Agonistic or antagonistic mucosal-associated invariant T (MAIT) cell activity is determined by the 6-alkylamino substituent on uracil MR1 ligands

Article abstract of DOI:10.1039/d0cc00247j

Mucosal-associated invariant T (MAIT) are a subset of innate-like T cells that are activated by uracil ligands presented by MR1. For the first time, we demonstrate that changes to the 6-aminoalkyl chain on uracil agonist 5-OP-RU can determine agonistic or antagonistic MAIT cell activity. Insomuch, a simplified agonist with a functional profile similar to 5-OP-RU, and a new structural class of antagonist that exhibits similar activity to known MAIT cell antagonist Ac-6-FP, were identified. This journal is

Full text of DOI:10.1039/d0cc00247j

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DOI: 10.1039/D0CC00247J.
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Agonistic or antagonistic mucosal-associated invariant T (MAIT)
cell activity is determined by the 6-alkylamino substituent on
uracil MR1 ligands
Received 00th January
20xx,
Accepted 00th January 20xx
Chriselle D. Braganza,a Chihiro Motozono,b Koh-hei Sonoda,c Sho Yamasaki,d,e,f,g Kensuke
DOI: 10.1039/x0xx00000x
Shibata,*c,e,h Mattie S. M. Timmer,*a Bridget L. Stocker*a
Mucosal-associated invariant T (MAIT) are a subset of innate-like T
cells that are activated by uracil ligands presented by MR1. For the
first time, we demonstrate that changes to the 6-aminoalkyl chain
on uracil agonist 5-OP-RU can determine agonistic or antagonistic
MAIT cell activity. Insomuch, a simplified agonist with a functional
profile similar to 5-OP-RU, and a new structural class of antagonist
that exhibits similar activity to known MAIT cell antagonist Ac-6-
FP, were identified.
O
N
O
N
HN
NH
OH
O
N
HN
N
OH
N
N
O
N
OH
HN
RHN
O
O
N
OH
H
OH OH
OH OH
Fig. 1. Representative MR1 ligands.
O
1a
R = H, 6-FP (
R = Ac, Ac-6-FP (
)
1b
2
3a
5-OP-RU ( )
)
RL-6,7-diMe (
)
it might be possible to develop better therapeutics for such
diseases.7 This is a pertinent goal given the highly conserved
nature of both the manipulation of MAIT cell function, via the
development of MAIT cell agonists or antagonists, it might be
possible to MAIT TCR α-chain and MR1,1,4 which allows for the
targeting of MAIT cells in all individuals.
6-Formylpterin (6-FP, 1a, Fig. 1), a metabolite of vitamin B9
(folic acid), and its synthetic derivative, acetyl-6-formylpterin
(Ac-6-FP, 1b) are MAIT cell antagonists,5,8 with the inhibitory
activity of these compounds being attributed to their ability to
covalently bind MR1 through formation of a Schiff base with
Lys43 of MR1, leading to MR1 upregulation.5,8,9 In contrast, the
MAIT cell agonists, the ribityl lumazines [e.g., 6,7-dimethyl-8-
ᴅ-ribityllumazine (RL-6,7-diMe, 2)], which are products of
vitamin B2 (riboflavin) metabolism,5 are unable to form a
Schiff base with Lys43, although the ribityl tail of these ligands
directly contacts Tyr95 of the MAIT TCR, thereby allowing for
moderate levels of MAIT cell activity.5,9 However, the most
potent MAIT cell agonist identified is the unstable
Mucosal-associated invariant T (MAIT) cells are a subset of
recently identified innate-like T lymphocytes bearing a T cell
receptor (TCR) with a highly conserved Vα19Jα33 α-chain in
mice or Vα7.2-Jα33 in humans, which pair with a limited array
of TCR β-chains.1,2 MAIT cells are abundant in human tissue
and blood,3 and are activated by ligands bound to the major
histocompatibility complex (MHC) class I-like protein MR1 on
antigen presenting cells.4,5 Upon activation, MAIT cells migrate
to sites of infection, rapidly proliferate, and secrete pro-
inflammatory cytokines or cytotoxic molecules.3,6 MAIT cells
have been implicated in a number of diseases such as
tuberculosis, HIV, diabetes, and cancer,3 and through the
manipulation of MAIT cell function, via the development of
agonists and antagonists,
a. School of Chemical and Physical Sciences, Victoria University of Wellington, PO
Box 600, Wellington, New Zealand
intermediate
5-(2-oxopropylideneamino)-6-ᴅ-
b. Department of Molecular Immunology, Research Institute for Microbial Diseases,
Osaka University, Osaka, Japan
ribitylaminouracil (5-OP-RU, 3a),10 and its activity has been
attributed to the combination of Schiff base formation with
Lys43 of MR1, as well as the presence of the ribityl tail, which
interacts directly with the MAIT TCR.
