Discovery and Optimization of a Series of Benzofuran Selective ERAP1 Inhibitors: Biochemical and in Silico Studies

Article abstract of DOI:10.1021/acsmedchemlett.1c00235

ERAP1 is a key aminopeptidase involved in peptide trimming before major histocompatibility complex (MHC) presentation. A single nucleotide polymorphism (SNP) in the ERAP1 gene can lead to impaired trimming activity and affect ERAP1 function. ERAP1 genetic variations have been linked to an increased susceptibility to cancer and autoimmune disease. Here, we report the discovery of novel ERAP1 inhibitors using a high throughput screening approach. Due to ERAP1 broad substrate specificity, the hit finding strategy included testing inhibitors with a range of biochemical assays. Based on the hit potency, selectivity, and in vitro absorption, distribution, metabolism, excretion, and toxicity, the benzofuran series was selected. Fifteen derivatives were designed and synthesized, the compound potency was improved to the nanomolar range, and the structure-activity relationship supported by modeling studies.

Full text of DOI:10.1021/acsmedchemlett.1c00235

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Letter
Discovery and Optimization of a Series of Benzofuran Selective
ERAP1 Inhibitors: Biochemical and In Silico Studies
Safia Deddouche-Grass, Cyrielle Andouche, Felix Bärenz, Célia Halter, Arnaud Hohwald,
Louison Lebrun, Nathalie Membré, Renaud Morales, Nicolas Muzet, Matthieu Poirot,
Morgane Reynaud, Véronique Roujean, Fabienne Weber, André Zimmermann, Rama Heng,*
and Nicolas Basse*
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ABSTRACT: ERAP1 is a key aminopeptidase involved in peptide trimming before major
histocompatibility complex (MHC) presentation. A single nucleotide polymorphism (SNP)
in the ERAP1 gene can lead to impaired trimming activity and affect ERAP1 function. ERAP1
genetic variations have been linked to an increased susceptibility to cancer and autoimmune
disease. Here, we report the discovery of novel ERAP1 inhibitors using a high throughput
screening approach. Due to ERAP1 broad substrate specificity, the hit finding strategy
included testing inhibitors with a range of biochemical assays. Based on the hit potency,
selectivity, and in vitro absorption, distribution, metabolism, excretion, and toxicity, the
benzofuran series was selected. Fifteen derivatives were designed and synthesized, the
compound potency was improved to the nanomolar range, and the structure−activity
relationship supported by modeling studies.
KEYWORDS: ERAP1, aminopeptidase, inhibitor, benzofuran, MHC, immunology
RAP1 (endoplasmic reticulum aminopeptidase 1) is a
Emultifunctional enzyme trimming amino acids from the
N-terminus of peptides and proteins.1 It is mostly known for
its endoplasmic reticulum (ER) role in generating MHC-I
presenting peptides. Here it acts as an antigenic peptide editor
Figure 2. Structures of short (L-Rho-(D)-Q) and long peptidic
substrates (L-Rho-Succ-FKARKF).
Table 1. Kinetic Parameters for Each Substrate Derived
from Fitting the Kinetics
compd
substrate
Km (μM)
Ki (μM)
a
LVAFKARKF
9
S3
L-Rho-(D)-Q
EFAPGNYPAL
L-Rho-Succ-KARKF
L-Rho-Succ-FKARKF
L-Rho-Succ-AFKARKF
31.8
100
3.7
3.1
2
335
S5c
S5b
S5a
812
151
563
a
Value from Evnouchidou et al.17
Received: April 20, 2021
Accepted: June 3, 2021
Published: June 4, 2021
Figure 1. Hit finding workflow.
