA.11 antibody was obtained from Covance-Berkeley Antibody Company (Richmond, CA). Murine IL-1 and TNF were purchased from PeproTech (Rocky Hill, NJ). Mouse TNF and IL-6 ELISA kits were purchased from BD Biosciences (San Jose, CA). Stable Cell Line and Culture Conditions. The stable HeLa line HeLa/CL3-4, which expresses two -lactamase fusion proteins, MyD88-Bla(a) and TLR4-Bla(b), was made using two expression constructs, pCDNA3.1/MyD88/Bla(a) and pEF6/TLR4/Bla(b), as described previously (Lee et al., 2004). The stable HeLa/full-Bla line, which expresses a fusion protein of MyD88 with full-length -lactamase, was made using pCDNA3.1/MyD88/full-length Bla. HeLa/ CL3-4 were grown in DMEM with 10% FCS containing 200 g/ml G418 and 10 g/ml blasticidin and HeLa/full-Bla in the same medium containing 200 g/ml G418 only. Both HeLa/CL3-4 and HeLa/ full-Bla were selected using FACSort (BD Biosciences) equipped with excitation at 408 nm and emission at 519/30 nm (CCF, green fluorescence) or 450/40 nm (cleaved CCF2, blue fluorescence). The primary blue-positive cells sorted by FACS were collected, grown in the media described above, and resorted. This sequential sorting step was repeated three times. Assay Optimization. HeLa/CL3-4 cells were plated into 384-well plates in a series of different concentrations (1 10, 0.5 10, 0.25 10, 0.125 10, and 0.06 10 cells/ml) using the FlexDrop Precision Reagent Dispenser (PerkinElmer Life and Analytical Sciences, Waltham, MA). Each well contained 30 l of cells. Cells were incubated at 37°C overnight. On the following day, cells were treated with different amounts of clavulanate (10, 3.3, 1.1, 0.37, 0.12, 0.04, 0.014, 0.0046, and 0.0015 g/ml) for 30 min at 37°C and then loaded with 1 M CCF2/AM for 2 h at room temperature. These two steps were performed using the BioRaptor FRD (Beckman Coulter, Fullerton, CA). The plates were then read using the EnVision multilabel reader (PerkinElmer Life and Analytical Sciences). The data were analyzed with Microsoft Excel (Redmond, WA) and Prism software (GraphPad Software, San Diego, CA). The response metric was the ratio of blue fluorescence intensity to green fluorescence intensity. To assess assay uniformity across multiple plates, HeLa/CL3-4 cells (30 l of 0.125 10 cells/ml) were plated into three 384-well plates with DMEM/10% FCS using the FlexDrop Precision Reagent Dispenser. Cells were incubated at 37°C overnight. On the following day, the three plates were treated with 10 g/ml (High clavulanate), 0.12 g/ml (Medium clavulanate), or control PBS (Low clavulanate) with three plate layouts, specifically plate 1 (HML), plate 2 (LHM), and plate 3 (MLH). After 30 min, the plates were loaded with 1 M CCF2/AM for 2 h at room temperature. The plates were then read using the EnVision multilabel reader (PerkinElmer Life and Analytical Sciences). Compound Screening and Hit Selection. HeLa/CL3-4 cells in DMEM/10% FCS (10 l of 0.22 10 cells/ml) were dispensed into low-volume, black clear bottom 384-well plates (Greiner Bio-One, Longwood, FL) on the FlexDrop Precision Reagent Dispenser (PerkinElmer Life and Analytical Sciences). Cells were incubated at 37°C overnight. On the following day, cells were treated with 10 M concentrations of various compounds (10 nl/well) delivered with a pintool (V&P Scientific, San Diego, CA) on a Beckman FX (Beckman Coulter, Fullerton, CA), except for the first two and the last two columns, which served as controls. The first two columns were treated with different amounts of clavulanate (10, 3.3, 1.1, 0.37, 0.12, 0.04, 0.014, 0.0046, and 0.0015 g/ml), whereas the last two columns were left untreated. The plates were incubated at 37°C for 30 min and then loaded with 1 M CCF2/AM for 2 h at room temperature. Thereafter, the plates were read using the EnVision multilabel reader (PerkinElmer Life