Transcript RICT RAF

Indole-pyridinyl-ethanones as novel inhibitors of indoleamine-2,3dioxygenase (IDO), a promising target for anti-cancer immunotherapy
Eduard Dolušić,a Pierre Larrieu,b Sébastien Blanc,c Laurence Moineaux,a Frédéric Sapunaric,d Jenny Pouyez,a
Delphine Colette,c Graeme Fraser,c Vincent Stroobant,b Luc Pilotte,b Didier Colau,b Jean-Marie Frère,d Bernard Masereel,a
Benoît Van den Eyndeb, Johan Wouters,a and Raphaël Frédéricka*
aDrug
design and Discovery Center, University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium ; bLudwig Institute for Cancer Research, Université Catholique de Louvain, 74 Avenue Hippocrate, 1200 Brussels, Belgium ;
cEuroscreen SA, 47 Rue Adrienne Boland, 6041 Gosselies, Belgium ; dCentre d'Ingénierie des Protéines, Université de Liège, Allée du 6 août, 4000 Liège, Belgium
E-mail : [email protected]
Introduction
Detailed binding analysis of 1a
 IDO is an extrahepatic, tryptophane (Trp)
metabolizing enzyme that catalyzes the initial and ratelimiting step along the kynurenine pathway.
0.7
0.6
1/Vmax
0.5
 Trp metabolism results in a local Trp depletion that
severely affects the proliferation of T lymphocytes and is
thereby profoundly immunosuppressive. Recently, the
team of Van den Eynde and colleagues showed that many
human tumours express IDO in a constitutive manner.1
IDO thus noticeably protects foreign cells against
immune rejection. Based on its implication in
immunosuppression, and particularly in tumors,2 IDO
clearly represents an attractive target for the development
of inhibitors.3
 The most frequently used IDO inhibitor,
1-methyl-tryptophane (1MT) has a reported
Ki of 34 µM. A phase 1 clinical trial is
currently investigating the safety, toxicity,
pharmacokinetics and efficacy of 1MT in
subjects with advanced malignancies.
Although more potent IDO inhibitors were
recently reported,4,5,6 only a limited number
of different scaffolds are available.
Representative inhibitors are derived from
the indole moiety (1-MT, brassinin and
methylthiohydantoine-tryprtophane (MTHTrp)), the naphtoquinone nucleus (annulin
B or C and exiguamine A), or the
phenylimidazole (PIM), a weak IDO
inhibitor.
O
0.4
0.3
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0.1
0
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-200
-150
-100
-50
0
50
-0.1
[Inhibiteur] µM
Possible pharmacomodulations for
preliminary SARs
OH
HN
S
S
 substitution of the indole in the 1-,
4and
5-position
through
introduction of small goups such as
halogens, methyl, methoxy or nitro
moieties
S
NH
NMe
NH2
 appraise the importance of the
ketone function through replacement
of the ethanone linker with an
ethylene, a hydroxyethylene or an
amide
O
N
Me
N
Me
MTH-Trp
1MT
O
O
N
H
Brassinin
O
OH
O
O
O
O
100
1a shows the best graphical match to
an uncompetitive inhibition mode
with a Ki value around 190 µM
O
OH
O
O
O
O
Annulin B
Annulin C
O
N
N
O
Chemical Synthesis and Biological Activities
NH2
O
O
HO
N
H
O
N
N
(d)
NH
Exiguamine A
PIM
Scheme 18. (i), LDA, THF /
hexanes, -78 to 0°C, 1 h; (ii) THF /
hexanes, 0°C to rt, 16-24 h; (iii),
H2NNH2, KOH, (CH2OH)2, mW, 1h
then aq. NH4Cl; (iv) HCO2NH4, Pd
black, MeOH, r. t, 3 days; (v)
SOCl2, D, 15 min; 3-aminopyridine,
DIPEA, THF, 0°C -> r. t, 30 min.
 The two three-dimensional structures of IDO, in complex with PIM and the cyanide ion
(CN¯), provide important results for the structure-based drug design of novel IDO
inhibitors.7 As the enzyme active site is now well known, it represents a very good tool to
undertake a direct approach of receptor-based drug discovery and design.
Pocket A
Pocket B
Biological activities of the newly designed IDO inhibitors
This preliminary SAR suggests, in
agreement with the docking study
reported above, that only rather small
and lipophilic groups such as halogens
are tolerated in the 5-position. Nmethylation of the indole is also
tolerated
In the present work, we performed a virtual screen to identify novel IDO inhibitors. From the
few scaffolds that display IDO inhibition, preliminary SARs were investigated around the
keto-indole scaffolds.
