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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.

Al-Riyami L, Pineda MA, Rzepecka J, Huggan JK, Khalaf AI, Suckling CJ, Scott FJ, Rodgers DT, Harnett MM, Harnett W - J. Med. Chem. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde , 161 Cathedral Street, Glasgow G4 0RE, U.K.

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.

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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 blottingfor expression of MyD88 and loading control GAPDH. Levels of expressionwere determined by densitometry using Image-J software and expressedas the ratio of MyD88:GAPDH and normalized to the RPMI value. (B)Data from six analyses revealed that while LPS significantly increasedMyD88 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 MyD88expression in permeabilised bmMs relative to isotype control (20,000bmMs/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, LPSupregulated levels of MyD88 (127% relative to MFI of RPMI-treatedbmMs). Moreover, bmMs pretreated with 11a exhibited lowerlevels of MyD88 expression (MFI for 11a + LPS is 95.7%of the level of the RPMI value: the RPMI trace is not shown due toits overlap with that of 11a + LPS) than cells treatedwith LPS alone. (D) Simple schematic of model of action of 11a. 11a downregulates MyD88 expression and hence inducesa partial uncoupling of TLR/IL-1R from NF-κB activation andconsequent pro-inflammatory cytokine production that both initiatespathogenic IL-17-mediated inflammation and perpetuates chronic vascularpermeability, inflammation, and pathology in the joints.54,55 Thus 11a-mediated downregulation of MyD88 impactingat one or more of these sites provides a molecular mechanism for theprotection afforded in CIA.
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fig8: 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 blottingfor expression of MyD88 and loading control GAPDH. Levels of expressionwere determined by densitometry using Image-J software and expressedas the ratio of MyD88:GAPDH and normalized to the RPMI value. (B)Data from six analyses revealed that while LPS significantly increasedMyD88 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 MyD88expression in permeabilised bmMs relative to isotype control (20,000bmMs/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, LPSupregulated levels of MyD88 (127% relative to MFI of RPMI-treatedbmMs). Moreover, bmMs pretreated with 11a exhibited lowerlevels of MyD88 expression (MFI for 11a + LPS is 95.7%of the level of the RPMI value: the RPMI trace is not shown due toits overlap with that of 11a + LPS) than cells treatedwith LPS alone. (D) Simple schematic of model of action of 11a. 11a downregulates MyD88 expression and hence inducesa partial uncoupling of TLR/IL-1R from NF-κB activation andconsequent pro-inflammatory cytokine production that both initiatespathogenic IL-17-mediated inflammation and perpetuates chronic vascularpermeability, inflammation, and pathology in the joints.54,55 Thus 11a-mediated downregulation of MyD88 impactingat one or more of these sites provides a molecular mechanism for theprotection afforded in CIA.

Mentions: Previous work published by our group has shown thatES-62’s mechanism of action involves downregulation of thekey TLR/IL-1R signaling adaptor MyD88 in Th17 cells9 and mast cells.53 This is alsotrue of macrophages (our unpublished results), the cell type usedfor our primary screen, and so we next investigated whether 11a was also able to cause downregulation of MyD88 in thesecells. As shown in Figure 8A,B, the SMA doesindeed cause significant downregulation of the signaling adaptor inthese cells. Figure 8A–C also showsthat, as expected, LPS causes an increase in MyD88 expression withinthe cells, but as demonstrated in Figure 8C,this is prevented by 11a. A simple schematic of the proposedmechanism of action of 11a is shown in Figure 8D and described in detail in the figure legend.


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.

Al-Riyami L, Pineda MA, Rzepecka J, Huggan JK, Khalaf AI, Suckling CJ, Scott FJ, Rodgers DT, Harnett MM, Harnett W - J. Med. Chem. (2013)

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 blottingfor expression of MyD88 and loading control GAPDH. Levels of expressionwere determined by densitometry using Image-J software and expressedas the ratio of MyD88:GAPDH and normalized to the RPMI value. (B)Data from six analyses revealed that while LPS significantly increasedMyD88 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 MyD88expression in permeabilised bmMs relative to isotype control (20,000bmMs/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, LPSupregulated levels of MyD88 (127% relative to MFI of RPMI-treatedbmMs). Moreover, bmMs pretreated with 11a exhibited lowerlevels of MyD88 expression (MFI for 11a + LPS is 95.7%of the level of the RPMI value: the RPMI trace is not shown due toits overlap with that of 11a + LPS) than cells treatedwith LPS alone. (D) Simple schematic of model of action of 11a. 11a downregulates MyD88 expression and hence inducesa partial uncoupling of TLR/IL-1R from NF-κB activation andconsequent pro-inflammatory cytokine production that both initiatespathogenic IL-17-mediated inflammation and perpetuates chronic vascularpermeability, inflammation, and pathology in the joints.54,55 Thus 11a-mediated downregulation of MyD88 impactingat one or more of these sites provides a molecular mechanism for theprotection afforded in CIA.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125414&req=5

fig8: 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 blottingfor expression of MyD88 and loading control GAPDH. Levels of expressionwere determined by densitometry using Image-J software and expressedas the ratio of MyD88:GAPDH and normalized to the RPMI value. (B)Data from six analyses revealed that while LPS significantly increasedMyD88 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 MyD88expression in permeabilised bmMs relative to isotype control (20,000bmMs/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, LPSupregulated levels of MyD88 (127% relative to MFI of RPMI-treatedbmMs). Moreover, bmMs pretreated with 11a exhibited lowerlevels of MyD88 expression (MFI for 11a + LPS is 95.7%of the level of the RPMI value: the RPMI trace is not shown due toits overlap with that of 11a + LPS) than cells treatedwith LPS alone. (D) Simple schematic of model of action of 11a. 11a downregulates MyD88 expression and hence inducesa partial uncoupling of TLR/IL-1R from NF-κB activation andconsequent pro-inflammatory cytokine production that both initiatespathogenic IL-17-mediated inflammation and perpetuates chronic vascularpermeability, inflammation, and pathology in the joints.54,55 Thus 11a-mediated downregulation of MyD88 impactingat one or more of these sites provides a molecular mechanism for theprotection afforded in CIA.
Mentions: Previous work published by our group has shown thatES-62’s mechanism of action involves downregulation of thekey TLR/IL-1R signaling adaptor MyD88 in Th17 cells9 and mast cells.53 This is alsotrue of macrophages (our unpublished results), the cell type usedfor our primary screen, and so we next investigated whether 11a was also able to cause downregulation of MyD88 in thesecells. As shown in Figure 8A,B, the SMA doesindeed cause significant downregulation of the signaling adaptor inthese cells. Figure 8A–C also showsthat, as expected, LPS causes an increase in MyD88 expression withinthe cells, but as demonstrated in Figure 8C,this is prevented by 11a. A simple schematic of the proposedmechanism of action of 11a is shown in Figure 8D and described in detail in the figure legend.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde , 161 Cathedral Street, Glasgow G4 0RE, U.K.

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.

Show MeSH
Related in: MedlinePlus