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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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Related in: MedlinePlus

Representative data of SAR compound screeningusing TLR4 transfectomas.Mouse TLR4 (A,C), and hTLR4 (B,D) HEK transfectomas were incubatedwith graded concentrations of the indicated compounds 1, 36, and 39 (A) or 11 and 12 (B) for 18 h. DMSO 0.5% served as the vehicle control.The specific activation of the reporter cell lines was measured bySEAP activity in the supernatant by absorption at 630 nm. Data shownare mean ± SEM of triplicate data.
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fig5: Representative data of SAR compound screeningusing TLR4 transfectomas.Mouse TLR4 (A,C), and hTLR4 (B,D) HEK transfectomas were incubatedwith graded concentrations of the indicated compounds 1, 36, and 39 (A) or 11 and 12 (B) for 18 h. DMSO 0.5% served as the vehicle control.The specific activation of the reporter cell lines was measured bySEAP activity in the supernatant by absorption at 630 nm. Data shownare mean ± SEM of triplicate data.

Mentions: The above assays were utilized to compare the derivativesof thelead pyrimidoindole for their ability to activate mouse and humanTLR4. Mouse TLR4 activation was assessed using primary mBMDC and IL-6release (Figure 4) and confirmed using mTLR4transfected HEK293 cells (Figure 5A, C). Thevalues shown in the Tables 1–3 for IL-6 release, and mTLR4 activation are areaunder the curve (AUC) values for titrated doses of compounds from312 nM to 10 μM. Each cytokine induction curve was first convertedto a percent activity curve, and then the AUC of the percent activitycurve was calculated. The process of converting to a percent activitycurve allowed subtracting background and adjusting for plate-to-platevariation. Finally, the AUC values were normalized to the activityof compound 1 within each experiment, set at 100. HumanTLR4 activation is shown for stimulation of PBMC (IL-8) and hTLR4HEK293 transfectomas (Figure 5B,D) at 10 μM,as these assays were not sufficiently sensitive at lower concentrationsto make AUC comparisons. The levels of hTLR4 activation by SAR derivativesare expressed relative to compound 1, set at 100.


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)

Representative data of SAR compound screeningusing TLR4 transfectomas.Mouse TLR4 (A,C), and hTLR4 (B,D) HEK transfectomas were incubatedwith graded concentrations of the indicated compounds 1, 36, and 39 (A) or 11 and 12 (B) for 18 h. DMSO 0.5% served as the vehicle control.The specific activation of the reporter cell lines was measured bySEAP activity in the supernatant by absorption at 630 nm. Data shownare mean ± SEM of triplicate data.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Representative data of SAR compound screeningusing TLR4 transfectomas.Mouse TLR4 (A,C), and hTLR4 (B,D) HEK transfectomas were incubatedwith graded concentrations of the indicated compounds 1, 36, and 39 (A) or 11 and 12 (B) for 18 h. DMSO 0.5% served as the vehicle control.The specific activation of the reporter cell lines was measured bySEAP activity in the supernatant by absorption at 630 nm. Data shownare mean ± SEM of triplicate data.
Mentions: The above assays were utilized to compare the derivativesof thelead pyrimidoindole for their ability to activate mouse and humanTLR4. Mouse TLR4 activation was assessed using primary mBMDC and IL-6release (Figure 4) and confirmed using mTLR4transfected HEK293 cells (Figure 5A, C). Thevalues shown in the Tables 1–3 for IL-6 release, and mTLR4 activation are areaunder the curve (AUC) values for titrated doses of compounds from312 nM to 10 μM. Each cytokine induction curve was first convertedto a percent activity curve, and then the AUC of the percent activitycurve was calculated. The process of converting to a percent activitycurve allowed subtracting background and adjusting for plate-to-platevariation. Finally, the AUC values were normalized to the activityof compound 1 within each experiment, set at 100. HumanTLR4 activation is shown for stimulation of PBMC (IL-8) and hTLR4HEK293 transfectomas (Figure 5B,D) at 10 μM,as these assays were not sufficiently sensitive at lower concentrationsto make AUC comparisons. The levels of hTLR4 activation by SAR derivativesare expressed relative to compound 1, set at 100.

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