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Identification of substituted pyrimido[5,4-b]indoles as selective Toll-like receptor 4 ligands.

Chan M, Hayashi T, Mathewson RD, Nour A, Hayashi Y, Yao S, Tawatao RI, Crain B, Tsigelny IF, Kouznetsova VL, Messer K, Pu M, Corr M, Carson DA, Cottam HB - J. Med. Chem. (2013)

Bottom Line: Synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide, N-3, and N-5 positions revealed differential TLR4 dependent production of NFκB and type I interferon associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively.Substitution at N-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound.Computational studies supported that active compounds appeared to bind primarily to MD-2 in the TLR4/MD-2 complex.

View Article: PubMed Central - PubMed

Affiliation: Moores Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, USA.

ABSTRACT
A cell-based high-throughput screen to identify small molecular weight stimulators of the innate immune system revealed substituted pyrimido[5,4-b]indoles as potent NFκB activators. The most potent hit compound selectively stimulated Toll-like receptor 4 (TLR4) in human and mouse cells. Synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide, N-3, and N-5 positions revealed differential TLR4 dependent production of NFκB and type I interferon associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively. Specifically, a subset of compounds bearing phenyl and substituted phenyl carboxamides induced lower IL-6 release while maintaining higher IP-10 production, skewing toward the type I interferon pathway. Substitution at N-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound. Computational studies supported that active compounds appeared to bind primarily to MD-2 in the TLR4/MD-2 complex. These small molecules, which stimulate innate immune cells with minimal toxicity, could potentially be used as adjuvants or immune modulators.

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Type I IFN inductionby compound 1. Wild-type mBMDCswere incubated with graded concentrations of compound 1 overnight. DMSO 0.5% served as the vehicle control. The levels oftype I IFN were determined using an L929-ISRE luciferase reportercell line and a mIFNβ standard (A). IP-10 levels were determinedby ELISA (B). Data shown are mean ± SEM of triplicates and representativeof two independent experiments showing similar results. * denotes p < 0.05 compared to vehicle using one way ANOVA withDunnett’s post hoc testing.
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fig3: Type I IFN inductionby compound 1. Wild-type mBMDCswere incubated with graded concentrations of compound 1 overnight. DMSO 0.5% served as the vehicle control. The levels oftype I IFN were determined using an L929-ISRE luciferase reportercell line and a mIFNβ standard (A). IP-10 levels were determinedby ELISA (B). Data shown are mean ± SEM of triplicates and representativeof two independent experiments showing similar results. * denotes p < 0.05 compared to vehicle using one way ANOVA withDunnett’s post hoc testing.

Mentions: The hTLR4 transfected HEK293 cell line(Figure 2A) also overexpresses MD-2 and CD14,which are TLR4 accessoryproteins.14,15 Compound 1, however, was notdependent on CD14 for either IL-6 or type I IFN production, as demonstratedusing CD14 deficient cells (Figure 2F, G).The supernatants from mBMDCs stimulated with graded doses of compound 1 were also tested for IP-10 as a surrogate marker of typeI IFN release. Results showed a dose-dependent response for type IIFN (Figure 3A) production, which paralleledthat of IP-10 (Figure 3B).


Identification of substituted pyrimido[5,4-b]indoles as selective Toll-like receptor 4 ligands.

Chan M, Hayashi T, Mathewson RD, Nour A, Hayashi Y, Yao S, Tawatao RI, Crain B, Tsigelny IF, Kouznetsova VL, Messer K, Pu M, Corr M, Carson DA, Cottam HB - J. Med. Chem. (2013)

Type I IFN inductionby compound 1. Wild-type mBMDCswere incubated with graded concentrations of compound 1 overnight. DMSO 0.5% served as the vehicle control. The levels oftype I IFN were determined using an L929-ISRE luciferase reportercell line and a mIFNβ standard (A). IP-10 levels were determinedby ELISA (B). Data shown are mean ± SEM of triplicates and representativeof two independent experiments showing similar results. * denotes p < 0.05 compared to vehicle using one way ANOVA withDunnett’s post hoc testing.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3722616&req=5

fig3: Type I IFN inductionby compound 1. Wild-type mBMDCswere incubated with graded concentrations of compound 1 overnight. DMSO 0.5% served as the vehicle control. The levels oftype I IFN were determined using an L929-ISRE luciferase reportercell line and a mIFNβ standard (A). IP-10 levels were determinedby ELISA (B). Data shown are mean ± SEM of triplicates and representativeof two independent experiments showing similar results. * denotes p < 0.05 compared to vehicle using one way ANOVA withDunnett’s post hoc testing.
Mentions: The hTLR4 transfected HEK293 cell line(Figure 2A) also overexpresses MD-2 and CD14,which are TLR4 accessoryproteins.14,15 Compound 1, however, was notdependent on CD14 for either IL-6 or type I IFN production, as demonstratedusing CD14 deficient cells (Figure 2F, G).The supernatants from mBMDCs stimulated with graded doses of compound 1 were also tested for IP-10 as a surrogate marker of typeI IFN release. Results showed a dose-dependent response for type IIFN (Figure 3A) production, which paralleledthat of IP-10 (Figure 3B).

Bottom Line: Synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide, N-3, and N-5 positions revealed differential TLR4 dependent production of NFκB and type I interferon associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively.Substitution at N-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound.Computational studies supported that active compounds appeared to bind primarily to MD-2 in the TLR4/MD-2 complex.

View Article: PubMed Central - PubMed

Affiliation: Moores Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, USA.

ABSTRACT
A cell-based high-throughput screen to identify small molecular weight stimulators of the innate immune system revealed substituted pyrimido[5,4-b]indoles as potent NFκB activators. The most potent hit compound selectively stimulated Toll-like receptor 4 (TLR4) in human and mouse cells. Synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide, N-3, and N-5 positions revealed differential TLR4 dependent production of NFκB and type I interferon associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively. Specifically, a subset of compounds bearing phenyl and substituted phenyl carboxamides induced lower IL-6 release while maintaining higher IP-10 production, skewing toward the type I interferon pathway. Substitution at N-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound. Computational studies supported that active compounds appeared to bind primarily to MD-2 in the TLR4/MD-2 complex. These small molecules, which stimulate innate immune cells with minimal toxicity, could potentially be used as adjuvants or immune modulators.

Show MeSH
Related in: MedlinePlus