Heteroaromatic Moieties in the Sphingosine Backbone of α-Galactosylceramides for Noncovalent Interactions with CD1d

Abstract

A series of α-GalCer analogues containing heterocyclic and aromatic moieties in the sphingosine backbone were synthesized to improve the selectivity in the Th1/Th2 cytokine profile via noncovalent interaction with three aromatic residues at the binding pocket of CD1d. In vitro and in vivo biological evaluations revealed the treatment of α-GalCer analogue (6) induced the selective stimulation of natural killer T cells to facilitate the secretion of Th2 cytokines.KEYWORDS: α-Galactosylceramide, CD1d, selectivity, cytokine secretion, noncovalent interaction α-Galactosylceramide (α-GalCer) is a typical antigen for invariant natural killer T (iNKT) cells that are a subset of T cells and play a critical role in regulating immune responses. 1−5 After α-GalCer is loaded onto the CD1d protein in antigenpresenting cells (APCs), the CD1d-α-GalCer bimolecular complex is recognized by the T cell receptor (TCR) and the complex activates iNKT cells. 6−8 Subsequently, the activated iNKT cells release many cytokines, including pro-inflammatory T helper 1 (Th1) cytokines [interferon-γ(IFN-γ), tumor necrosis factor-α (TNF-α), etc.] and anti-inflammatory T helper 2 (Th2) cytokines [interleukin-4 (IL-4), interleukin-10 (IL-10), etc.]. 1−5,9,10 The secreted cytokines are recognized by other cells in the immune system, which affect a wide variety of immune responses, including the host response to parasites and bacteria, antitumor responses, and the protection against autoimmune diseases. 1−5 KRN7000 (1) is a well-known molecule of the α-GalCer family (Figure 1), and its therapeutic effects have been extensively studied in various disease models. 11−15 However, the in vivo efficacy of 1 has been somewhat limited because of its dual antagonizing effects on both Th1 and Th2 cytokines, and these dual effects are known to be polar opposites in immune response. 1 In other words, 1 can stimulate the secretion of both pro-and anti-inflammatory cytokines and can result in no net changes in immunity. Thus, many recent studies have focused on the synthesis of KRN7000 analogues that can selectively activate iNKT cells to secrete certain cytokines. 1 For example, the structural modification of the sugar moiety in α-GalCer revealed that the α-anomeric galactose is considerably better than other sugars, such as glucose, mannose, and β-galactose without selectivity. 12 However, the structural modification at the C6 position of the galactosyl moiety in α-GalCer with a 4Cl-3CF 3 -benzamide substituent (2) showed a promising

Anti-Inflammatory Effects of α-Galactosylceramide Analogs in Activated Microglia: Involvement of the p38 MAPK Signaling Pathway

“…Therefore, we studied the systematic structural modification of α-GalCer to selectively polarize either the T H 1 or T H 2 immune response. For example, we recently described a series of α-GalCer analogs containing heterocyclic and phenyl moieties in the sphingosine backbone and found that treatment with a T H 2-biased α-GalCer analog selectively stimulates the secretion of anti-inflammatory cytokines in iNKT cells ( Figure 1 ) [25] , [26] . With this selective T H 2-biased α-GalCer analog in hand, we envisioned the development of new α-GalCer-based anti-inflammatory agents that control microglial activation as a potential therapy for neurodegenerative diseases.…”

“…Unlike our previous report [25] , we aimed to introduce various alkyl moieties in the sphingosine backbone at the late stage of synthesis via simple N- alkylation of pyrazole. The key intermediate 13 can be prepared from commercially available d -galactal in eight steps with a 12% overall yield ( Figure 3 ).…”

“…For example, N- alkylation of pyrazole on 13 with 1-bromoundecane or 1-bromoethane yielded compounds 14a or 14b , whose structure was designed to keep backbone length similar to KRN7000 or OCH, respectively. N- alkylation of 13 with 4- n -hexylbenzylbromide led to compound 14c containing a phenyl group as well as pyrazole in its sphingosine backbone, originated from our T H 2-biased α-GalCer analog [25] . The subsequent migration of the phenyl group in the sphingosine backbone yielded compounds 14d and 14e .…”

“…As an isostere of amide bonds, triazole moiety was introduced via copper-mediated click reaction of the azide group in 14a – e with hexacos-1-yne at the acyl chain position to yield compounds 5a–e . Compound 1c is our reported T H 2-selective α-GalCer analog [25] , and the systematic diversification of this compound provided a collection of 25 α-GalCer analogs ( 1a – 5e ) as a focused library to identify novel anti-inflammatory agents for the treatment of neurodegenerative diseases.…”

Development of α-GalCer Analogues with an α-Fluorocarbonyl Moiety as Th2-Selective Ligands of CD1d

“…3,27 Briefly, HMNCs were incubated with individual α-GalCer analogues (25 nM) for 72 h, and the effects of these analogues on cytokine induction levels were detected by ELISA. 28,29 Prototype α-GalCer was used as a positive control. The bioassay results of each analogue were compared to that of the positive control, α-GalCer, along with that of OCH (Figure 1a), known as the Th2-biasing ligand, 30 for comparison.…”