https://doi.org/10.1021/acsmedchemlett.1c00235
© 2021 American Chemical Society
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Table 2. Benzofuran Hits Identified from the HTS
TSA ΔTm
(°C) @10
μM
hepatocyte stability
Clint
CYP 3A4
Inhib IC50
(μM)
ERAP1 (L-Rho-Succ-
compd FKARKF) IC50 (nM)
MS (EFAPGNYPAL)
(% inhib @10 μM)
Caco-2 PA2B
microsomal stability
LE
(10−7cm·s−1
)
Clint (μL·min−1·mg−1
)
(mL/h/106 cells)
a
b
M: >30;
c
1
377
0.32
ND
0.82
0.94
0.5
85
<20 (rat);
0.25
31 (mouse);
32 (human)
T: >30
a
b
M: >30;
c
2
698
0.31
53%
108
25 (rat);
ND
<20 (mouse);
33 (human)
T: >30
a
a
a
3
4
1620
2680
0.30
0.25
37%
10%
184
ND
ND
ND
ND
ND
ND
a
a
a
a
a
ND
ND
a
b
c
Not determined. Midazolam substrate for CYP3A4 inhibition assay. Testosterone substrate for CYP3A4 inhibition assay.
catalytic activity (Figure 1). The primary assay was a
fluorescence-based assay using a physiologically relevant
peptide length. ERAP1 trims peptides from up to 16 residues
to a 9-mer for MHC presentation.10 The primary assay was
used to run a high-throughput screen of almost 1 million
compounds. Active compounds were titrated and further
confirmed by an orthogonal mass spectrometric assay using a
previously described natural peptidic substrate.11 Hit series
were selected based on selectivity against ERAP2 and APN as
well as chemical structure attractivity. We report here the
chemical optimization of the benzofuran series.
Scheme 1. General Synthesis for 7-Benzofuran Amide
Variations
As the ERAP1 active site can accommodate a large variety of
peptide substrates and inhibitors, we decided to use multiple
biochemical assays to ensure ERAP1 inhibition. This should
allow us to reduce substrate-specific bias. The compound effect
on ERAP1 N-terminal trimming activity was measured in two
in vitro fluorescent based enzymatic assays. In addition to the
previously described N-terminal leucine short peptidic
substrate used to detect aminopeptidase activity,12 we
developed more natural-like peptidic substrates based on the
optimized sequence published by Evnouchidou et al.13 Its
synthesis is described in Supporting Information (SI) Figure
S1. ERAP1 trimming activity is sequence dependent; in
particular, the C-terminal residue of the peptides influences its
affinity.14 Small molecules could bind this regulatory site and
modulate ERAP1 activity. Long peptide substrates might be a
better tool to detect such inhibitory mode of action.
Fluorescence Assay Development. To measure catalytic
activity, we incorporated a rhodamine fluorophore in the P1
position to track peptidic cleavage. The rhodamine fluo-
rescence signal is quenched by the N-terminal leucine in the
P1 position and the rest of the peptidic sequence in P2′ after a
succinic linker (Figure 2). To optimize substrate length based
on enzymatic parameter, three long fluorescent peptides were
synthesized and evaluated. The three substrates showed similar
Km values in the low micromolar range. Measured Km values
are equivalent to the one obtained with the nonlabeled peptide
(Table 1). The main differences between the three substrates
are in the signal intensity and the inhibitory effect observed at
high substrate concentration in the plot of relative fluorescence
intensity versus substrate concentration (SI Figure S3). In
particular, for L-Rho-Succ-FKARKF, this inhibitory effect is
more notable and has been previously observed with peptidic
influencing the peptide repertoire displayed at the cell surface
to circulating CD8+ T cells and natural killer (NK) cells.2
ERAP1 is a polymorphic gene; SNPs are associated with
immune disorders, modulating the protein level and the
substrate specificity of the aminopeptidase.3 In addition, it has
been established that manipulating ERAP1 expression levels in
tumor cells can lead to increased antigenicity and tumor
rejection in mouse models.4 Therefore, the modulation of
ERAP1 activity by specific molecules is of therapeutic interest,
particularly in cancer.5 Humans possess nine M1 amino-
peptidases including ERAP1. They are characterized by the
presence of two conserved sequence motifs: a zinc binding
motif HEXXH-(X18)-E and the GXMEN exopeptidase motif.6
Aminopeptidase sequence homology represents a challenge to
the identification of selective ERAP1 inhibitors. The first
ERAP1 inhibitors described, such as DG013 or leucinethiol,
lack selectivity.7 More recent ERAP1 inhibitors are zinc
chelators like peptidic phosphonic molecules8 or more recently
sulfonamide, presenting a better selectivity profile.9 In this
report, we describe the identification and biological evaluation
of benzofuran derivatives as specific inhibitors of ERAP1. We
developed orthogonal biochemical assays to detect ERAP1
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Table 3. DG013 Reference Inhibitors and Benzofuran SAR in Position 7
a
Not determined.
