Design and evaluation of ω-hydroxy fatty acids containing α-GalCer analogues for CD1d-mediated NKT cell activation

Article abstract of DOI:10.1021/ml400517b

CD1d molecules recognize glycolipid antigens with straight chain fatty acid moieties. Although most of the residues in the CD1d binding groove are hydrophobic, some of the amino acids can form hydrogen bonds. Consequently, we have designed ω-hydroxy fatty acid-containing glycolipid derivatives of the prototypical CD1d ligand α-GalCer. The potency of the ω-hydroxy analogues of the proper length is comparable to that of α-GalCer. We propose, based on the biological results and molecular modeling studies, that a hydrogen bonding interaction is involved between the ω-hydroxy group and a polar amino acid residue in the hydrophobic binding groove.

Full text of DOI:10.1021/ml400517b

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Letter
Design and Evaluation of #-Hydroxy Fatty Acids Containing
#-GalCer Analogues for CD1d-Mediated NKT Cell Activation
Chaemin Lim, Jae Hyun Kim, Dong Jae Baek, Joo-Youn Lee, Minjae Cho, Yoon-
Sook Lee, Chang-Yuil Kang, Doo Hyun Chung, Won-Jea Cho, and Sanghee Kim
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml400517b • Publication Date (Web): 04 Feb 2014
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Chaemin Lim,† Jae Hyun Kim,† Dong Jae Baek,† Joo-Youn Lee,† Minjae Cho,† Yoon-Sook Lee,†
Chang-Yuil Kang,† Doo Hyun Chung,‡ Won-Jae Cho,§ Sanghee Kim*,†
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†College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea
‡College of Medicine, Seoul National University, 28 Yongon, Chongno-gu, Seoul 110-799, Korea
§College of Pharmacy, Chonnam National University, Yongbong, Buk-gu, Kwangju 500-757, Korea.
KEYWORDS: -Galactosylceramide, CD1d, hydrogen bonding interaction, NKT cell activator, drug design
ABSTRACT: CD1d molecules recognize glycolipid antigens with straight chain fatty acid moieties. Although most of the
residues in the CD1d binding groove are hydrophobic, some of the amino acids can form hydrogen bonds. Consequently,
we have designed -hydroxy fatty acid-containing glycolipid derivatives of the prototypical CD1d ligand -GalCer. The
potency of the -hydroxy analogues of the proper length is comparable to that of -GalCer. We propose, based on the
biological results and molecular modeling studies, that a hydrogen bonding interaction is involved between the -
hydroxy group and a polar amino acid residue in the hydrophobic binding groove.
cles around the central pole formed by residues Phe70
and Cys12 in the A’ pocket (Figure 2a). Various types of
interactions hold -GalCer in the correct position for
recognition by the TCR. While hydrogen-bonding is the
predominant interaction between the polar portion of -
GalCer and the surface residues of CD1d,10 hydrophobic
interactions are the principal factors generating the bind-
ing energy between the two lipid chains and the CD1d
binding groove.17
-GalCer (also known as KRN7000, 1, Figure 1) is a syn-
thetic glycolipid composed of -linked galactose, phyto-
sphingosine, and hexacosanoic acid. This glycolipid is the
first agonist discovered for natural killer T (NKT) cells1,2
and has helped elucidate NKT cell biology.3–5 In addition,
its structure has served as a basic guide for the design and
synthesis of various analogues.6–8
-GalCer activates NKT cells in a CD1d-restricted man-
ner. First, -GalCer binds to the CD1d molecule of anti-
gen-presenting cells to form a glycolipid/CD1d binary
complex.2,9 This complex is recognized by the T cell re-
ceptor (TCR) on the NKT cells, forming a ternary complex
that eventually activates the NKT cells.10,11 The activated
NKT cells promptly secrete T helper 1 and 2 (Th1 and Th2)
cytokines, such as interferon- (IFN-) and interleukin 4
(IL-4); these cytokines help induce a series of cellular ac-
tivation events during immune response.12,13 In this pro-
cess, -GalCer induces the secretion of Th1 and Th2 cyto-
kines indiscriminantly.14–16
Most of the residues in the CD1d deep binding groove
are hydrophobic amino acids. Interestingly, the binding
groove also contains a few amino acids able to form hy-
drogen bonds, such as Cys12, Gln14, Ser28, Thr37, His38,
and Arg74.21 It was envisioned that these amino acids
might be utilized to construct additional interactions and
stabilize the glycolipid/CD1d binary complex.22 Therefore,
we introduced a heteroatom into the -GalCer fatty acid
chain with the hope of providing a new rational basis for
the design of CD1-mediated immune modulating agents.
