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Inhibition of TLR2 Signaling by Small Molecule Inhibitors Targeting a Pocket Within the TLR2 TIR Domain

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Inhibition of TLR2 Signaling by Small Molecule Inhibitors Targeting a Pocket Within the TLR2 TIR Domain Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain Pragnesh Mistrya, Michelle H. W. Lairda, Ryan S. Schwarza, Shannon Greeneb, Tristan Dysona, Greg A. Snyderc, Tsan Sam Xiaod, Jay Chauhanb, Steven Fletcherb, Vladimir Y. Toshchakova, Alexander D. MacKerell Jr.b,1, and Stefanie N. Vogela,1 aDepartment of Microbiology and Immunology and cInstitute of Human Virology, School of Medicine, University of Maryland, Baltimore (UMB), Baltimore, MD 21201; bDepartment of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201; and dDepartment of Pathology, Case Western Reserve University, Cleveland, OH 44106 Edited by Shizuo Akira, Osaka University, Osaka, Japan, and approved March 17, 2015 (received for review November 25, 2014) Toll-like receptor (TLR) signaling is initiated by dimerization of in- during TLR signaling (19). Two adapter proteins implicated in tracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs TLR2 signaling are MyD88 and TIRAP (Mal). A conserved Pro except TLR3, recruitment of the adapter, myeloid differentiation [e.g., P681 in human TLR2 (hTLR2), P712 in murine TLR4 primary response gene 88 (MyD88), to TLR TIR domains results in (mTLR4), P674 in hTLR10, P804 in mTLR11] within the BB downstream signaling culminating in proinflammatory cytokine loop of almost all TIR domains is critical for signaling (20–27). production. Therefore, blocking TLR TIR dimerization may amelio- More importantly, the BB loop P681H mutation in hTLR2 rate TLR2-mediated hyperinflammatory states. The BB loop within abolished recruitment of MyD88 and signaling (20, 26). Based on the TLR TIR domain is critical for mediating certain protein–protein this evidence, the BB loop within the TLR2 TIR domain appears interactions. Examination of the human TLR2 TIR domain crystal to be an ideal target for attenuation of TLR2 signaling. structure revealed a pocket adjacent to the highly conserved P681 Visual inspection of the crystal structure of the hTLR2 TIR and G682 BB loop residues. Using computer-aided drug design β domain (26) revealed a pocket formed by residues on the -B INFLAMMATION (CADD), we sought to identify a small molecule inhibitor(s) that strand and α-B helix that includes the highly conserved Pro and IMMUNOLOGY AND would fit within this pocket and potentially disrupt TLR2 signaling. Gly residues of the BB loop. We hypothesized that targeting this In silico screening identified 149 compounds and 20 US Food and pocket with a small molecule might inhibit interaction of TLR2 Drug Administration-approved drugs based on their predicted abil- with MyD88, and thereby blunt TLR2 signaling. We identified ity to bind in the BB loop pocket. These compounds were screened C H NO (C29) and its derivative, ortho-vanillin (o-vanillin), in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-medi- 16 15 4 which inhibit mTLR2 and hTLR2 signaling initiated by synthetic ated IL-8 mRNA. C H NO (C29) was identified as a potential TLR2 16 15 4 and bacterial agonists without cytotoxicity. Interestingly, mutation inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 of the BB loop pocket residues revealed a differential requirement agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 sig- for TLR2/1 vs. TLR2/6 signaling. Our data indicate that computer- naling in murine macrophages. C29 failed to inhibit signaling in- aided drug design (CADD) is an effective approach for identifying duced by other TLR agonists and TNF-α. Mutagenesis of BB loop small molecule inhibitors of TLR2 signaling and has the potential pocket residues revealed an indispensable role for TLR2/1, but not to identify inhibitors for other TLR signaling pathways. TLR2/6, signaling, suggesting divergent roles. Mice treated with Results o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an Screening of Potential TLR2 Inhibitors. Visual inspection of the crys- effective approach for identification of TLR2 signaling inhibitors. tal structure of the TLR2 TIR domain (Protein Data Bank ID small molecule inhibitor | BB loop | TLR2 pocket | CADD Significance oll-like receptors (TLRs) are type I transmembrane receptors Excess Toll-like receptor 2 (TLR2) signaling has been implicated Tthat detect conserved “pathogen-associated molecular pat- in numerous inflammatory diseases, yet there is no TLR2 inhib- terns” from microbes, as well as host-derived “danger-associated itor licensed for human use. Using computer-aided drug design molecular patterns” (1). TLR2 heterodimerizes with TLR6 or (CADD), we identified a compound, C16H15NO4 (C29), and a de- TLR1 to recognize diacyl lipopeptides or triacyl lipopeptides, rivative, ortho-vanillin, that