A novel high throughput virtual screening protocol to discover new indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors

Article abstract of DOI:10.1248/cpb.c16-01010

Indoleamine 2,3-dioxygenase 1 (IDO1) plays an important role in the immune escape of tumors and has emerged as a promising target for cancer immunotherapy. Despite its potential in immuno-oncology, very few chemotypes have been reported to date. Here, we disigned a novel high throughput virtual screening (HTVS) cascade protocol, combining both pharmacophore modeling and molecular docking and it was employed to query commercially available compounds to identify novel inhibitors. Among the 23 compounds selected for the in vitro IDO1 inhibitory activity assay, five compounds exhibit greater than 20% inhibition at a test concentration of 10μM, with two compounds having an IC₅₀ value of 23.8 and 8.8μM, respectively. The novel scaffold together with a ligand efficiency of 0.28 kcal/mol per heavy atom makes both compounds as suitable starting points for future chemistry elaboration. Our HTVS protocol was validated and could be employed in discovery of IDO1 inhibitors.

Full text of DOI:10.1248/cpb.c16-01010

714
Chem. Pharm. Bull. 65, 714–717 (2017)
Vol. 65, No. 8
Regular Article
A Novel High Throughput Virtual Screening Protocol to Discover New
Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors
,b
,a
Haojie Xu,a Yunlong Song,* and Qing Yang*
a Department of Pathogenobiology, College of Basic Medical Sciences, Jilin University; Changchun, Jilin 130021,
b
China: and School of Pharmacy, Second Military Medical University; Shanghai 200433, China.
Received December 25, 2016; accepted May 8, 2017
Indoleamine 2,3-dioxygenase 1 (IDO1) plays an important role in the immune escape of tumors and
has emerged as a promising target for cancer immunotherapy. Despite its potential in immuno-oncology,
very few chemotypes have been reported to date. Here, we disigned a novel high throughput virtual screen-
ing (HTVS) cascade protocol, combining both pharmacophore modeling and molecular docking and it was
employed to query commercially available compounds to identify novel inhibitors. Among the 23 compounds
selected for the in vitro IDO1 inhibitory activity assay, five compounds exhibit greater than 20% inhibition
at a test concentration of 10µM, with two compounds having an IC50 value of 23.8 and 8.8µM, respectively.
The novel scaffold together with a ligand efficiency of 0.28 kcal/mol per heavy atom makes both compounds
as suitable starting points for future chemistry elaboration. Our HTVS protocol was validated and could be
employed in discovery of IDO1 inhibitors.
Key words indoleamine 2,3-dioxygenase 1; inhibitor; immuno-oncology; virtual screening
Tumor escape is a hallmark of cancer, which brings many col combining both pharmacophore modeling and molecular
difficulties and troubles in the cancer therapy.1) Indoleamine docking with the help of many basic studies after 2012 such
2,3-dioxygenase 1 (IDO1) has been proved to be closely asso- as structure–activity relationship (SAR), binding modes and
ciated with the immune escape of cancer.2,3) IDO1 is a mono- fresh released crystal structure. Sucessfuly, we report on the
meric 45kDa extrahepatic heme-containing dioxygenase. It is discovery of two novel IDO1 inhibitors by our HTVS protocol
the rate-limiting enzyme in the kynurenine pathway that plays (Fig. 2).
an essential role in tumor escape.4) While IDO1 is silent in
most issues, it is highly up-regulated to acquire peripheral tol-
Experimental
Processing and Preparation of Protein The X-ray crys-
erance, e.g., tolerance to placenta.5) Many studies demonstrate
that the abnormal high level of IDO1 is found in patients with tallographic structural details of the indoleamine 2, 3-dioxy-
quite a few carcinomas, such as ovarian carcinoma, hepatocel- genagse 1 in complex with NLG919 analogue with a resolu-
lular carcinoma, invasive cervical carcinoma, metastatic mela- tion factor of 2.21Å (PDB code: 5EK3) was retrieved from
noma, non-small cell lung carcinoma and metastatic prostate RCSB Protein Data Bank (PDB).15) The 5EK3.pdb file was
carcinoma.6) Apart from cancer, IDO1 has been connected to imported to Discovery Studio 3.0 Client (DS 3.0) (Accelrys
common diseases such as Alzheimer’s disease and melancho- Inc., San Diego, CA, U.S.A.) for the process to ensure that the
lia.7)
retrieved structure was reliable and qualitatively considerable
Design of selective and highly active IDO1 inhibitors has for further in silico studies. The missing hydrogen atoms were
attracted increasing interest. The first-generation inhibitors
were tryptophan and indole derivatives, with 1-methyl-L-tryp-
tophan (1-MT) having an IC50 value of 380µM identified in
1991.8,9) The 1-MT has been used as a tool compound in many
studies involving IDO1 and immunotherapy. However, little
progress has been made on the tryptophan and indole deriva-
tives, due to their poor cellular activity.3) IDO inhibitors from
natural sources were identified to show moderate inhibitory
activities, such as Norharman Tryptanthrin, a natural product
from the Chinese medicinal plants. Its derivatives can reach
nM inhibitory activity on IDO1, and suppress tumor growth
on Lewis lung cancer (LLC) tumor-bearing mice.10) The most
promising IDO1 inhibitors are Epacadostat11) and NLG919,
both advanced in clinical trials (Fig. 1).
Despite the intensive interest on IDO1 inhibitors, only few
chemotypes have been reported to date.12) Although there were
many basic studies on the IDO1 and IDO1 inhibitors, the stud-
ies on high throughput virtual screening (HTVS) for IDO1
inhibitors remained very few and most of them were done
before 2012.13,14) Here, we designed an effective HTVS proto-
Fig. 1. Known IDO1 Inhibitors
*To whom correspondence should be addressed. e-mail: ylsong@smmu.edu.cn; yangq@jlu.edu.cn
© 2017 The Pharmaceutical Society of Japan

