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

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.

ERAP1 (endoplasmic reticulum aminopeptidase 1) is a multifunctional 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 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 aminopeptidases 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 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.

Figure 1.

Hit finding workflow.

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 fluorescence 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 K_m values in the low micromolar range. Measured K_m 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 substates.¹⁵ L-Rho-Succ-FKARKF substrate was selected for the primary screen.

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)
	LVAFKARKF	9a
S3	L-Rho-(D)-Q	31.8	335
	EFAPGNYPAL	100
S5c	L-Rho-Succ-KARKF	3.7	812
S5b	L-Rho-Succ-FKARKF	3.1	151
S5a	L-Rho-Succ-AFKARKF	2	563

^aValue from Evnouchidou et al.¹⁷

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 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 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 T_m 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).

Table 2. Benzofuran Hits Identified from the HTS.

compd	ERAP1 (L-Rho-Succ-FKARKF) IC50 (nM)	LE	MS (EFAPGNYPAL) (% inhib @10 μM)	TSA ΔTm (°C) @10 μM	Caco-2 PA2B (10–7cm·s–1)	microsomal stability Clint (μL·min–1·mg–1)	hepatocyte stability Clint (mL/h/106 cells)	CYP 3A4 Inhib IC50 (μM)
1	377	0.32	NDa	0.82	85	<20 (rat); 31 (mouse); 32 (human)	0.25	M: >30;b T: >30c
2	698	0.31	53%	0.94	108	25 (rat); <20 (mouse); 33 (human)	NDa	M: >30;b T: >30c
3	1620	0.30	37%	0.5	184	NDa	NDa	NDa
4	2680	0.25	10%	NDa	NDa	NDa	NDa	NDa

^aNot determined.

^bMidazolam substrate for CYP3A4 inhibition assay.

^cTestosterone substrate for CYP3A4 inhibition assay.

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).

Scheme 1. General Synthesis for 7-Benzofuran Amide Variations.

Table 3. DG013 Reference Inhibitors and Benzofuran SAR in Position 7.

^aNot 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 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.

Table 4. Scaffold Substitution SAR.

compd	ERAP1 (L-Rho-Succ-FKARKF) IC50 (μM)	MS (EFAPGNYPAL) IC50 (μM)	ERAP2 IC50 (μM)	APN IC50 (μM)
2	0.698	15.26	>30	>30
11	0.034	NDa	>30	>30
16	1.027	25.18	>30	>30
17	0.120	2.17	>30	>30
18	1.760	>30	>30	>30
19	0.151	4.37	>30	>30

^aNot determined.

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.

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.

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, respectively).

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.

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 substitution is moved away from the lipophilic pocket, unlike its enantiomer compound 13 (SI Figure S4).

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.

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.

Glossary

Abbreviations

ERAP1

endoplasmic reticulum aminopeptidase 1

ERAP2

endoplasmic reticulum aminopeptidase 2

APN

aminopeptidase N

MHC

major histocompatibility complex

SNP

single nucleotide polymorphism

ADMET

absorption, distribution, metabolism, excretion, and toxicity

NK

natural killer

SAR

structure–activity relationship

TSA

thermal shift assay

T_m

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

dimethylformamide

NBS

N-bromosuccinimide

HATU

hexafluorophosphate azabenzotriazole tetramethyl uronium

DIEA

N,N-diisopropylethylamine

HPLC

high performance liquid chromatography

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00235.

• Analytic experimental methods, synthetic procedures for substrates and compounds 1–19, analytical data, and biological and in silico methods (PDF)

The authors declare no competing financial interest.