inhibit TLR2 signaling in vitro and respectively (2, 3), present in gram-positive and gram-negative in vivo. Our findings also revealed unexpected differences be- bacteria (4–9). tween TLR2/1 and TLR2/6 signaling in mice vs. humans. Impor- Ligand engagement of TLR2/1 or TLR2/6 activates the myeloid tantly, our data provide proof of principle that the CADD- differentiation primary response gene 88 (MyD88)-dependent targeted BB loop pocket residues are critical for TLR2 signaling pathway (i.e., nuclear translocation of NF-κB, activation of and may be targeted therapeutically. MAPKs), resulting in production of proinflammatory cytokines (10). Dysregulated TLR2 signaling has been implicated in nu- Author contributions: P.M., M.H.W.L., R.S.S., G.A.S., T.S.X., S.F., V.Y.T., A.D.M., and S.N.V. designed research; P.M., M.H.W.L., R.S.S., S.G., T.D., G.A.S., J.C., and V.Y.T. performed merous diseases (e.g., sepsis, atherosclerosis, tumor metastasis, research; A.D.M. and S.N.V. contributed new reagents/analytic tools; and P.M. and ischemia/reperfusion injury) (11–14). Several inhibitors of TLR2 S.N.V. wrote the paper. signaling have been developed (15–18), yet none is licensed for The authors declare no conflict of interest. human use. A better understanding of the Toll/IL-1 receptor This article is a PNAS Direct Submission. resistance (TIR) domain interactions involved in TLR2 signaling 1To whom correspondence may be addressed. Email: [email protected] or could lead to novel therapeutic agents. [email protected] Both TLRs and adapter proteins contain a cytoplasmic TIR This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. domain that mediates homotypic and heterotypic interactions 1073/pnas.1422576112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1422576112 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 code 1FYW) revealed the BB loop pocket (Y647, C673, D678, consistently decreased P3C-induced IL-8 mRNA by ≥50% (Fig. F679, I680, K683, D687, N688, D691, and S692) adjacent to the S2A), and eight compounds (C14, C29, and C32 as seen in P3C- conserved P681 and G682 residues of the BB loop (Fig. S1). Over induced signaling, as well as C24, C25, C26, C30, and C33) de- 1 million commercially available small compounds, as well as US creased P2C-induced IL-8 mRNA by ≥50% (Fig. S2B). These Food and Drug Administration (FDA)-approved drugs, were results suggest that CADD is an effective first step for the iden- screened in silico for those compounds that could potentially fit tification of potential small molecule inhibitors of TLR2 signaling. into the pocket. CADD analysis identified ∼1,000 compounds based on predicted favorable interactions with the TLR2 BB loop C29 Blunts hTLR2/1 and hTLR2/6 Signaling in HEK-TLR2 Stable Trans- pocket (SI Materials and Methods). Of these compounds, 149 fectants and THP-1 Cells. We next tested these potential TLR2 in- chemically diverse small molecules and 20 FDA-approved drugs hibitors to assess dose dependency and cytotoxicity. Compound with physiochemical properties that should maximize bio- C29 (Fig. 1A) blocked P3C- and P2C-induced IL-8 mRNA dose- availability and ranked highest for their potential to fit into the dependently in HEK-TLR2 stable transfectants, although it had TLR2 TIR pocket were screened for their ability to inhibit TLR2- no effect on TNF-α–induced signaling or on cytotoxicity (Fig. 1B mediated signaling. and Fig. S3). We next investigated the effect of C29 on TLR2 Initially, 34 compounds (C1–C34) were tested in stably transfected signaling in the human THP-1 macrophage-like cell line (28). C29 HEK-hTLR2 cells for the ability to block N-Palmitoyl-S-[2,3- also inhibited P3C- and P2C-induced IL-1β gene expression sig- bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-(S)-seryl-(S)-lysyl-(S)- nificantly at both 1 h and 4 h following stimulation (Fig. 1C), as lysyl-(S)-lysyl-(S)-Lys (P3C)–induced and S-[2,3-bis(palmitoyloxy)- well as both P3C- and P2C-induced NF-κB–luciferase activity in (2RS)-propyl]-(R)-cysteinyl-(S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-Lys transiently transfected HEK293T cells expressing hTLR2 and an (P2C)–induced IL-8 mRNA via TLR2/1 and TLR2/6 signaling NF-κB–sensitive luciferase reporter construct (Fig. S4). Thus, C29 pathways, respectively. Four compounds (C10, C14, C29, and C32) inhibits both TLR2/1 and TLR2/6 signaling in human cell lines. Fig. 1. Differential effect of C29 on gene expression in human cell lines and murine peritoneal macrophages. (A) Structure of C29. (B) Total RNA was extracted from HEK-TLR2 cells pretreated for 1 h with media, vehicle (65 μM NaOH), or C29 (10 μMor50μM) and then stimulated with P3C (200 ng/mL), P2C (200 ng/mL), or human TNF-α (300 ng/mL) for 1 h in the presence of media, vehicle, or C29. IL-8 mRNA was measured by qRT-PCR and was normalized to the expression of the GAPDH housekeeping gene.
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