Research ArticleHEALTH AND MEDICINE

Modulation of lymphocyte-mediated tissue repair by rational design of heterocyclic aryl hydrocarbon receptor agonists

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Science Advances  15 Jan 2020:
Vol. 6, no. 3, eaay8230
DOI: 10.1126/sciadv.aay8230
  • Fig. 1 Identification of AHR agonist and SAR.

    (A) Experimental design demonstrating AHR agonist hit-to-lead selection cascade. (B) Structure of focused library and lead compound PY10. (C) Activity of compound PY10 and ITE, measured by XRE-driven luciferase reporter assay in the human HepG2 cell line (n = 3). (D) Library screen reveals stringent structural requirements for AHR activation. (E) General scheme for SAR-based modifications of the PY10 backbone. Series I represents interrogation of the pyridine-2-carboxylate ester group and series II represents interrogation of the indole moiety. (F) Pyridine esters with increased bulkiness displayed substantial loss of AHR activity. (G) AHR activity is sensitive to indole substitution. (H) Superimposed AHR homology model before (green) and after (gray) MD simulation. Secondary structures including four α-helices (Cα, Dα, Eα, and Fα) and four β-strands (Aβ, Gβ, Hβ, and Iβ) are labeled. The molecular surface (Connolly) volume of the LBD is displayed in blue. (I) Detailed view of the ligand binding domain (LBD) in complex with PY10 of the molecular dynamics (MD) homology model. Amino acid residuals forming the LBD are labeled. (J) Detailed view of the water-mediated H-bond network near the indole moiety of PY10 (red line). (K) Detailed view of the Met340 subdomain occupied by the methyl ester group of PY10. Color for heteroatoms are yellow for sulfur (S), red for oxygen (O), and blue for nitrogen (N). Data represent mean ± SEM.

  • Fig. 2 Characterization of compounds with improved stability.

    (A) Compound stability in mouse plasma and (B) liver microsomes was measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS; n = 3). Plasma t1/2 (in minutes): ITE (44); PY10 (45); PY108 (>60); PY109 (>60). Liver microsome t1/2 (in minutes): ITE (11); PY10 (17); PY108 (69); PY109 (60). (C) Structure of lead compounds PY108 and PY109. (D and E) Activity of PY108 and PY109 measured by XRE-driven luciferase reporter assay in human HepG2 and mouse Hepa-1c1c7 cell lines. (F) Docking pose (delta E = 10.4 kcal/mol) of PY109 in the MD homology model. (G) Docking pose (delta E = 10.4 kcal/mol) of PY108 in the MD homology model. Data represent mean ± SEM. The 95% confidence interval of reported EC50 values is available in table S2.

  • Fig. 3 Biological characterization of lead compounds in vitro and after oral administration.

    (A) Nuclear translocation of AHR induced by ITE and lead compounds PY108 and PY109 at 90 min. AHR (green), actin (red), and nucleus (blue). (B and C) Hepa-1c1c7 was incubated with 1 μM compound, followed by immunoprecipitation of AHR complex. AHR-associated binding partner level was assessed by LC-MS/MS for ARNT (B) or Western blots for ARNT and AIP (C) (n = 3). (D) Biological and in silico physiochemical parameters of PY108 and PY109. *Polar surface area (PSA) and octanol/water partition coefficient (cLogP) were calculated using ChemAxon software. LLE, lipophilic ligand efficiency; calculated from EC50 in human HepG2 cells by the equation: LLE = pEC50 − cLogP (pEC50 = −log10EC50). (E) Lead compounds PY108 and PY109 induced AHR-dependent Cyp1a1 expression in mouse Hepa-1c1c7 cells (n = 4). (F) Quantitative polymerase chain reaction (qPCR) analysis of Cyp1a1 induction in liver and colon, 12 hours after oral gavage of PY109 (n = 3). (G) Plasma concentration of PY109 and PY108 after oral administration of 1 mg (PO) of PY109 and PY108. Compounds were quantified in plasma using LC-MS/MS (n = 4). Data represent mean ± SEM. *P < 0.05 versus control, #P < 0.05 versus ITE by Student’s t test (D and F) or ANOVA and Bonferroni’s multiple comparison test (B).

  • Fig. 4 PY109 attenuates DSS-induced colitis in mice.