and Analytical Sciences). Z was calculated as described previously (Zhang et al., 1999). Active wells were defined as those with percentage of inhibition greater than 3 S.D. from the low control. Percentage of inhibition was defined as 100 (1 (Well Median High Control)/(Median Low Control Median High Control), where High Control was the wells treated with 10 g/ml clavulanic acid, and Low Control was treated with vehicle alone. A total of 24 wells met this definition, and 21 more with borderline activity were selected for repeat. Cherry-picked compounds were tested in the primary screening assay in triplicate. Microscopy. Microscopy was performed as described previously (Lee et al., 2004). In brief, cells were cultured in a 12-well plate overnight. Cells were washed twice with a modified physiological saline buffer (10 mM HEPES, 6 mM sucrose, 10 mM glucose, 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1% probenecid, pH 7.35) and loaded with 1 M CCF2/AM for 1 h at room temperature. Cells were washed with physiological saline buffer and analyzed by fluorescence microscopy [Zeiss Axiovert 100TV (Carl Zeiss, Thornwood, NJ) with a Diagnostic Instruments SPOT-cooled charge-coupled device camera (Diagnostic Instruments, Sterling Heights, MI)] using a filter set from Omega Optical XF12-2 (Brattleboro, VT): 405 nm excitation, 420 nm dichroic mirror, and 435 nm long-pass emission. Electrophoretic Mobility Shift Assay. RAW264.7 cells (0.5 10 cells/ml) were grown in 12-well tissue culture plates with DMEM/10% FCS for 24 h and pretreated with 10 M inhibitory compounds for 30 min. Cells were then stimulated with LPS (0.1 g/ml), MALP-2 (50 ng/ml), pIpC (20 g/ml), CpG (20 g/ml), IL-1 (50 ng/ml), or TNF (50 g/ml) for 1 h. Cells were lysed with buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 1% Nonidet P-40) followed by centrifugation. The pellet was treated with buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride) to prepare nuclear extracts. Electrophoretic mobility shift assay was performed using NFB oligonucleotide probe (Promega, Madison, WI) labeled with [ -P]ATP (GE Healthcare) using T4 polynucleotide kinase (New England BioLabs, Ipswich, MA) as described previously (Lee et al., 2006). Luciferase Reporter Assay. The luciferase reporter assay was performed as described previously (Lee et al., 2004). In brief, HeLa cells (0.5 10 cells/ml) were grown in 12-well plates with DMEM/10% FCS. On the following day, cells were transiently transfected with 0.01 g/ml TLR4, CD14, and MD-2 vectors, along with 0.05 g/ml pIL8-promoter-Luc vector and pSV-galactosidase vector (Promega). After 24 h, cells were pretreated with different compounds for 30 min and then stimulated with LPS for 6 h. Cell extracts were prepared using the cell culture lysis buffer (Promega), and the luciferase activity was measured using a Luciferase Reporter Assay System (Promega), and -galactosidase activity was measured using O-nitrophenyl-D-galactopyranoside as substrate. Luciferase activity reported in the figures is normalized for transfection efficiency using the -galactosidase activity. -Lactamase Enzyme Complementation-Based HTS 869 at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from Immunoprecipitation and Western Blotting. HEK293T cells (0.5 10 cells/ml) were transiently transfected with 0.5 g of TLR4 transmembrane-cytoplasmic domain (TLR4CD) (Lee et al., 2004) and MyD88 vectors. Twenty-four hours after transfection, cells were treated with different compounds at the different time points as indicated in the figure legends. Cells were lysed with lysis buffer (150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, and 50 mM Tris-HCl, pH 7.5) containing protease inhibitor cocktails (Roche Applied Science, Indianapolis, IN). The supernatants were incubated with antiFLAG M2-agarose (Sigma-Aldrich) and incubated for 3 h at 4°C. The mixtures were washed four times with lysis buffer, separated on a 4 to 12% Bis-Tris NuPAGE gel (Invitrogen) and transferred to nitrocellulose membrane. Western blotting was performed by probing the membrane with both anti-FLAG and anti-HA antibodies according to standard protocols. Cytokine ELISA. RAW264.7 cells (0.5 10 cells/ml) were cultured in a 12-well plate for 24 h and preincubated with different concentrations of compounds for 1 h as indicated in the figures. Cells were then washed once with DMEM/10% FCS and stimulated with 0.05 g/ml LPS for 16 h. TNF and IL-6 in the culture supernatants were measured by mouse TNF and IL-6 ELISA according to the manufacturer’s instructions (BD Biosciences). Cell Viability Test. RAW264.7 cells (0.5 10 cells/ml) were cultured in a 12-well plate for 24 h. Cells were washed with PBS once and treated with different compounds at 20 M or DMSO only, or 70% ethanol for 1 h. Cells were then washed with PBS and treated with 2 M calcein AM and 4 M EthD-1 solution (Invitrogen) for 30 min. Cells were analyzed by fluorescence microscopy at 495 and 528 nm. Fig. 1. Strategy for screening of TLR4 signaling inhibitors and protein expression patterns in HeLa/CL3-4 cell line. A, schematic representation of inhibitor screening strategy using -lactamase fragment fusion proteins. TLR4 and MyD88 in HeLa/CL3-4 spontaneously interact with each other and bring two -lactamase fragments into proximity, resulting in a functional form of -lactamase. Upon the addition of -lactamase substrate CCF2/AM and excitation at 409 nm, HeLa/CL3-4 cells emit blue fluorescence at 447 nm (A, top). If an inhibitor breaks TLR4-MyD88 binding resulting in disruption of -lactamase fragment complementation, HeLa/CL3-4 cells emit green fluorescence at 520 nm (A, bottom). B, intact or permeabilized HeLa or HeLa/CL3-4 cells were stained with anti-FLAG [TLR4/Bla(b) detection] or anti-HA antibody [MyD88/Bla(a) detection] and analyzed by FACS. Fig. 2. Compound screening and hit selection. A, EC50 evaluation using clavulanic acid as a model inhibitor. HeLa/CL3-4 cells in DMEM/10% FCS (0.125 10 cells/ml) were dispensed into 384-well plates using the FlexDrop Precision Reagent Dispenser. After 24 h, cells were treated with a series of different concentrations of clavulanic acid for 30 min and incubated with CCF2/AM substrate for 2 h. The plates were then read using the EnVision multilabel reader (PerkinElmer Life and Analytical Sciences). EC50 value of clavulanic acid was analyzed using Prism software. The table beside the graph summarizes the S/B ratio and Z factor achieved. B, the HeLa/CL3-4 cell line-based complementation assay was screened against 16,000 compounds. The figure includes controls and standards and represents 19,200 wells in total. The well number versus percentage of inhibition of the high (10 g/ml clavulanic acid) and low (vehicle alone) controls is shown. The average Z for each plate of the screening campaign was 0.64. C, elimination of false-positive compounds in 10 positive hits using stable HeLa line expressing full-length -lactamase. HeLa/CL3-4 or HeLa/full Bla cells were plated in 12-well plates. After 24 h, cells were treated with 10 g/ml clavulanic acid or 10 M compound for 30 min as indicated. Cells were then incubated with CCF2/AM substrate for 1 h and analyzed by fluorescence microscopy. The five compounds kept for further study are underlined. 870 Lee et al. at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from

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@inproceedings{Lee2007ApplicationO, title={Application of -Lactamase Enzyme Complementation to the High-Throughput Screening of Toll-Like Receptor Signaling Inhibitors}, author={Hyun-Ku Lee and Steven J. Brown and Hugh Rosen and Peter S. Tobias}, year={2007} }