Discovery of Selective Inhibitors of Endoplasmic Reticulum Aminopeptidase 1

Abstract

ERAP1 is an endoplasmic reticulum-resident zinc aminopeptidase that plays an important role in the immune system by trimming peptides for loading onto major histocompatibility complex proteins. Here, we report discovery of the first inhibitors selective for ERAP1 over its paralogues ERAP2 and IRAP. Compound 1 (N-(N-(2-(1H-indol-3-yl)­ethyl)­carbamimidoyl)-2,5-difluorobenzenesulfonamide) and compound 2 (1-(1-(4-acetylpiperazine-1-carbonyl)­cyclohexyl)-3-(p-tolyl)­urea) are competitive inhibitors of ERAP1 aminopeptidase activity. Compound 3 (4-methoxy-3-(N-(2-(piperidin-1-yl)-5-(trifluoromethyl)­phenyl)­sulfamoyl)­benzoic acid) allosterically activates ERAP1’s hydrolysis of fluorogenic and chromogenic amino acid substrates but competitively inhibits its activity toward a nonamer peptide representative of physiological substrates. Compounds 2 and 3 inhibit antigen presentation in a cellular assay. Compound 3 displays higher potency for an ERAP1 variant associated with increased risk of autoimmune disease. These inhibitors provide mechanistic insights into the determinants of specificity for ERAP1, ERAP2, and IRAP and offer a new therapeutic approach of specifically inhibiting ERAP1 activity in vivo.

Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism

“…To critically test the role of the C404–C443 disulfide in coupling domain closure to enzymatic activation, we tested the effect of C404S and C443S mutation on allosteric activation induced by compound 3 (Supplementary Fig. S1 ), a previously characterized non-peptidomimetic small-molecule allosteric modulator 20 . Compound 3 activates ERAP1 for Leu-AMC hydrolysis; the activation has been hypothesized to act by stabilizing the closed conformer 20 , and this is confirmed below.…”

“…S1 ), a previously characterized non-peptidomimetic small-molecule allosteric modulator 20 . Compound 3 activates ERAP1 for Leu-AMC hydrolysis; the activation has been hypothesized to act by stabilizing the closed conformer 20 , and this is confirmed below. The C404S and C443S mutations abrogated the allosteric activation effect of compound 3 (Fig.…”

“…Compound 3 docks in a pocket at the junctions of domains II, III, and IV formed by the closed conformation of ERAP1 (Fig. 4a ), and acts as an allosteric activator of ERAP1 by increasing hydrolysis activity towards the dipeptide substrate Leu-AMC 20 (Fig. 4b ).…”

“…These findings, along with data showing preferential hydrolysis of substrates longer than eight amino acids 16 , support a model that an optimal length substrate peptide simultaneously binds the active site and a distal regulatory site that activates catalysis 12 , 17 . Binding sites within the overall substrate-binding cavity have been identified for two small-molecule ERAP1 allosteric modulators 19 , 20 , one of which overlaps with the binding site identified for the C-terminus of peptides with a hydrophobic terminal residue 21 , 22 , but how occupancy of these sites induces activation, and whether activation is related to domain reorganization, remains unknown.…”

Investigating the phosphinic acid tripeptide mimetic DG013A as a tool compound inhibitor of the M1-aminopeptidase ERAP1

“…However, to interpret data from chemical probes, their cellular permeability and protein selectivity profiles must be robustly established. Several chemotypes have been reported as inhibitors of ERAP1; 13 , 14 , 15 however, only the phosphinic acid, transition-state mimic compounds, exemplified by the tripeptide mimetic DG013A 1 ( Scheme 1 ), have demonstrated sub-micromolar affinity for the recombinant protein. 16 As part of a recent study into the role of ERAP1 in cancer, we now report our investigation into the use of 1 as tool compound to study ERAP1 inhibition.…”