c. Department of Ocular Pathology and Imaging Science, Graduate School of
Medical Sciences, Kyushu University, Fukuoka, Japan
d. Department of Molecular Immunology, Research Institute for Microbial Diseases,
Osaka University, Osaka, Japan
e. Laboratory of Molecular Immunology, Immunology Frontier Research Center,
Osaka University, Suita 565-0871, Japan
Although studies have focussed on developing more stable
analogues of 5-OP-RU (3a),11 much still remains unknown
about the effects of the ribityl side chain on MAIT TCR binding
and activity. However, we recently demonstrated that the
incorporation of different sugar motifs at the 6-position of 5-
OP-RU (3a) can affect MAIT cell activity,12 and that removal of
the 2′-hydroxy group of the ribityl tail significantly decreased
f. Division of Molecular Immunology, Medical Institute of Bioregulation, Kyushu
University, Fukuoka 812-8582, Japan
g. Division of Molecular Immunology, Medical Mycology Research Center, Chiba
University, Chiba 260-8673, Japan
h. Systems Biochemistry in Pathology and Regeneration, Graduate School of
Medicine, Yamaguchi University, Ube, Japan
Electronic Supplementary Information (ESI) available: Experimental procedures and
characterisation data, including copies of 1H- and 13C-NMR spectra of all new
compounds. See DOI: 10.1039/x0xx00000x
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via flow cytometry to determine ligand acVtiievwitAyrt.icleHOenrlinee,
hydroxyethyl analogue 3b led to the highest activity from the
O
O
N
O
N
DOI: 10.1039/D0CC00247J
HN
NH
HN
NH
HN
NH
OH
n
O
O
O
N
O
N
O
N
n
O
R
NH2
H
H
H
HNO3, H2SO4
66%
N
HN
NH
Cl
6a-h
HN
NH
HN
NH
Cl
R
N
H
NO2
NaOH
EtOH:H2O
(78-86%)
O
O
O
NO2
O
O
O
4
5
7a-h
3b
3c
3d
3e
3f
,
3g
3h
, n = 1
, n = 2
, n = 4
n = 1
, n = 3
, n = 8
Na2S2O4, KOH, H2O
or
OH OH
DMSO:EtOH:H2O
(67-77%)
OH
a R =
O
OH
Fig. 2. Proposed 6-alkylamino analogues of 5-OP-RU (3a).
O
HN
NH
HCl
R
b R = (CH2)2OH
c R = (CH2)3OH
d R = (CH2)5OH
e R = CH3
methylglyoxal
HN
NH
R
O
N
H
in situ
O
N
N
H
f R = (CH2)2CH3
g R = (CH2)4CH3
h R = (CH2)9CH3
NH2
MAIT cell activation. In this study, we sought to investigate the
effects of gross structural changes to the 6-alkylamino side
chain on MAIT cell activation through the synthesis and
subsequent biological evaluation of 5-OP-RU analogues
hydroxyethylamine 3b,13 hydroxypropylamine 3c, hydroxy-
pentylamine 3d, methylamine 3e, propylamine 3f,
pentylamine 3g, and decylamine 3h (Fig. 2). From this work,
we were able to demonstrate how changes to the 6-
aminoalkyl chain can switch the function of the ligands from
agonist to antagonist.