substates.15 L-Rho-Succ-FKARKF substrate was selected for
the primary screen.
on ERAP1 (up to 3 μM IC50) in the long substrate fluorescent
assay L-Rho-Succ-FKARKF. Among all these first hits, 4000
compounds were tested in the orthogonal MS assay at 10 μM.
The selection was made to allow a significant representation of
all clusters. In this second set of results, many clusters were
found to be inactive on the MS assay, independently of the
measured IC50 in the primary fluorescent biochemical assay (SI
Figure S2A). Substrate-specific inhibition could be due to the
differences between their peptidic sequence and/or the small
molecule binding in regulatory pockets.18 Some other clusters
gave acceptable correlation between the inhibitory activities
observed in the orthogonal MS and fluorescent assays (SI
Figure S2B). To ensure a stronger impact on the
immunopeptidome, compounds with activity against both
substrates were prioritized. This filter combined with the
chemical structural attractivity and the Ligand Efficiency
metric criteria enabled us to reduce the number of clusters
of interest down to 19 (with 306 compounds).
In addition, to compare long versus short peptidic substrates
with the same fluorophore, we synthesized a short rhodamine
derived substrate L-Rho-(D)-Q. (D)-Q was introduced to
increase substrate solubility and the D-stereoisomer was chosen
as ERAP1 substrate specificity prevents cleavage of D-amino
acids and trims the N-terminus of peptides with a preference
for hydrophobic residues.14
RapidFire MS assay. To reduce any potential artifacts risk,
we used an orthogonal biochemical HPLC mass spectrometry
(MS) assay in the screening tree (Figure 1). The MS assay was
designed to follow the trimming activity of the natural
antigenic peptide EFAPGNYPAL.16
In addition, a biophysical thermal shift assay (TSA) was
used to confirm direct binding to ERAP1.
Hits Triage. After completion of the high throughput
screening (HTS), more than 6000 compounds, grouped in 215
different clusters, were identified as having inhibitory activity
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Table 4. Scaffold Substitution SAR
Figure 4. In silico docking site and docking pose of compound 11 in
an allosteric binding site described by Giatas et al.18 Blue surface
represents the hydrophobic pocket, and dotted lines represent
hydrogen bonds.
MS
ERAP2
IC50
(μM)
APN
IC50
(μM)
ERAP1 (L-Rho-Succ- (EFAPGNYPAL)
compd FKARKF) IC50 (μM)
IC50 (μM)
Chemistry and Structure−Activity Relationship (SAR)
Studies. From initial hits, compound 2 was resynthesized
from the available intermediate and its activity was confirmed.
The difference in the enzymatic activity of compounds 1 and 4
implied that the chlorophenyl moiety might be important for
the ERAP1 inhibition compared to the trifluoromethyl
substitution. We first investigated the possibility to modify
the carboxylic acid function, that could be responsible for high
intrinsic clearance observed on human hepatocyte, through
conjugation mechanisms of phase II metabolism. Analogues of
compounds 1 and 2 were synthesized, following the general
synthesis for 7-benzofuran amide variations (Scheme 1). A
similar synthesis was carried out by modifying the benzofuran
core for compounds 16−19 (see synthetic procedure in the
SI). After testing on the primary biochemical assay, we found
that neither the methyl ester (5) nor the carboxamide (6) was
able to maintain any activity. Classical bioisoster replacement
of the carboxylic acid with a tetrazole moiety (7) led to a 2.5-
fold increase in activity (Table 3).
2
0.698
0.034
1.027
0.120
1.760
0.151
15.26
ND
25.18
2.17
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
a
11
16
17
18
19
4.37
a
Not determined.
