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

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.

Figure 1

Structure of hit compound 1.

Figure 2

Target identification of compound 1 using human TLR HEK293 reporter cell lines and genetically deficient cells. (A) Human TLR2, TLR3, TLR4/MD-2/CD14, TLR5, TLR7, TLR8, and TLR9 HEK 293 Blue cells or NF-κB/SEAPorter cells were incubated with compound 1 (10 μM) for 20–24 h, and activation was evaluated by SEAP secretion in the culture supernatants by colorimetric assay at OD₄₀₅. Data shown are mean ± SEM of triplicates and representative of two to three independent experiments showing similar results. p < 0.05 was considered significant compared to the vehicle control using Student’s t test. (B,C) Mouse (B) or human (C) TLR4 HEK transfectomas were incubated with graded concentrations of compound 1. TLR4 mediated NFκB activation was measured by SEAP secretion in the culture supernatant. (D) WT and Tlr4^–/– mBMDC were incubated with compound 1 (10 μM) for18 h. IL-6 in the culture supernatants was measured by ELISA. (E) Mouse TLR4 transfectoma cells were incubated with 2.5 μM compound 1 in the presence or absence of TLR4 antagonist LPS-RS (12, 111, 1000 ng/mL). Activation of the TLR4/NFκB pathway was evaluated by SEAP secretion in the culture supernatants. * denotes p < 0.05 considered as significant compared to vehicle using one way ANOVA with Dunnett’s post hoc testing. (F,G) WT and Cd14^–/– mBMDC were incubated with compound 1 (3.7 μM) overnight. IL-6 in the culture supernatants was measured by ELISA (F) and type I IFN (IFN-β) measured by luciferase release in from an ISRE reporter cell line (G). p < 0.05 was considered as significant compared to vehicle using Student’s t test. NS denotes “not significant”. Data shown are mean ± SEM of triplicates and representative of two independent experiments showing similar results.

Scheme 1. General Synthetic Route

Reagents and conditions: (a) BrCH₂COOEt, NaHCO₃, EtOH, reflux; (b) tert-BuOK, THF, <30 °C; (c) R¹-NCS, EtOH, reflux; (d) PPA, 110 °C; (e) ClCH₂COOH, KOH/EtOH, reflux; (f) R²-NH₂ (for R² substitutions refer to Table 1), HATU, DMF, room temp.

Figure 3

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

Figure 4

Representative data of SAR compound screening using mouse primary dendritic cells. Biological screening of SAR compounds was conducted using primary mBMDC. The cells were incubated with 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. IL-6 levels in culture supernatants were measured by ELISA. Data shown are mean ± SEM of triplicate.

Figure 5

Representative data of SAR compound screening using TLR4 transfectomas. Mouse TLR4 (A,C), and hTLR4 (B,D) HEK transfectomas were incubated with 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 by SEAP activity in the supernatant by absorption at 630 nm. Data shown are mean ± SEM of triplicate data.

Figure 6

SAR regions of modification of hit compound 1.

Figure 7

Assessment of cytotoxicity of compounds 1 and 42. Cytotoxicity of the compounds was evaluated by MTT assay as a measure of viability. mBMDC (10⁵/well) were incubated with graded concentrations of the compounds for 18 h and compared with vehicle (0.5% DMSO) and MPLA (1 μg/mL). (A) IL-6 levels in the culture supernatants were determined by ELISA. (B) The cells were lysed after the overnight incubation with MTT reagents, and absorbance at 570 nm was measured, subtracting the reference absorbance at 650 nm. Data shown are mean ± SEM of triplicates and are representative of two independent experiments showing similar results.

Scheme 2. N-5 Derivatives

(a) NaH, DMF, room temp, then CH₃I; (b) NaH, DMF, then Br(CH₂)₃COOCH₃

Scheme 3. N-5 Alkyl Derivatives

(a) ClCH₂COO t-Bu, KOH, DMA,H₂O; (b) NaH, DMF, then R³X (iodopropane or iodopentane or bromododecane or bromoacetonitrile); (c) TFA, DCM or CH₃CN; (d) cyclohexyl-NH₂, HATU, DMF, room temp; (e) compound 51, H₂SO₄, H₂O.

Figure 8

Correlation plot of IL-6 versus IP-10 induced by SAR derivatives. IL-6 (AUC) and IP-10 values in Tables 1–3 were plotted. Two distinct groups, compounds in the dotted circled (upper right) and in solid (left) circled areas, were selected based on the induction of IL-6 and IP-10. Numbers in the legends indicate compound ID. These clusters were selected based on mean ± SD of the normalized IL-6 and IP-10 values of all SAR compounds. For IL-6. the mean ± SD was 32 ± 37. For IP-10, the mean ± SD was 37 ± 33. The group at the upper right area comprises compounds that induce high IL-6 and high IP-10 production, including compounds 1, 9, 10, 28, and 42, whose values were at least one SD above the mean for both cytokines. The other cluster is composed of compounds (13, 16, 17, 29, 30, and 33) that induce low IL-6 but relatively higher IP-10 production, whose values were below the mean for IL-6 and were above the mean for IP-10.

Figure 9

Predicted binding mode of compound 28 to mouse TLR4/MD-2 complex.

Figure 10

Predicted binding interactions of compound 28 with mouse TLR4/MD-2 complex (PDB 2Z64).