Discovery of the First Selective Nanomolar Inhibitors of ERAP2 by Kinetic Target-Guided Synthesis

Abstract

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a key enzyme involved in the trimming of antigenic peptides presented by Major Histocompatibility Complex class I. It is a target of growing interest for the treatment of autoimmune diseases and in cancer immunotherapy. However, the discovery of potent and selective ERAP2 inhibitors is highly challenging. Herein, we have used kinetic target-guided synthesis (KTGS) to identify such inhibitors. Co-crystallization experiments revealed the binding mode of three different inhibitors with increasing potency and selectivity over related enzymes. Selected analogues engage ERAP2 in cells and inhibit antigen presentation in a cellular context. 4 d (BDM88951) displays favorable in vitro ADME properties and in vivo exposure. In summary, KTGS allowed the discovery of the first nanomolar and selective highly promising ERAP2 inhibitors that pave the way of the exploration of the biological roles of this enzyme and provide lead compounds for drug discovery efforts.

Keywords: ERAP2; Isoform Selectivity; Medicinal Chemistry; Metalloenzymes; Protein-Templated Reactions.

Figure 1

Function of ERAP2 in antigen trimming leading to either epitope destruction or generation.

Figure 2

Design of the kinetic target‐guided synthesis (KTGS) of ERAP2 ligands. a) function of ERAP2 in antigen trimming leading to either epitope destruction or generation; b) Principle of KTGS; c) Clusters of azides 1  a–f (Z1–Z4); d) Clusters of alkynes (C1–C18); Misc. miscellaneous

Figure 3

Discovery of ligands of ERAP2 by kinetic target‐guided synthesis (KTGS). a) Flow chart for hit selection including KTGS, selectivity, confirmatory, dose‐response assay and resynthesis. Selection criteria and number of compounds are given for each step. Overall 1050 combinations of azide‐alkyne producing potentially 2100 triazoles including 1,4‐ and 1,5‐ regioisomers. b) Example of LCMS‐MS chromatograms from KTGS (top); un‐templated reaction mixture in buffer (middle); and 1,4‐triazole synthetic control (bottom) c) Dose‐response curves and structure for resynthesized hits 2  a–c; 3  a–b.

Figure 4

3  a in the active site of ERAP2 (PDB: 7NUP), 3  a in chain A of ERAP2 (a) or in chain C (b) of the asymmetric unit, at 3.1 Å resolution. ERAP2 is shown in cartoon representation in light gray. c) Structure of 3  a. 3  a (C: yellow, S: dark yellow, O: red, N: blue) and selected residues (C: green, S: dark yellow, O: red, N: blue) in close proximity to 3  a are shown as sticks. Zinc ion is shown as a magenta sphere. The GAMEN loop, pockets S1, S′1 and S′2 are indicated. Hydrogen‐bond interactions are represented as dashed lines. Images were generated using PyMOL^TM Molecular Graphics System v1.3.

Figure 5

4  c in the active site of ERAP2 (PDB: 7NSK). 8  c in chain A of ERAP2 (a) or in chain B (b) of the asymmetric unit, at 3.1 Å resolution. ERAP2 is shown in cartoon representation in light grey. 4  c (C: yellow, S: dark yellow, O: red, N: blue) and selected residues (C: green) in close proximity to 4  c are shown as sticks. Zinc ion is shown as a magenta sphere. The GAMEN loop, pockets S1, S′1 and S′2 are indicated. Hydrogen‐bond interactions are represented as dashed lines. Images were generated using PyMOL^TM Molecular Graphics System v1.3.

Figure 6

Compound 4  e in the active site of ERAP2 and superimposition with ERAP1 and IRAP. a,b) 8  e in chain B of the asymmetric unit of ERAP2 (PDB code 7HS0) at 3.2 Å; c,d) Superimposition of active‐site residues of ERAP2‐4  e (PDB code 7HS0, in green) with homologous c) ERAP1 (PDB code 6Q4R, purple) and d) IRAP (PDB code 5MJ6, cyan) with views similar to a) and b). ERAP2 is shown in cartoon representation in grey and 8e is shown in yellow sticks (C: yellow, S: dark yellow, O: red, N: blue). The zinc ion is shown as a magenta sphere. Selected residues of ERAP2/ ERAP1/IRAP in close proximity to 4  e are shown in sticks (C: green (ERAP2) or purple (ERAP1) or cyan (IRAP), O: red; N: blue). The GAMEN loop and approximate location of the specificity pockets S1, S1′, and S2′ are indicated. The black arrows indicate differences in key residue between the enzymes that could impact inhibitor binding. Hydrogen‐bond interactions are represented as dashed lines. Images were generated using PyMOL^TM Molecular Graphics System v1.3.

Figure 7

Selectivities towards related and distant metalloproteases. a) Selectivities towards ERAP1 and IRAP. IC₅₀ >100 μm; SI=selectivity index; IC₅₀ on IRAP/IC₅₀ on ERAP2; b) Selectivities of 4  d–f on a panel of metalloproteases; ND=not determined; c) Dose‐response curves for 4  e. IC₅₀ are mean of two to four independent measurements.

Figure 8

a–c) Target engagement of ERAP2 by 4  d–f in HEK cells using CETSA (Cellular Thermal Shift Assay), a) Representative western blots showing thermostable ERAP2 following indicated heat shocks in the presence of DMSO, 4  d, 4  e and 4  f at 30 μM. b) Quantification of thermostable ERAP2 obtained by three independent experiments (n=3) +/−SD. Stabilization of ERAP2 in the presence of 4  e is expressed as the ΔT _m. c) Dose‐dependent stabilization of ERAP2 by CETSA ITDRF (isothermal dose‐response fingerprint) at 56 °C; OC₅₀=23 μM concentration at which 50 % of ERAP2 is occupied by 4  d in cell. n=3, normalized to tubulin. d) Dose‐dependent effect of 4  d and 4  f, and Leucinethiol on SIINFEKL OVA‐derived antigen presentation in HEK cells at 24 h, from at least three independent experiments (n≥3) +/−SD. Asteriks shows statistical significance assessed by one‐way ANOVA, Post‐Hoc Dunnett for multiple comparisons with DMSO control, **** p<0.0001, *** p<0.001, ** p<0.01, ** p<0.05.