O
3a-h
8a-h
Scheme
1.
Synthesis
of
10000
8000
6000
4000
2000
0
****
****
****
**
***
****
****
No 1b 3a 3b 3c 3d 3e 3f 3g 3h
To synthesise 5-OP-RU (3a) and analogues 3b-h, 6-
chlorouracil (4) was first nitrated under the agency of fuming
HNO3 and H2SO4 to produce 6-chloro-5-nitrouracil (5) (Scheme
2).12 Substitution of the chloride in 4 was then undertaken
under basic conditions12 using 1-amino-1-deoxy-ᴅ-ribitol (6a),
2-aminoethanol (6b), 3-amino-1-propanol (6c), 5-amino-1-
pentanol (6d), methylamine (6e), 1-aminopropane (6f), 1-
aminopentane (6g) and 1-aminodecane (6h) to give the
corresponding nitrouracils 7a-h in good yields (74-86%). Next,
reduction of the nitro groups was performed to give the
desired diaminouracil products ready for condensation with
methylglyoxal. Here, reductions of hydroxyalkyl nitrouracils 7a,
7c and 7d were carried out under the agency of KOH and
Na2S2O4 in H2O,12,14 and upon completion of the reactions, as
determined by high resolution mass spectrometry (HRMS)
analyses, the reaction mixtures were directly loaded onto C18
reversed-phase columns. Elution with water, and acidification
of the product fractions with 1M HCl, followed by
lyophilization, then gave diaminouracils 8a, 8c and 8d in good
yields (71-75%) as HCl salts, in order to improve their
stability.14 The poor aqueous solubility of hydroxyalkylated
nitrouracil 7b and alkylated nitrouracils 7e-h required
reductions to be performed in DMSO:EtOH:H2O (1:1:1). After
completion of these reactions, the mixtures were acidified
with 1M HCl, dried using a flow of argon to remove EtOH,
followed by lyophilisation to remove DMSO and H2O.
Purification of the residue by C18 reversed-phase
chromatography and acidifycation of the product fractions
gave the desired diaminouracils 8b, 8e-h as HCl salts in good
yields of 67-77%.
hydroxyalkyl and alkyl analogues.
Fig. 3. MAIT cell activation assay for 5-OP-RU analogues 3b-h. The MAIT cell line
6C2 was cultured for 24 h on NiH.cl9 cells overexpressing mouse MR1 in the
presence of 3b-h (10 µM) and the mean fluorescence intensity (MFI) of CD137
expression was measured after gating on viable TCRβ+ cells. Ac-6-FP (1b, 100
µM) and 5-OP-RU (3a, 10 µM) were used as negative and positive controls,
respectively. Data represents two independent experiments (n = 3). (**) P ≤
0.01; (***) P ≤ 0.001; (****) P ≤ 0.0001 compared to unstimulated cells.
series of synthesised analogues (Fig. 3), with this activity being
comparable to that elicited by agonist 5-OP-RU (3a). The
activity of the hydroxyalkyl analogues decreased as chain
length increased, with hydroxypentyl analogue 3d being
unable to activate MAIT cells. Conversely, removal of the
terminal hydroxy group abolished all activity, as no MAIT cell
activation was observed for the alkylated analogues 3f-h. In
this assay, 3d, 3f and 3g behaved in a similar manner to Ac-6-
FP (1b) as they led to significant downregulation of CD137
when compared to the no-ligand control. This data indicated
that 3d, 3f and 3g represent potential MAIT cell antagonists.
To understand more about the ability of the compounds to
bind MR1, we then examined the effect of these ligands on
MR1 surface expression. Here, compounds were incubated at
two concentrations (10 µM and 1 µM) with NiH.cl9 cells for 4
hours before the surface expression of MR18 was assessed
using flow cytometry. Overall, antagonist Ac-6-FP (1b) induced
the highest upregulation of MR1 surface expression at both
concentrations (Fig. 4A). Consistent with the MAIT cell
activation assay, hydroxyethyl analogue 3b showed similar
activity in this assay to that observed for 5-OP-RU (3a).
