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.

Distinct modulation of cellular immunopeptidome by the allosteric regulatory site of ER aminopeptidase 1

“…Although ERAP1 inhibitors targeting the active site have been developed 17 , recurring limitations with selectivity versus other aminopeptidases in the family due to the conservation of active site pharmacophores have raised concerns about their clinical usefulness. As an alternative, allosteric inhibitors are being explored [18][19][20] . Due to its mechanistic relevance, of particular interest are compounds that target a regulatory site in ERAP1 that accommodates the C-terminus of some peptides and helps self-activate their trimming 26 .…”

“…These would include partial target engagement by the inhibitor and possible off-target effects by either the inhibitor or the KO. Furthermore, we have not addressed whether other chemical scaffolds that target the regulatory site have the same effects 20 . In addition, little is known about how the inhibitor affects the kinetics of antigen presentation.…”

“…Due to its roles in antigen processing, ERAP1 is currently an emerging target for cancer immunotherapy and HLA-associated autoimmunity 15,16 . Several inhibitors have been developed 17 including inhibitors that target allosteric sites with enhanced selectivity [18][19][20][21] , although no clinical results have been reported yet.…”

ERAP1 Controls the Interaction of the Inhibitory Receptor KIR3DL1 With HLA-B51:01 by Affecting Natural Killer Cell Function

“…Leu-SH is a potent inhibitor of ERAP1 activity and it has been successfully used to reproduce the effects of genetic ERAP1 suppression ( 28 ) albeit it is not ERAP1 specific. A novel class of more selective inhibitors has been recently described ( 44 – 47 ). These new compounds are effective in targeting in vitro ERAP1 inside the ER at the nmol/L level, and modulate cytotoxic T cell responses, suggesting their potential use for pharmacologic manipulation of NK cell and T cell antitumor activity.…”

Phosphinic Peptides as Tool Compounds for the Study of Pharmacologically Relevant Zn-Metalloproteases

“…Interestingly, the central region of DG055 was found disordered which suggests a conformational freedom that may have important implications in the catalytic mechanism. Notably, this interaction between the peptides’ C-terminal moieties and the regulatory (or allosteric) site of ERAP1 revealed an extremely powerful alternative for the development of small-molecule allosteric inhibitors that may prove advantageous drug candidates as compared to their active-site counterparts. − …”