We report here, as a part of our work on this subject, the
synthesis and preliminary evaluation of the -hydroxy -
GalCer analogues.
Since the discovery of -GalCer, various analogues have
been designed and synthesized.6–8,13,23 Previous structural
alterations of the -GalCer fatty acid chain have focused
on modulating the hydrophobic interactions. Our -
hydroxy -GalCer analogue represents the first example
that introduces a hydrogen bonding interaction within
the binding groove of the A’ pocket.24
Figure 1. Structures of glycolipids 1–6.
The binding mode of -GalCer within CD1d was clearly
defined during X-ray crystallographic studies.10,17–20 The
phytosphingosine chain of -GalCer fit into the F’ pocket
of the binding groove and the fatty acid chain filled the A’
pocket. In this binding complex, the fatty acid chain cir-
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docking complex displaying the hydrogen bonding be-
Using the CD1d X-ray crystal structure, we designed -
hydroxy fatty acid-containing -GalCer analogues. In this
study, the mouse CD1d structure (Protein Data Bank code
3GML)25 was used instead of the human analogue because
our biological assay was based on the murine system. The
optimal carbon chain length between the carbonyl carbon
and the terminal hydroxyl group appears to be ten to
eleven atoms, placing the hydroxyl group close to the
Cys12, Gln14, and Ser28 residues (Figure 2b). Therefore, -
hydroxy -GalCer analogues 2 and 3 (Figure 1) were de-
signed. To probe the influence of the hydroxyl group on
the interactions within the binding groove, additional
analogues were designed. Analogue 4 contains an -
hydroxy fatty acid with a 16-carbon-atom chain that
seemed too long to facilitate a good hydrogen bonding
interaction with the desired residues. Analogues 5 and 6
have a saturated fatty acid with the same chain length as
in analogues 2 and 3 but without the -hydroxy group.
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tween the -hydroxy group and Gln14 had a better dock-
ing score than that with a hydrogen bond at Cys12 or
Ser28.26 The -hydroxy-group-lacking analogue 6 adopted
a docking conformation very similar to that of analogue 3,
but with a lower docking score (Figure S1 in the Support-
ing Information). These modeling studies suggest that the
additional hydrogen bonding interaction caused by the -
hydroxy group may increase the affinity for CD1d and
strengthen the glycolipid/CD1d complex. To validate the
docking results, we additionally performed docking simu-
lation with a small set of virtual analogues. As expected,
the docking scores of the analogues with hydrogen-
bonding capability were higher than that of analogue 6
(Table S1 in the Supporting Information).
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Figure 3. Molecular docking simulation of -hydroxy -
GalCer analogue 3 within the mCD1d (PDB code 3GML)
binding groove. H-bonds are displayed as green dotted lines.
For clarity, only the key residues are visible in line model and
are labeled using the 3-letter amino acid code.
To synthesize the designed compounds, known -
galactosyl phytosphingosine 727 was employed (Scheme 1).
The amino group of 7 was acylated with an -benzyloxy
fatty acid followed by the global removal of the benzyl
protecting groups in 8 via hydrogenolysis, affording the
desired -hydroxy -GalCer analogues 2–4. The -
hydroxy group-lacking analogues 5 and 6 were prepared
in a similar manner using fatty acids with chain lengths of
11 and 12 carbon atoms.