Hydroxypentyl analogue 3d induced slightly higher MR1
expression at 10 µM when compared to 3a, whereas the
propyl (3f) and pentyl (3g) analogues led to lower MR1
expression levels. Altogether, these results confirmed that the
agonistic hydroxyethyl analogue 3b binds MR1.
With the diaminouracil analogues 8a-h in hand, we then
sought to assess their ability to activate MAIT cells. To this end,
analogues 8a-h (10 µM) were combined with methylglyoxal in
situ to obtain 5-OP-RU analogues 3a-h,12,14 immediately before
incubation with mouse MR1-overexpressing NiH.cl9 cells12 and
the mouse MAIT cell line 6C2.2,12 The expression of MAIT cell
activation marker CD13712 was then measured after 24 hours
Further evidence for MR1 binding and MAIT cell interaction
was provided by the ability of the compounds to bind MR1
2 | J. Name., 2012, 00, 1-3
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tetramers and stain MAIT cells. To this end, compounds were
loaded onto empty MR1 tetramers12 and the resulting antigen-
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DOI: 10.1039/D0CC00247J
Fig. 5. A. MAIT cell competitive inhibition assay. NiH.cl9 cells were incubated
with Ac-6-FP (1b, 100 µM or 0 µM) for 1 h before the addition of 10 µM 5-OP-RU
(3a) or 3b and 6C2 cells for 24 h. MFI of CD137 expression was measured after
gating on viable TCRβ+ cells. Data represents two independent experiments (n =
3). (****) P ≤ 0.0001. B. Dose-response assay for 5-OP-RU (3a) and 3b. 6C2 cells
were cultured for 24 h on NiH.cl9 cells with 3a or 3b at 100 µM, 10 µM, 1 µM,
0.1 µM, 0.01 µM and 0.001 µM and MFI of CD137 expression was measured
after gating on viable TCRβ+ cells. Data represents % maximal activation of 6C2
cells from two independent experiments (n = 3).
agonist 5-OP-RU (3a). Moreover, analogue 3b can be obtained
from commercially available 2-aminoethanol via a three-step
synthesis that is shorter than the current synthesis of 5-OP-RU
(3a), which requires the generation of ribitylamine prior to
substitution of the uracil moiety.
Fig. 4. A. MR1 surface expression assay. NiH.cl9 cells overexpressing mouse MR1
were stimulated with compounds 1b, 3a, 3b, 3d, 3f and 3g at 10 µM and 1 µM
for 4 h, after which time the MFI of MR1 expression was measured. Data
represents two independent experiments (n = 2). B. MR1–analogue tetramer
staining of MAIT cells. Empty MR1 tetramers were loaded with 1b, 3a, 3b, 3d, 3f
and 3g and assessed for their ability to stain the MAIT cell line 6C2 and the
parental cell line TG40 (negative for TCR). Data represents two independent
experiments.
Given the observations that hydroxypentyl analogue 3d,
propyl analogue 3f and pentyl analogue 3g induced MR1
surface expression and showed tetramer staining of MAIT
cells, but not MAIT cell activation, we sought to further
investigate the potential antagonistic activity of these
compounds. Accordingly, the compounds were tested for their
ability to inhibit 5-OP-RU-mediated MAIT cell activation,
whereby NiH.cl9 cells were incubated with titrated amounts of
compounds 3d, 3f or 3g for 1 hour before the addition of 5-OP-
RU (3a) and 6C2 cells for 24 hours. Flow cytometric analysis
was then undertaken to determine CD137 expression levels
and the known antagonist Ac-6-FP (1b) was used as a positive
control. All three compounds showed concentration
dependent inhibition of 5-OP-RU-mediated MAIT cell
activation akin to 1b (Fig. 6), with pentyl analogue 3g inhibiting
activity to the greatest extent at a concentration of 100 µM,
although the antagonistic activity of this compound was less
than 1b at 10 µM. Altogether, these results suggest that we
have identified a new class of MAIT cell antagonists with a
structural scaffold that is different to known antagonists 6-FP
(1a) and Ac-6-FP (1b), and that is instead structurally more
similar to agonist 5-OP-RU (3a).
