4“‐O‐Alkylated α‐Galactosylceramide Analogues as iNKT‐Cell Antigens: Synthetic, Biological, and Structural Studies

Abstract

Invariant natural killer T-cells (iNKT) are a glycolipid-responsive subset of T-lymphocytes that fulfill a pivotal role in the immune system. The archetypical synthetic glycolipid, α-GalCer, whose molecular framework is inspired by a group of amphiphilic natural products, remains the most studied antigen for iNKT-cells. Nonetheless, the potential of α-GalCer as an immunostimulating agent is compromised by the fact that this glycolipid elicits simultaneous secretion of Th1- and Th2-cytokines. This has incited medicinal chemistry efforts to identify analogues that are able to perturb the Th1/Th2-balance. In this work, we present the synthesis of an extensive set of 4”-O-alkylated α-GalCer analogues, which were evaluated in vivo for their cytokine induction. We have found that conversion of the 4”-OH to ether moieties reduces the immunogenic potential in mice as compared to α-GalCer. Yet, the benzyl-modified glycolipids are able to produce a distinct pro-inflammatory immune response. The crystal structures suggest an extra hydrophobic interaction between the benzyl moiety and the α2-helix of CD1d.

Amide-Linked C4″-Saccharide Modification of KRN7000 Provides Potent Stimulation of Human Invariant NKT Cells and Anti-Tumor Immunity in a Humanized Mouse Model

“…Significant anti-tumor activity could also be demonstrated for compound 10 , but not for the other two C4″-amides, apparently reflecting a fine-tuning of the overall immune response that depends on subtle variations in the glycolipid structure. It has been suggested previously that appending phenyl substituents to the C4″-position could alter the affinity of TCR interaction with the glycolipid-CD1d complex, either through π–π stacking as suggested by Zhang et al or through an extra hydrophobic interaction between the phenyl moiety and the α2-helix of CD1d . However, our modeling and molecular dynamics analysis of ternary complexes containing 8 , 9 , and 10 did not suggest a uniform effect for the phenyl modification in enhancing interactions within the ternary complex.…”

“…The group of van Calenbergh reached similar conclusions in their studies of 4″- O -alkylated α-GalCer analogs, initially identifying a C4″-linked p -chlorobenzyl ether as an analog with promising immunostimulating properties, although less potent than KRN7000 based on studies in mice . More recently, they reported synthesis and biologic evaluation in mice of a large panel of 4″- O -alkylated derivatives, concluding that while ether-linked substitutions to the C4″-position decreased the immunogenic potential in mice relative to KRN7000, phenyl modified glycolipids with this structure are able to produce a distinct pro-inflammatory immune response that may be useful for particular applications …”

“…All of the compounds were able to stimulate human iNKT cells in vitro , again with only methyl ether 4 being slightly more antigenic than KRN7000. More recently, based on the polarization of iNKT cell cytokine responses seen with ethers 4 and 5 , van Calenbergh and co-workers prepared an extensive library of 4″-O-alkylated α-GalCers with aromatic and cycloaliphatic moieties with differing tether lengths . None was as potent a stimulus as KRN7000, although several induced a proinflammatory response, suggesting that there could be applications for such compounds as cancer immunotherapeutics or as vaccine adjuvants.…”

Natural and synthetic carbohydrate-based vaccine adjuvants and their mechanisms of action

“…2a ). Notable exceptions were the 3- O -sulfo-α-GalCer 66 , 67 and the 4- O -phenylpropyl-α-GalCer analogues 68 , which induced comparable T H 1/T H 2 responses, as well as benzyl-modified 4- O variants that promoted T H 1-biased immunity 69 , 70 . Thus, while deletion or modifications at the 2 position abolished activity, particular variations at the 3-OH (sulfation) and especially the 4-OH (phenyl groups) were less critical for immunostimulation (Fig.…”

Chemical Strategies to Boost Cancer Vaccines

“…The significant cytokine type change of that high IFN-γ/IL-4 secretion was achieved by α-5-thio-galactopyranosyl-N-perfluorooctanoyl phytosphingosine. , Cerundolo and others synthesized and improved the iNKT activating ability of the nonglycosidic compound threitolceramide (ThrCer) . Calenbergh’s team modified αGalCer analogues with C4″-, C5′′-, C6′′, and phytosphingosine modification, that improved the iNKT cell stimulating ability in vitro and in vivo. − Park’s group recently synthesized α-fluorocarbonyl-modified αGalCer analogues through an alkyne–alkyne cross coupling strategy. In contrast, the screened molecule showed high Th2 type preference in the iNKT cell test .…”