Scheme 1. Synthesis of the designed analogues 2–6.a
Figure 2. (a) Overview of the binding mode of -GalCer (1)
within mCD1d (PDB code 3HE6). (b) Schematic representa-
tion of design concept for -hydroxyl group containing -
GalCer analogues. Red dotted arrow indicates their ability to
form hydrogen bonds with designed ligands.
To simulate our design, molecular modeling studies
were performed on the interaction between the analogues
and CD1d. In our docking model (Figure 3), the -hydroxy
group of 3 was located close to the entrance of the A’
pocket and engaged in hydrogen bonding with the sur-
rounding residues, such as Cys12, Gln14, and Ser28. The
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aReagents and conditions: a) -benzyloxy fatty acids or
saturated fatty acids, EDCI, DMAP, CH2Cl2, rt, 69–80%. b) H2,
Pd(OH)2, EtOH/CH2Cl2 (3:1), rt, 2 d, 73–85%.
with the proper chain lengths can be accommodated
within the CD1d binding groove. By comparing the IL-2
production levels, we deduced the binding involves hy-
drogen bonding with the -group. If hydrogen bonding
interactions of the -groups were not involved, relying
only on the hydrophobic interactions in the binding
groove, -hydroxy analogues 2 and 3 would not induce
more IL-2 secretion than analogues 5 and 6. This hydro-
gen bonding interaction might be strong enough to com-
pensate for the loss of a full-length fatty acid chain from
the -GalCer 1 because the potencies of -hydroxy ana-
logues 2 and 3 were maintained.
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To evaluate obtained analogues, initially we used
mouse CD1d-specific NKT hybridoma cells (DN32.D3)
that produce IL-2 upon stimulation. The parent -GalCer
1 was also tested for comparison. As presented in Figure
4a, the rationally designed -hydroxy analogues 2 and 3
were as effective as -GalCer when promoting IL-2 pro-
duction. Analogue 4 was less efficient than -GalCer. The
-hydroxy group lacking analogues 5 and 6 elicited the
production of IL-2, but they were less efficient than -
GalCer or the corresponding -hydroxy analogues 2 and 3.
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During a second biological evaluation, we measured the
levels of the cytokines released from intact NKT cells.
While NKT hybridoma cells tend to produce IL-2 when
stimulated, primary NKT cells produce IFN- (Th1) and
IL-4 (Th2) with various other cytokines. The IFN- and IL-
4 levels were measured in the supernatant of mouse sple-
nocytes cultured with 32 ng/mL analogues 2–6. Figure 4b
presents the relative IFN- and IL-4 production levels of
2–6 compared with those of 1. The NKT stimulation activ-
ity for -hydroxy analogue 3 was very close to that of the
parent -GalCer 1, while the activity of 2 was slightly low-
er than that of 1. The cytokine release profiles for 2 and 3
were similar to that of -GalCer. However, 5 and 6 biased
cytokine secretion toward the Th2 response; these ana-
logues demonstrated a stronger stimulatory effect on IL-4
production than -GalCer 1 while promoting less IFN-
production than 1. The analogue with longer-alkyl chain
(4) also induced NKT cell cytokine responses with a Th2
bias.
Figure 4. Biological evaluation of compounds 1–6. (a) IL-2
secreted by the DN32.D3 NKT hybridoma cells. IL-2 produc-
tion was measured from the co-cultured supernatants of
NKT hybridoma DN32.D3 and mouse CD1d transfected RBL
cells after 16 h. The representative data of two individual
experiments are expressed as the means ±SD of the dupli-
cates. (b) IFN- and IL-4 secretion by mouse splenocytes
after individual treatment with compounds 1–6. Cytokine
production was measured after 72 h of culture. The results
are expressed as the relative activity. The representative data
of two individual experiments are expressed as the means
±SD of the triplicates.
Figure 5. Serum (a) IL-4 and (b) IFN- levels from mice in-
jected (i.v.) with -GalCer (1) or -hydroxy -GalCer ana-
logue 2 (0.5 g/mouse). Representative data of two individu-
al experiments are shown as the means ±SD of three mice.