MR1 complexes were tested for their ability to stain the 6C2
MAIT cell line and the TG40 cell line,15 which was used as a
negative control as it lacks TCR-α and β chains. Here, all
compounds were capable of specifically staining MAIT cells, as
indicated by the co-staining of cells bearing the T cell marker
CD3ε (Fig. 4B). 5-OP-RU (3a)-loaded tetramers showed the
highest level of staining, with 96.8% of cells co-staining with
both the MR1-ligand complex and CD3ε. As expected,
hydroxyethyl analogue 3b showed a high level of MAIT cell
staining (94.5%), which was comparable to 5-OP-RU (3a). Non-
agonistic compounds 3d, 3f and 3g were also capable of
staining MAIT cells, albeit at slightly lower levels of 78-90%.
This data suggests that analogues 3b, 3d, 3f and 3g bind MR1
and interact with MAIT cells in a similar manner to 5-OP-RU
(3a).
To determine whether lead agonist hydroxyethyl analogue
3b binds in the same binding site as Ac-6-FP (1b), a
competitive inhibition experiment was carried out whereby
NiH.cl9 cells were incubated with antagonist Ac-6-FP (1b) for 1
hour before the addition of 5-OP-RU (3a) or 3b and 6C2 cells
for 24 hours. The expression of CD137 was then determined by
flow cytometry. Here, a significant reduction in MAIT cell
activity for both 3a and 3b was observed when the cells were
pre-incubated with antagonist 1b (Fig. 5A), thereby indicating
that analogue 3b binds the known MR1-MAIT TCR binding site.
Next, we sought to directly compare the agonistic profile of 3b
to 5-OP-RU (3a). A dose-response assay was undertaken where
6C2 cells were cultured for 24 hours on NiH.cl9 cells in the
presence of 3a or 3b at titrated concentrations. Hydroxyethyl
agonist 3b was found to have a similar activation profile to 3a
(Fig. 5B), with similar EC50 values of 0.4 µM and 0.6 µM being
observed for 3b and 3a, respectively. Taken together, these
results demonstrate that hydroxyethyl analogue 3b is a new,
fully water soluble, MAIT cell agonist with potent activity and a
functional profile similar to the current best known MAIT cell
To better understand our observations that the hydroxy-
ethyl (3b) and hydroxypropyl (3c) analogues are MAIT cell
agonists, whereas the hydroxypentyl (3d), propyl (3f) and
pentyl (3g) analogues act as antagonists, these structures were
docked
Fig. 6. MAIT cell inhibition assay for analogues 3d, 3f and 3g. NiH.cl9 cells were
incubated with Ac-6-FP (1b), 3d, 3f or 3g at 100 µM, 10 µM, 1 µM, 0.1 µM and 0
µM for 1 h before the addition of 5-OP-RU (10 µM) and 6C2 cells for 24 h. MFI of
CD137 expression was measured after gating on viable TCRβ+ cells. Data
represents two independent experiments (n = 3).
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J. Name., 2013, 00, 1-3 | 3
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into the MR1-MAIT TCR binding site and their binding and Ac-6-FP. Using computational docking studies, it was
View Article Online
DOI: 10.1039/D0CC00247J
interactions were compared to those observed in the crystal determined that the agonistic compounds contact the MAIT
structure of MR1-5-OP-RU complexed with a human MAIT TCR TCR directly, while the antagonistic analogues lack such
(Fig. 7). For all compounds, the formation of a Schiff base interactions. Thus, for the first time, we have demonstrated
between the ketone on the 5-position of the uracil ring and that changing the 6-amino side chain of MR1 ligands can
Lys43 of MR1 caused the uracil ring to sit in a slightly different regulate MAIT cell agonistic or antagonistic activity.
orientation to 5-OP-RU (3a, yellow). The uracil ring of 5-OP-RU Additionally, we have identified a new class of MAIT cell
(3a) showed van der Waals interactions with Thr34, Ser24 and antagonists which are structurally distinct from known
Arg9, and aromatic interactions with Tyr7 of MR1. These antagonist Ac-6-FP. By providing further insight into the
interactions were also observed for 3b, 3d, 3f and 3g, thereby structural requirements for MR1-MAIT TCR binding, this work
allowing them to occupy the MR1-MAIT TCR binding site. Here, will undoubtedly aid in the development of therapeutics
the 2-hydroxyethylamino (pink) and 3-hydroxypropylamino targeting the MR1-MAIT cell signalling axis.