Virtual screening
(i)
(ii)
(iii)
(iv)
Replacement of the ethanone linker with
an ethylene (2) or even a hydroxylethylene (3) totally suppresses the IDO
inhibitory potency thus confirming the
importance
of
the
ketone
pharmacophore for IDO inhibition in
this series. Only its replacement with an
amide (4) afforded a compound that is
still active against IDO.
Lipinski-style rules
Goldscore > 50
Cscore ≥ 4, (iv)
Visual analysis and
commercial availability
Concerning the ability of these
derivatives to inhibit tryptophan
degradation in cells expressing murine
IDO, 1c, 1d and 1e are the most potent
derivatives with ~25% inhibition of
tryptophane degradation at 20 µM. With
the best hits being as potent as 1MT,
these data corroborate the potency of
this series for further structure-based
design effort.
The biological activity of the 39 compounds was evaluated using a colorimetric in vitro IDO
inhibition assay.1
CN
N
90
N
80
70
N
N
O
(1a)
60
50
40
30
20
10
0
zi
nc
zi 002
nc 7
6
zi 010 33
nc 8 1
1
zi 002 36
n 3 3
zi c00 77
nc 6 0 2
0 10
zi 030 34
nc 8 3
1
zi 001 57
nc 5 1
0 55
zi 432 63
n 3
zi c00 38
nc 3 3
6
zi 000 59
nc 8 7
3
zi 000 55
nc 7 9
9
zi 052 82
nc 3 0
4
zi 015 72
nc 9 5
9
zi 001 00
nc 1 1
7
zi 024 59
nc 9 5
2
zi 010 42
nc 7 9
6
zi 010 09
nc 5 0
2
zi 006 35
nc 1 5
7
zi 005 31
nc 7 8
1
zi 004 95
nc 4 6
8
zi 004 87
nc 4 9
0
zi 002 37
nc 7 6
6
zi 002 84
nc 7 7
6
zi 002 10
nc 7 6
5
zi 002 98
nc 3 6
7
zi 002 70
nc 7 2
2
zi 002 61
nc 4 3
0 47
zi 009 02
nc 9
zi 04 02
nc 0 1
9
zi 020 96
nc 2 6
3
zi 016 28
nc 8 5
9
zi 004 95
nc 4 2
8
zi 015 87
nc 9 9
9
zi 046 00
nc 6 1
7
zi 041 84
nc 0 0
0
zi 045 31
nc 6 2
6
zi 004 58
nc 4 4
8
zi 003 87
nc 8 9
7
zi 043 47
nc 2 8
08 33
76 83
31
02
1M
T
IDO inhibition (%) @ [I] = 100 µM
Discussion and conclusion
H
N
100
In conclusion, a virtual screen combining various filters including high-throughput docking was used to search
for new IDO inhibitors.9-10 From the 39 final compounds identified and assayed, six derivatives displayed an
inhibitory potency > 30% at a concentration of 100µM, 2 of them possessing more than 50% IDO inhibition at
this concentration. Detailed kinetics revealed an uncompetitive inhibition profile for the best hit 1a. Its binding
mode inside the IDO binding cleft was evaluated by means of docking and revealed essential features
responsible for the IDO inhibition potency in this series. Preliminary SARs around 1a corroborated this putative
binding orientation and support the interest of this series for further drug design effort.
Bibliography
1) Uyttenhove, C. et al. Nat Med 2003, 9, 1269-1274; (2) Löb, S. et al. Nat Rev Cancer 2009, 9, 445-452; (3) Macchiarulo, A. et al. Amino
Acids 2009, 37, 219-229; (4) Röhrig, U. et al. J Med Chem 2010, 53, 1172-1189; (5) Kumar, S. et al. J Med Chem 2008, 51, 4968-4977;
(6) Yue, E. et al. J Med Chem 2009, 52, 7364-7367; (7) Sugimoto, H. PNAS 2006, 103, 2611-2616; (8) Sundberg, R. J Org Chem 1978,
43, 4859-4865; (9) Dolusic, et al. Bioorg Med Chem 2011, 19(4), 1550-61; (10) Dolusic, et al. Eur J Med Chem 2011, 46, 3058-65
This work is supported in part by the FNRS and Biowin (Cantol : convention n°5678). RF is greatly indebted to the Belgian “Fonds
de la Recherche Scientifique – FNRS” for the award of a postdoctoral research grant.