As an in vivo evaluation, the serum cytokine levels were
measured after intravenous injection of analogue 2 (0.5
g/mouse) into naïve C57BL/6 mice. The -GalCer 1 was
also tested for in vivo comparison. As shown in Figure 5,
analogue 2 exhibited comparable activity to -GalCer 1.
Both 1 and 2 induced a rapid rise of IL-4 with the peak
value at 2 h and a delayed elevation of IFN- with the
peak value at 12 h after treatment. Analogue 2 induced
nearly an equal amount of IL-4 secretion as -GalCer 1
IL-2 secretion is dependent on antigen-loaded CD1d in
this assay system.28,29 Therefore, the -hydroxy analogues
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did, while the effect of 2 on IFN- secretion was slightly
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lower than that of 1. These in vivo cytokine release pro-
files for 2 were very similar to that in vitro.
* Tel: 82-2-880-2487. Fax: 82-2-888-0649. E-mail: penn-
kim@snu.ac.kr
Analogues 4–6, which showed a weaker stimulatory ef-
fect on IL-2 production than -GalCer 1 as well as ana-
logues 2 and 3, induced NKT cell cytokine responses with
a Th2 bias. The Th1-Th2 balance is determined by numer-
ous factors;5,16,30 one of the important factors was the sta-
bility of the glycolipid/CD1d complex.31 It has been gener-
ally believed that the less stable complex induces a biased
Th2 response. In this respect, the Th2 biasing analogues
4–6 are expected to form less stable complexes than ana-
logues 2 and 3.32 Conversely, -hydroxy analogues 2 and 3,
which provide responses that are less Th2-biased than
analogues 4–6, should form more stable glycolipid/CD1d
complexes. The only structural difference between -
hydroxy analogues 2 and 3 and truncated analogues 5 and
6 is the presence of a hydroxyl group at the end of each
acyl chain. Thus, the hydroxyl group is thought to play an
important role in strengthening the glycolipid/CD1d
complex, presumably via hydrogen bonding with an ami-
no acid residue in the A’ pocket, as the docking experi-
ments suggest. The structural difference between -
hydroxy analogues 2–3 and 4 is that the latter has a longer
space chain. Analogue 4 was designed to have a poor hy-
drogen-bonding interaction with the amino-acid residue
in the A’ pocket. Its pure Th2-like cytokine release profile
suggests that it forms less stable complexes than ana-
logues 2 and 3, which may be due to the absence of hy-
drogen-bonding interactions. Together with the observed
IL-2 production levels, these cytokine-polarizing proper-
ties support our hypothesis that -hydroxy analogues
with proper chain lengths can bind to CD1d using hydro-
gen bonds.
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The authors declare no competing financial interest.
9
This work was supported by a Bio & Medical Technology
Development Program (NRF-2013M3A9B8031389) NRF grant
funded by the Ministry of Science, ICT and Future Planning
(MSIP) and a Seoul National University Brain Fusion Pro-
gram research grant. This work was also supported by the
project of Global Ph. D. Fellowship of the NRF conducts from
2012.
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acid chain in -GalCer with a shorter -hydroxy fatty acid
chain maintains the NKT cell stimulation activity. After
combining these results with our molecular modeling
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Supporting Information. This section contains additional
figure displaying the molecular docking simulation of ana-
logue 6 (Figure S1), Table S1, detailed information regarding
the experimental procedures, characterization data, and cop-
1
ies of H and 13C spectra for final compounds. “This material
is available free of charge via the Internet at
http://pubs.acs.org.”
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in mouse CD1d.
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Y.; Bachi, A.; Kronenberg, M.; Wilson, I. A.; Degano, M. Crystal
Structure of Mouse CD1d Bound to the Self Ligand Phosphati-
dylcholine: A Molecular Basis for NKT Cell Activation. J. Immu-
nol. 2005, 175, 977–984.