(blue) side chains of agonists 3b and 3c, respectively, were
oriented towards, and formed interactions with, Tyr95 of the
Conflicts of interest
MAIT TCR (Fig. 7A). The hydroxypentyl side chain (green) of 3d
was oriented in the same direction as the ribityl tail of 5-OP-RU
(3a, yellow), however, the hydroxy group was located further
away from Tyr95 and could not interact with the MAIT TCR
(Fig. 7B). Similarly, the lack of hydroxy groups in the propyl (3f,
black) and pentyl (3g, orange) analogues prevented
interactions with Tyr95, with the side chains oriented away
from the MAIT TCR. Thus, in summary, although all the
aminouracils bound MR1, antagonists 3d, 3f and 3g are unable
to interact with the MAIT TCR, whilst agonists 5-OP-RU (3a), 3b
and 3c interact directly with Tyr95 of the MAIT TCR. Previously,
we observed that for glyco-analogues of 5-OP-RU, ligand
activity depended on the number of interactions between the
sugar backbone and Tyr95,12 and that an interaction between
the 2′-hydroxy group on the sugar backbone enhanced MAIT
cell activation. In this study, we show that an interaction
between the 2′-OH of agonist 3b and Tyr95 is favourable for
MR1-MAIT TCR binding, and sufficient for potent agonistic
activity.
Herein, a series of hydroxyalkyl and alkyl analogues of 5-
OP-RU were synthesised and their MAIT cell activity assessed.
Hydroxyethyl analogue 3b exhibited MAIT cell activation that
was similar to the most potent agonist 5-OP-RU (3a). MR1
surface expression and dose-response assays revealed that 3b
also has a similar functional profile to 5-OP-RU (3a). However,
an advantage of 3b over 3a is that it can be synthesised easily
in three steps from commercially available 2-aminoethanol.
Furthermore, the hydroxypentyl (3d), propyl (3f) and pentyl
(3g) analogues of 5-OP-RU were found to exhibit antagonistic
activity similar to Ac-6-FP, with competitive inhibition assays
confirming that 3b, 3d, 3f and 3g bind the same site as 5-OP-
RU
There are no conflicts to declare.
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M. Ler, W. Xu, J. Y. W. Mak, L. Liu, P. V. Bernhardt, D. P. Fairlie.
Chemistry, 2019, 25, 15594-15608.
14 K. Li, C. K. Vorkas, A. Chaudhry, D. L. Bell, R. A. Willis, A. Rudensky, J.
D. Altman, M. S. Glickman and J. Aubé, PLOS ONE, 2018, 13,
e0191837.
15 T. Yokosuka, K. Takase, M. Suzuki, Y. Nakagawa, S. Taki, H.
Takahashi, T. Fujisawa, H. Arase and T. Saito, J. Exp. Med., 2002, 195,
991-1001.
Fig. 7. Docking of analogues 3b, 3c, 3d, 3f and 3g in the hMR1- MAIT TCR
complex (PDB: 4PJ7). A. Contacts between agonistic ligands containing side
chains: Hydroxyethyl (3b, pink) and hydroxypropyl (3c, blue) overlaid onto the
crystal structure of 5-OP-RU (3a, yellow). B. Contacts between antagonistic
ligands containing side chains: Hydroxypentyl (3d, green), propyl (3f, black) and
pentyl (3g, orange) overlaid onto the crystal structure of 5-OP-RU (3a, yellow).
Hydrogen bonds are shown as grey lines.
4 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/D0CC00247J
The 6-alkylamino side chain of aminouracil MR1 ligands controls MAIT cell agonistic or antagonistic
activity.