Targeting the Regulatory Site of ER Aminopeptidase 1 Leads to the Discovery of a Natural Product Modulator of Antigen Presentation

Abstract

ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that generates antigenic peptides and is an emerging target for cancer immunotherapy and the control of autoimmunity. ERAP1 inhibitors described previously target the active site and are limited in selectivity, minimizing their clinical potential. To address this, we targeted the regulatory site of ERAP1 using a high-throughput screen and discovered a small molecule hit that is highly selective for ERAP1. (4aR,5S,6R,8S,8aR)-5-(2-(Furan-3-yl)­ethyl)-8-hydroxy-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid is a natural product found in Dodonaea viscosa that constitutes a submicromolar, highly selective, and cell-active modulator of ERAP1. Although the compound activates hydrolysis of small model substrates, it is a competitive inhibitor for physiologically relevant longer peptides. Crystallographic analysis confirmed that the compound targets the regulatory site of the enzyme that normally binds the C-terminus of the peptide substrate. Our findings constitute a novel starting point for the development of selective ERAP1 modulators that have potential for further clinical development.

Common allotypes of ER aminopeptidase 1 have substrate-dependent and highly variable enzymatic properties

“…To better understand the mechanism behind the variation of trimming rates, we utilized a recently developed assay that is suitable for Michaelis-Menten analysis of peptide trimming and follows the trimming of the N-terminal residue of a 9mer antigenic epitope with the sequence YTAFTIPSI (31). Using this assay we calculated kcat and KM for each ERAP1 allotype ( Figure 3C and Table 3).…”

“…DG013A is a transition state analogue that targets the active site of the enzyme with high potency (27). GSK849 is a natural product that targets the regulatory site of ERAP1 and while is an apparent activator for small substrates, it inhibits peptide hydrolysis by interfering with C-terminus recognition (31). DG013A was able to inhibit all 10 allotypes with high potency (pIC50 between 7.2 and 7.6) ( Figure 5A).…”

“…Reactions were stopped after 60 minutes incubation by addition of 25 μL of 0.75 % TFA in water containing 5 μM Ac-YTAFTIPSI as internal standard. Mass signal intensities corresponding to the product (TAFTIPSI) and internal standard (Ac-YTAFTIPSI) were measured on a Rapidfire autosampler (Agilent) equipped with a C18 solid phase cartridge, coupled to a Sciex 4000 Q-trap MS (AB Sciex), using Multiple reaction monitoring (MRM), as described previously (31). Integrated product intensity signals were normalized to the respective internal standard intensity signals, before conversion to product concentration using a TAFTIPSI standard curve.…”

“…The corrected intensity data were used to calculate % turnover, from which product concentration was calculated. Data were normalized to % enzymatic activity between high (uninhibited) and low (100 μM DG13) controls and fitted to a 4 parameter logistic expression as described previously (31).…”

IRAP Inhibitors: M1-Aminopeptidase Family Inspiration

“…By analogy, Liddle et al recently described an allosteric ligand for ERAP1 (23) that activates hydrolysis of small substrates (such as Leu-AMC) while inhibiting cleavage of longer substrates by competing with the extended peptide binding site such as the antigen precursor, YTAFTIPSI. It is postulated to achieve this by stabilizing the dynamics of active site residues and/or facilitating conformational change to a partially closed, more active conformation (Liddle et al, 2020).…”