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Reagents and conditions: (i) NaHCO3, BrCN, Et2O, r.t., 36h, 56%; (ii) NH4Cl,
NaN3, DMF, 90°C, 5h, 83%; (iii) HCl, NaNO2, H2O, 4°C, 0.5h; (iv) Zn, HOAc,
H2O, 10°C, 2h, 24% (2 steps); (v) EtOH, r.t., 1h, 88%.
Chart 1. Synthetic Route of Compound 9
ADMET_BBB_Level=5 or ADMET_Absorption_Level=3
would be eliminated by ligand filter. Only the compounds pass
Lipinski’s Rule of five would be retained for the following
investigation.
Fig. 2. Flowchart of the Virtual Screening Cascade Protocol
corrected and the water molecules were removed. We also
Molecular Docking CDOCKER module of DS 3.0 was
built loops and assigned suitable protonation states at pH 7.4 applied for docking studies. Protein and ligands were prepared
and minimized the energy of the structure and so on.
as described above. A 15Å radius sphere was defined around
Preparation of Ligands Compounds in Specs database the bounded ligand to compose the active site of the IDO1
were selected in the in silico study, since it is open-accessible protein.
and the compounds could be purchased for further in vitro
Enzyme-Based IDO1 Inhibition Assay The enzymatic
experiment easily. A slice of reported active ligands were inhibition assays were performed as described by Su-ying Wu
selected for pharmocophore modeling and investigating their with some minor modifications.15) Briefly, 50nM of IDO1 and
interactions with the IDO1 protein. All the ligands were pre- required concentration of compounds were added to 100µL
pared by using DS3.0 Client and were in a ready-to-dock 3D of standard assay medium, which includes 50mM potassium
format.
phosphate buffer (pH 6.5), 10mM ascorbic acid (neutralized
Generation of Pharmacophore Pharmacophore model- with 1M NaOH solution), 10µM methylene blue, 100g/mL cata-
ing is an effective approach to discover original and novel lase, 200µM L-tryptophan in 96-well black plate. The prepared
scaffolds for a particular target. There are several ways to reaction was incubated at 37°C for 30min. To stop, 50µL 30%
generate pharmacophores automatically in DS3.0, such as (w/v) trichloroacetic acid was added to the reaction. The mix-
from bioactive conformation, from a receptor–ligand complex, ture was incubated for 30min at 52°C, and then centrifuged at
from a set of ligands and so on. In view of the huge distinct of 12000×g at room temperature for 10min. One hundred micro-
the reported active scaffolds, pharmacophores were generated liters supernatant was mixed with 100µL Ehrlich’s reagent.
by four structurally distinct and potent IDO1 inhibitors11,15,16) The amount of kynurenine production from reaction was
(Fig. 1) and modified to search database. Due to the short measured by the emission of fluorescence at 480nm using a
bonding distances between the ligands and the neighboring Victor 2 multi-label reader (PerkinElmer, Inc.). The inhibition
residues and the heme of the IDO1 active site, excluded vol- percentage was determined as [100−(A/B) 100]/100, where A
umes were created manually to avoid potential collisions and was the IDO1 activity with the test compounds added to the
eliminate unsatisfactory compounds.
protein and B was the absence of the test compounds.
Database Search Based on Pharmacophore Model The
Chemisitry Compound 9 was resynthesized according to
prepared ligands in specs datebase were used in the compound Chart 1.
search. It has been shown that direct coordination to the heme
iron is vital to the IDO1 inhibitory activity, so hydrogen bond
acceptor was set as a required feature. The other parameters
Results and Discussion
The four structurally distinct and potent IDO1 inhibitors
in protocol were set as default. Molecules which fit value >3.5 (Fig. 1) were used to generate a 3D pharmacophore model,
were retrieved as candidate compounds.
which briefly consists of two hydrophobic regions, one hydro-
ADMET Prediction and Lipinski’s Rule of Five The gen bond acceptor, and one hydrogen bond donor (Fig. 3). In
failure of many drug candidates in clinical trials can be addition, excluded volumes were added manually to avoid ste-
attributed to poor ADMET (absorption, distribution, me- ric collisions, upon inspection of the crystallographic structure
tabolism, excretion, toxicity) properties. Computer-aided of IDO1 in complex with an NLG919 analogue (PDB code:
ADMET prediction allows us to eliminate compounds with 5EK3) (Fig. 3E). It has been shown that direct coordination
unfavorable ADMET properties early to avoid expensive to the heme iron is vital to the IDO1 inhibitory activity, so
development costs. DS 3.0 ADMET was employed in our hydrogen bond acceptor was set as a required feature when
study. Compounds with the value of ADMET_Solubility=0, we screened datebase The original Specs compounds library

