Designing anti-inflammatory drugs from parasitic worms: a synthetic small molecule analogue of the Acanthocheilonema viteae product ES-62 prevents development of collagen-induced arthritis

Abstract

In spite of increasing evidence that parasitic worms may protect humans from developing allergic and autoimmune diseases and the continuing identification of defined helminth-derived immunomodulatory molecules, to date no new anti-inflammatory drugs have been developed from these organisms. We have approached this matter in a novel manner by synthesizing a library of drug-like small molecules based upon phosphorylcholine, the active moiety of the anti-inflammatory Acanthocheilonema viteae product, ES-62, which as an immunogenic protein is unsuitable for use as a drug. Following preliminary in vitro screening for inhibitory effects on relevant macrophage cytokine responses, a sulfone-containing phosphorylcholine analogue (11a) was selected for testing in an in vivo model of inflammation, collagen-induced arthritis (CIA). Testing revealed that 11a was as effective as ES-62 in protecting DBA/1 mice from developing CIA and mirrored its mechanism of action in downregulating the TLR/IL-1R transducer, MyD88. 11a is thus a novel prototype for anti-inflammatory drug development.

Figure 1

PC-BSA protects against CIA and targets IL-17 and IFNγ responses. Arthritis scores (BSA, n = 7; PC-BSA, n = 6 (A)) and hind paw width (B), expressed as mean scores ± SEM for BSA- or PC-BSA-treatment groups where n = number of individual mice exposed to collagen and disease incidence (C,D), indicated by the % of mice developing a severity score ≥2 (C) or ≥4 (D). Serum IL-17 levels are plotted as mean values of triplicate IL-17 analyses of serum from individual mice (naïve, n = 3; BSA, n = 6; PC-BSA, n = 6 (E)). (F,G) Exemplar plots of gating strategy of intracellular IL-17 and IFNγ expression by DLN (draining lymph node) cells pooled from BSA- and PC-BSA-treated mice with CIA show CD4 or γδ expression on the x-axis versus cytokine expression on the y-axis, with the relevant % cytokine positive cells annotated. The numbers of cytokine-expressing CD4⁺ T cells (H,J), γδ T cells (I,L), and CD8⁺ T cells (K) present in the pooled DLN cells from the naïve (not exposed to collagen), BSA, and PC-BSA groups are shown. For statistical analysis, *p < 0.05.

Figure 2

Design of small molecule analogues (SMAs) of ES-62 based upon a tyrosyl-phosphoryl choline peptide.

Scheme 1. Synthesis of Phosphorus-Containing SMAs

Scheme 2. Synthesis of Aminoethylsulfones

Secondary amines were Me₂NH, pyrrolidine, and morpholine. Substituents X and R of compounds evaluated are given in the tables.

Scheme 3. Synthesis of Aminopropylsulfones and Phenyl Sulfones

Secondary amines were Me₂NH, pyrrolidine, and morpholine. Substituents X and R of compounds evaluated are given in the tables.

Scheme 4. Synthesis of Sulfonamides

Substituents X and R of compounds evaluated are given in the tables. (19) is referred to as the CSN type, (21) as the CNS type, and (23) as the NSC type in the text.

Scheme 5. Synthesis of Carboxamides

Substituents X and R of compounds evaluated are given in the tables.

Figure 3

11a-related changes in macrophage cytokine production and signaling of p65 NFκB in response to LPS, BLP, and CpG. BmMs were preincubated with 11a at 5 μg/mL for 18 h prior to stimulation with 100 ng/mL LPS, 10 ng/mL BLP, or 0.01 μM CpG for 24 h and analysis of levels of IL-12p40 (A) and IL-6 (B) performed by ELISA. (C) Stimulation with PAMPs as above for LPS and BLP, and 1 μM CpG, but for 1 h and the level of p65 activation in duplicate samples measured by TransAM. For statistical analysis for (A) and (B), *p < 0.05.

Figure 4

Lack of toxic effect of 11a on macrophages. BmMs in ultralow binding tissue culture plates were rested in RPMI complete medium for 24 h before culturing with fresh medium or medium containing 11a (5 μg/mL) or ES-62 (2 μg/mL). After 18 h, the macrophages were stimulated with medium containing 100 ng/mL LPS or 10 ng/mL BLP or 0.01 μM CpG for an additional 24 h before staining with 7-AAD to assess their viability. The samples were analyzed by flow cytometry, and the data are presented as density plots with frequency of 7-AAD positive (dead) cells indicated in the gates. The results shown are from a single experiment representative of two independent experiments.