We then decided to initiate a SAR study around the amino
acid alpha substitution (compounds 8−14). These compounds
could be easily synthesized by introducing the commercially
available enantiopure amino acid, protected with a methyl ester
in the last steps.
The slight increase of affinity observed between hit
compounds 1 and 2 could be due to the more hindered and
lipophilic α-amino acid, which was confirmed with compounds
9−12 (Table 3). We were then able to obtain more than one
log increase in activity, with compounds as potent as the well-
described inhibitor DG013A. An interesting enantiomeric
effect was observed between the non-natural phenyl alanine
substitution (13) and the natural amino acid (14), with an
important increase of activity for the D-amino acid compared
to the L-amino acid. One example of a tertiary amide was made
(15), which proved to be quite detrimental for the activity,
when compared to the secondary amide counterpart (2).
In the course of our investigations, the fluorine atom in the
5-position demonstrated a positive effect on the compound
potency, compared to their nonsubstituted benzofuran match
pairs (1/16 and 11/17), and the 3-bromo substituted
derivatives (18 and 19) were also found to be almost as
potent as their nonsubstituted counterparts (16 and 17)
(Table 4). When the selectivity of these compounds was
assessed, they were found to be very selective over ERAP2 and
APN, showing no inhibition at the highest dose tested (Tables
Figure 3. Potent ERAP1 inhibitor in long substrate biochemical assay
showing similar inhibition potency on short substrate assay with
known zinc binder reference compounds activation, no effect, or trend
of inhibition at high doses on short substrate assays with benzofuran
compounds.
In parallel with the MS assay, these 306 compounds were
tested for ERAP1 binding capacities by TSA. Here, 130
compounds induced a shift of the ERAP1 Tm greater than 0.5
°C, including the benzofuran cluster (Table 2). Based on
biochemical selectivity against ERAP2 and APN, and early
ADME profiling, the benzofuran series was selected for further
investigation. The starting point identified (compounds 1−4)
displayed good biochemical potency on the primary assay and
trends on the MS assay and confirmed binding by TSA. In
addition, the ligand efficiency metric (>0.3) and the first
preliminary ADME data were suitable values as starting points
(Table 2).
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3 and 4). As a preliminary evaluation of the selectivity of the
benzofuran hits was 80-fold for compound 1, through the
optimization process selectivity was tested for some analogues
and for compound 11 a selectivity of almost a 1000-fold was
observed against APN and ERAP2.
While DG013A and leucinethiol showed similar potent
inhibition with both short and long substrates in the
fluorescent assays (Figure 3), the compounds from our hit
series returned in the short substrate fluorescent assay either as
low inhibitor, inactive compounds, or even as activators
(Figure 3). Small modifications on the R side chain amino acid
led to a drastic change of behavior, when comparing effects of
compounds 10 and 1. Compound 10 was identified as a clear
activator of ERAP1, whereas compound 1 showed almost no
activation or inhibition of ERAP1 in the tested range. Some
other compounds, such as compound 19, showed a minor
inhibition effect only at the highest dose.
These differences in behavior between the short and long
substrate assays could be due to a binding of our compounds
in an allosteric pocket, far from the catalytic site, and could also
explain the selectivity observed over ERAP2 and APN, as the
corresponding assays used short substrates (Arg-7-amido-4-
methyl coumarin and Ala-7-amido-4-methyl coumarin, respec-
tively).
substitution is moved away from the lipophilic pocket, unlike
its enantiomer compound 13 (SI Figure S4).
In conclusion, from a high throughput screen against
ERAP1, a series of benzofurans were identified and modified
to increase potency. The described inhibitors have a good
selectivity profile against close peptidase homologues.
Benzofurans represent an interesting chemotype worth
developing as potent ERAP1 inhibitors. This study confirmed
the potential of allosteric inhibition as suggested by docking
studies and biochemical behavior.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00235.