(23) Wu, D.; Fujio, M.; Wong, C.-H. Glycolipids as im-
munostimulating agents. Bioorg. Med. Chem. 2008, 16, 1073–1083.
(24) Hydroxylated KRN7000 analogues with multiple hydroxyl
groups on the phytosphingosine main chain and/or amide side
chain of -GalCer were recently reported; see Shiozaki, M.; Ta-
shiro, T.; Koshino, H.; Shigeura, T.; Watarai, H.; Taniguchi, M.;
Mori, K. Synthesis and biological activity of hydroxylated ana-
logues of KRN7000 (-galactosylceramide). Carbohydr. Res. 2013,
370, 46–66.
(25) Schiefner, A.; Fujio, M.; Wu, D.; Wong, C.-H.; Wilson, I. A.
Structural Evaluation of Potent NKT Cell Agonists: Implications
for Design of Novel Stimulatory Ligands. J. Mol. Biol. 2009, 394,
71–82.
(26) The docking scores of the complexes exhibiting hydrogen
bonding between the -hydroxy group and Gln14, Cys12, or
Ser28 are –11.955, –10.544, and –9.775, respectively.
(27) Du, W.; Gervay-Hague, J. Efficient Synthesis of -
Galactosyl Ceramide Analogues Using Glycosyl Iodide Donors.
Org. Lett. 2005, 7, 2063–2065.
(28) Prigozy, T. I.; Naidenki, O.; Qasba, P.; Elewaut, D.;
Brossay, L.; Khurana, A.; Natori, T.; Koezuka, Y.; Kulkarni, A.;
Kronenberg, M. Glycolipid Antigen Processing for Presentation
by CD1d Molecules. Science 2001, 291, 664–667.
(29) Jiang, S.; Game, D. S.; Davies, D.; Lombardi, G.; Lechler, R.
I. Activated CD1d-restricted natural killer T cells secrete IL-2:
innate help for CD4+CD25+ regulartory T cells? Eur. J. Immunol.
2005, 35, 1193–1200.
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Design and Evaluation of -Hydroxy Fatty Acids Containing -GalCer Ana-
logues for CD1d-Mediated NKT Cell Activation
Chaemin Lim, Jae Hyun Kim, Dong Jae Baek, Joo-Youn Lee, Minjae Cho, Yoon-Sook Lee, Chang-Yuil Kang, Doo Hyun
Chung, Won-Jae Cho, Sanghee Kim*
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Figure 2. (a) Overview of the binding mode of αꢀGalCer (1) within mCD1d (PDB code 3HE6). (b) Schematic
representation of design concept for ωꢀhydroxyl group containing αꢀGalCer analogues. Red dotted arrow
indicates their ability to form hydrogen bonds with designed ligands.
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Figure 3. Molecular docking simulation of ωꢀhydroxy αꢀGalCer analogue 3 within the mCD1d (PDB code
3GML) binding groove. Hꢀbonds are displayed as green dotted lines. For clarity, only the key residues are
visible in line model and are labeled using the 3ꢀletter amino acid code.
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Figure 4. Biological evaluation of compounds 1–6. (a) ILꢀ2 secreted by the DN32.D3 NKT hybridoma cells.
ILꢀ2 production was measured from the coꢀcultured supernatants of NKT hybridoma DN32.D3 and mouse
CD1d transfected RBL cells after 16 h. The representative data of two individual experiments are expressed
as the means ±SD of the duplicates. (b) IFNꢀγ and ILꢀ4 secretion by mouse splenocytes after individual
treatment with compounds 1–6. Cytokine production was measured after 72 h of culture. The results are
expressed as the relative activity. The representative data of two individual experiments are expressed as
the means ±SD of the triplicates.
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Figure 5. Serum (a) ILꢀ4 and (b) IFNꢀγ levels from mice injected (i.v.) with αꢀGalCer (1) or ωꢀhydroxy αꢀ
GalCer analogue 2 (0.5 ꢁg/mouse). Representative data of two individual experiments are shown as the
means ±SD of three mice.
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