Figure 5

11a protects against CIA. Disease is shown by each of mean arthritis score ((A) PBS, n = 13; 11a, n = 13, data pooled from two independent experiments), hind paw width ((B) n = 7, data from a single experiment), and incidence (C,D) indicated by the % of mice developing a severity score ≥2 ((C) cumulative incidence) or ≥4 ((D) high score incidence). Results are expressed as mean scores ± SEM for PBS or 11a-treatment groups of mice exposed to collagen. The numbers of each of total (E), CD4⁺ (F), CD8⁺ (G), and γδ (H) T cells in DLN of individual mice from the naïve (n = 7), PBS-treated (n = 13) and 11a-treated (n = 13) groups are shown. For statistical analysis, *p < 0.05 and **p < 0.01.

Figure 6

11a suppresses IFNγ and IL-17 responses in CIA. Exemplar plots of gating strategy of intracellular IFNγ (A) and IL-17 (B) expression by DLN cells from representative individual PBS-treated mice with CIA show forward scatter (FSC) or CD4, CD8, and γδ expression on the y-axis versus cytokine expression on the x-axis as indicated. The number of (A) IFNγ-expressing total DLN cells, CD8⁺ T cells, and CD4⁺ T cells and (B) IL-17-expressing total DLN cells, CD4⁺ T cells, and γδ T cells following stimulation with PMA/ionomycin from individual mice are shown (naïve, n = 7; PBS, n = 13; 11a, n = 13). For statistical analysis, **p < 0.01 and ***p < 0.001.

Figure 7

11a inhibits Th17 polarization. (A) Serum IL-17 levels are plotted as mean values of triplicate IL-17 analyses from individual mice (PBS, n = 12; 11a, n = 13). (B) BmDCs from C57BL/6 mice were preincubated with or without (11a) (5 μg/mL) for 2 h prior to stimulation with LPS for 24 h, and TNFα, IL-6, and IL-23 levels were then analyzed. Data are the mean values (of triplicate samples) ± SEM pooled from 4 independent experiments. (C) BmDCs from BALB/c mice preincubated with or without 11a (5 μg/mL) were pulsed with the indicated concentration of OVA peptide and cocultured with naive OVA-specific CD4⁺ T cells (DO.11.10/BALB/c) for 3 days before measuring IL-17 release. Data are the mean values ± SD of duplicate samples pooled from two independent experiments (n = 4). (D) Pooled normalized data from four independent experiments analyzing the effect of 11a on IL-17 release from bmDC (C57BL/6)-OVA-specific CD4⁺T cell (OT-II/C57BL/6) cocultures. Data are presented as the means of the mean percentage maximum (LPS) response ± SEM where data were normalized to the LPS response at 10 nM (left), 100 nM (middle), and 300 nM (right) OVA, respectively. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 8

11a downregulates MyD88 expression in macrophages. (A) BmMs treated with medium (RPMI), 11a at 1 and 5 μg/mL (11a-1 or 11a-5), or LPS (100 ng/mL) for 20 h were analyzed by Western blotting for expression of MyD88 and loading control GAPDH. Levels of expression were determined by densitometry using Image-J software and expressed as the ratio of MyD88:GAPDH and normalized to the RPMI value. (B) Data from six analyses revealed that while LPS significantly increased MyD88 expression (*p < 0.05), both 11a-1 and 11a-5 reduced it (*p < 0.05) relative to the RPMI control. In addition, the level of MyD88 in cells treated with either 11a-1 or 11a-5 was significantly different (^†††p < 0.001) to that in those exposed to LPS. (C) Flow cytometric analysis of MyD88 expression in permeabilised bmMs relative to isotype control (20,000 bmMs/treatment group). BmMs were treated with RPMI or 11a (at 5 μg/mL) for 2 h prior to exposure to LPS (100 ng/mL) for a further 18 h, and consistent with the Western blot data, LPS upregulated levels of MyD88 (127% relative to MFI of RPMI-treated bmMs). Moreover, bmMs pretreated with 11a exhibited lower levels of MyD88 expression (MFI for 11a + LPS is 95.7% of the level of the RPMI value: the RPMI trace is not shown due to its overlap with that of 11a + LPS) than cells treated with LPS alone. (D) Simple schematic of model of action of 11a. 11a downregulates MyD88 expression and hence induces a partial uncoupling of TLR/IL-1R from NF-κB activation and consequent pro-inflammatory cytokine production that both initiates pathogenic IL-17-mediated inflammation and perpetuates chronic vascular permeability, inflammation, and pathology in the joints.^, Thus 11a-mediated downregulation of MyD88 impacting at one or more of these sites provides a molecular mechanism for the protection afforded in CIA.