    Mice in colitis model were recorded for survival (A), body weight (B), and disease activity score (C). Data represent three independent trials with n = 7 per group per trial. (D) Colon lengths at day 11 (n = 12 to 13). (E) Hematoxylin and eosin staining (100×) and (F) histological score at day 11 (n = 8). (G and H) Colon goblet cells at day 11 (n = 7 to 8). (I) RNA sequencing (RNA-seq) analysis of colon from untreated mice (n = 4) and dextran sulfate sodium (DSS)–treated mice ± PY109 (n = 5) at day 11. Heatmap demonstrates colitis transcriptome counter-regulated by PY109. (J) Volcano plot of differentially expressed genes by PY109. Red, increased genes; green, decreased genes; blue, AHR target genes. (K) Heatmap of reported AHR target mediators and Il22 or Il17a regulated barrier genes with red denoting increase and green denoting decrease. (L and M) qPCR expression level of Il22 and Il17a in the whole colon at days 7 and 11 of the DSS model. (N to Q) Flow cytometry staining of LPMC. IL-22+ and IL-17A+ were stained in combination with T helper staining (CD3+CD4+; N to O) or ILC3 staining (CD3RORrt+; P and Q). (R and S) Absolute number of ILC3 (R) or intraepithelial CD3+CD45+ TCR γδ+ cells (S). (T to V) qPCR of the epithelial fraction was performed for detection of antimicrobial peptides S100a8 (T), S100a9 (U), and RegIIIγ (V). qPCR data represent n = 4 to 8 per group. Flow cytometry data represent n = 4 to 10 per group. Data represent mean ± SEM, *P < 0.05 versus control (Student’s t test).

  • Fig. 5 PY109 promotes epithelial AMP expression via lymphocyte-derived IL-22 and IL-17A.

    (A) Flow cytometry scatterplots of IL-22 and IL-17A in spleen CD4+ T cells activated in Th17 media with or without PY109. (B and C) IL-22+ and IL-17+ frequency in CD4+ cells (n = 3). (D and E) Enzyme-linked immunosorbent assay (ELISA) measurement of IL-22 and IL-17A levels in the supernatant of Th17-polarized CD4+ T cells (n = 3). (F and G) IL-22+ and IL-17+ frequency in CD4+ cells from colon-derived LPMCs activated in Th17 media (n = 3). (H and I) IL-22+ and IL-17+ frequency in (CD3RORrt+) ILC3 cells from colon-derived LPMCs treated with IL-1β and IL-23 (n = 3). (J) Western blot detection of AHR, AIP, and ARNT in immunoprecipitated AHR complex from spleen-derived Th17 cells treated with or without PY109 for 1 hour (n = 3). (K) Quantification of band intensity in Western blots. (L) IP-MS determination of AHR-associated proteins. Proteins with more than twofold change in response to PY109 and 5% intensity of AHR are highlighted. (M) qPCR detection of S100a9 levels in mouse colon epithelial CMT-93 cells treated with IL-22 or IL-17A or both (n = 4). (N) qPCR detection of S100a9 level in CMT-93 cells treated with conditioned medium from spleen-derived CD4+ cells polarized in Th17 with or without PY109 and blocking antibodies (n = 4). Data represent mean ± SEM, *P < 0.05 versus control by Student’s t test (B to I) or ANOVA and Bonferroni’s multiple comparison test (K, M, and N).

  • Fig. 6 PY109 induces IL-22 in human T cells from healthy donors and patients with IBD.

    CD4+ T cells were purified from PBMCs of healthy participants (H.P.) and patients with IBD. Cells were activated with anti-CD3 and anti-CD28 antibodies in the presence of Th17-polarizing cytokines (TGF-β1, IL-1β, and IL-6) with or without 5 μM of PY109 for 5 days. Cells were re-stimulated with PMA, ionomycin, and brefeldin A and RNA expression of AHR (A), CYP1A1 (B), IL22 (C), and IL17A (D) analyzed by qPCR using 18S as an internal control. (E) Representative scatterplots of IL-22 and IL-17A staining from one donor. Flow cytometry quantification of IL-22 (F) and IL-17A (G). Dot plots represent 7 to 9 healthy donors and 10 to 12 patients with IBD. *P < 0.05, PY109 versus control (paired t test).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/3/eaay8230/DC1

    Supplementary Materials and Methods

    Fig. S1. AHR homology model.

    Fig. S2. Conformational analyses of ligands.

    Fig. S3. Lead optimization for metabolic stability.

    Fig. S4. Biological characterization of lead compounds in vitro and after oral administration.

    Fig. S5. Toxicity study of PY109.

    Fig. S6. Lead compounds attenuate DSS-induced colitis in mice.

    Fig. S7. Effect of PY109 on Th17 and CMT-93 cells.

    Table S1. Representative SARs for screening library.

    Table S2. SARs of pyridine series.

    Table S3. Pathway analysis for PY109 counter-regulated genes.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. AHR homology model.
    • Fig. S2. Conformational analyses of ligands.
    • Fig. S3. Lead optimization for metabolic stability.
    • Fig. S4. Biological characterization of lead compounds in vitro and after oral administration.
    • Fig. S5. Toxicity study of PY109.
    • Fig. S6. Lead compounds attenuate DSS-induced colitis in mice.
    • Fig. S7. Effect of PY109 on Th17 and CMT-93 cells.
    • Table S1. Representative SARs for screening library.
    • Table S2. SARs of pyridine series.
    • Table S3. Pathway analysis for PY109 counter-regulated genes.

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