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Chem. Pharm. Bull.
Vol. 65, No. 8 (2017)
Fig. 3. The Chemical Features of the Best Pharmacophore Hypothesis Aligned with (A) Compound 1, (B) Compound 2, (C) Compound 3, (D) Com-
pound 4 with Hydrogen Acceptor Indicated as Green, Hydrogen Acceptor Indicated as Pink and Hydrophobic Areas Indicated as Cyan, (E) Hypothesis
with Excluded Volumes Indicated as Grey
Table 1. Activity of the Five Novel IDO1 Inhibitors Identified by Virtual
Screening
Fig. 4. Predicted Binding Modes of Compounds 8 (A) and 9 (B) in the
Active Site of IDO1
The coordination to heme iron was highlighted by a blue dash line and hydrogen
bonds were shown in red dash lines.
selected for in vitro IDO1 inhibitory assay.
contains over 200000 molecules. The generated 3D pharma-
The enzymatic inhibition assay was performed as previ-
cophore model efficiently reduces the number to 19537. A ously reported15) with minor modifications. Among the 23
recent analysis of the attrition of drug candidates from four compounds tested at a single concentration of 10µM, five
major pharmaceutical companies indicates a link between the exhibited greater than 20% inhibition (Table 1, Table S1). The
physicochemical properties of compounds and clinical failure IC50 values of compounds 8 and 9 were further determined
due to safety issues.17) As such, compounds with violation of by a 7-dose response measurement. Notably, compound 9
Lipinski’s Rule of 5 were removed. Moreover, compounds showed an IC50 value of 8.8µM with a ligand efficiency of
with poor predicted ADMET properties were further re- 0.28kcal/mol per heavy atom.
moved. The 12143 passed compounds were then subject to
The interaction of both compounds 8 and 9 with IDO1 is
molecular docking by CDOCKER. The top 200 compounds characterized by the direct coordination to the Heme iron, a
ranked by the docking energies were visually inspected for typical feature among IDO1 inhibitors (Fig. 4). Compound 8
the detailed molecular interactions. As mentioned early on, was the first IDO1 inhibitors to bind the heme iron with the
only compounds capable of forming a direct coordination with nitrate directly. Compound 8 is further hydrogen bonded to
the heme in the active site were kept. Further accounting for Ser167 side chain by its nitro as an acceptor, to Ala264 back-
structural novelty and diversity, finally 23 compounds were bone amide by its sulfonamide oxygen as an acceptor, and

Vol. 65, No. 8 (2017)
Chem. Pharm. Bull.
717
to Gly262 backbone oxygen by its sulfonamide nitrogen as a to Q.Y.), and the Health and Family Planning Commission of
donor. The phenyl ring attached with the nitro group estab- Jilin Province (No. 2014Z068 to Q.Y.).
lishes a π–π stacking interaction with Phe163 side chain (Fig.
4A). The IDO1 inhibitors with tetrazole group like compound
Conflict of Interest The authors declare no conflict of
9 remain very few, while it is the first time to discover a interest.
compound with the naphthalene group bound to tetrazole. The
naphthalene of compound 9 forms not only establishes a π–π
Supplementary Materials The online version of this ar-
stacking interaction with Phe163 side chain, but aslo it forms ticle contains supplementary materials.
hydrophobic interactions with the surrounding hydrophobic
residues (Fig. 4B).
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In summary, the subject of this manuscript is to design a
high throughput virtual screening cascade protocol combin-
ing both pharmacophore modeling and molecular docking to
search for potential IDO1 inhibitors. We have designed an
effective HTVS protocol with the help of many basic studies
after 2012 such as SAR, binding modes and fresh released
crystal structures. We successfully identify two novel IDO1
inhibitors of moderate activity, and 5 out of 23 compounds
displayed an inhibitory potency >20% at a concentration of
10 µM meaning that our protocol was the most effective virtual
screening protocol for IDO1 inhibitors up to date. Given their
structural novelty and moderate ligand efficiency, compounds
8 and 9 may serve as interesting starting points for future
chemistry elaboration in the rising field of immuno-oncology.
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Acknowledgments Support for the study was provided
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