■
sı
Analytic experimental methods, synthetic procedures for
substrates and compounds 1−19, analytical data, and
biological and in silico methods (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Rama Heng − Evotec, 31036 Toulouse cedex, France;
Email: rama.heng@evotec.com
Nicolas Basse − Sanofi, 67080 Strasbourg, France;
orcid.org/0000-0002-0009-601X; Email: nicolas.basse@
sanofi.com
The results recently published by Stratikos et al.18 lend
support to our hypothesis, as they succeeded in cocrystallizing
a natural product modulator (GSK849), binding in a
regulatory site 25 Å away from the catalytic center. Similarly
to our benzofuran compounds, GSK849 shows remarkable
selectivity versus highly homologous aminopeptidases, can
inhibit the processing of a long substrate (a 15mer peptide
based on the SIINFEKL epitope) in a RapidFire MS based
assay, and can activate trimming of a short substrate using Leu-
AFC (a fluorinated derivative of Leu-AMC), in a fluorescent
enzymatic assay.
Authors
Safia Deddouche-Grass − Sanofi, 67080 Strasbourg, France
Cyrielle Andouche − Sanofi, 67080 Strasbourg, France
Felix Bärenz − Sanofi, D-65926 Frankfurt am Main,
Germany
Célia Halter − Domain Therapeutics, 67400 Illkirch, France
Arnaud Hohwald − Sanofi, 67080 Strasbourg, France
Louison Lebrun − Domain Therapeutics, 67400 Illkirch,
France
Nathalie Membré − Sanofi, 67080 Strasbourg, France
Renaud Morales − Sanofi, 67080 Strasbourg, France
Nicolas Muzet − Sanofi, 67080 Strasbourg, France
Matthieu Poirot − Sanofi, 67080 Strasbourg, France
Morgane Reynaud − Biosynex, 67400 Illkirch, France
Véronique Roujean − Sanofi, 67080 Strasbourg, France
Fabienne Weber − Sanofi, 67080 Strasbourg, France
André Zimmermann − Sanofi, 67080 Strasbourg, France
Docking studies were then carried out to see if our
benzofuran compounds could bind in the regulatory site
identified in the cocrystal structure of ERAP1 with
GSK849.19,20
All benzofuran compounds could show key interactions of
their carboxylate group with Arg 807 and Lys 685. The most
potent compound of the series (compound 11) fills the
lipophilic pocket the most efficiently, compared to the other
benzofuran analogues (Figure 4). The carboxylic moiety forms
hydrogen bonds with Lys685 and Arg807. The fluorine atom is
pointed toward Gln881 and Arg841. The para-chlorophenyl
moiety and the cyclohexyl are interacting with the hydrophobic
zones generated by a set of lipophilic residues including
Phe674, Leu677, Ile681, Leu733, Leu734, Val737, and Phe803.
The potential binding identified could be well superimposed
with the cocrystal structure with GSK849, showing a perfect
match between the oxygen atoms (between the two carboxylic
acids, or between the alcohol of GSK849 and the amide
function of the benzofuran) and in the lipophilic filling as
observed in the docking superposition (SI Figure S4). These
docking structures could also explain the loss of activity of the
methyl ester compared to the carboxylic acid (compound 2 vs
compound 5) or even the higher inhibitory effect of compound
13 compared to its enantiomer, compound 14. In order to
keep this key interaction between the carboxylic acid and Arg
807 and Lys 685, compound 14 needs to take another
conformation where the phenyl ring from the phenylalanine
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00235
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
ERAP1, endoplasmic reticulum aminopeptidase 1; ERAP2,
endoplasmic reticulum aminopeptidase 2; APN, amino-
peptidase N; MHC, major histocompatibility complex; SNP,
single nucleotide polymorphism; ADMET, absorption, dis-
tribution, metabolism, excretion, and toxicity; NK, natural
killer; SAR, structure−activity relationship; TSA, thermal shift
assay; Tm, melting temperature; MS, mass spectrometry;
CYP3A4, cytochrome P450 3A4; Rho, rhodamine; AMC,
amido-4-methyl coumarin; HTS, high throughput screening;
Inhib, inhibition; Conc, concentration; IC50, half maximal
inhibitory concentration; ND, not determined; DMF,
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