Rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses

Abstract

Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2' pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway. molecular structure | adaptive immunity | major histocompatibility molecules | specificity | kinetics

Screening Identifies Thimerosal as a Selective Inhibitor of Endoplasmic Reticulum Aminopeptidase 1

“…8 The V max of the reaction was unaffected by the presence of the inhibitor, whereas K M was increased according to the expected inhibition, indicating that thimerosal acts as a competitive inhibitor of ERAP1 ( Figure 4A,B).…”

“…8,9 In an effort to discover novel, nonpeptidic scaffolds that inhibit ERAP1 as leads for preclinical development we applied a combination of structure-based, ligand-based, and knowledge-based virtual screening approaches, taking advantage of key structural characteristics revealed in the recent crystal structures of ERAP1 and ERAP2 and their complexes with 1 and 2, respectively. 8,10,11 Toward this goal, we compiled a library of more than 265,000 compounds from selected collections of chemical vendors that are focused on drug-likeness and structural diversity (Table S1). The library was enriched with the National Cancer Institute's diversity set II (1364 compounds) and the DrugBank database comprising 6590 FDA-approved and experimental small-molecule drugs 12 We also performed a 3D pharmacophore search against the purchasable subset of the ZINC database (more than 20 million compounds) 13 using the online interface of ZINCPharmer.…”

Stereoselective Synthesis of α-Amino-C-phosphinic Acids and Derivatives

“…In a similar way, Stratikos et al [84] reported the synthesis of the phosphinic tripeptide (R,S,S)-94, which was tested in the inhibition of the endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), involved in regulation of cytotoxic cellular responses. Thus, the reaction of N-Boc H-phosphinic acid analogue of homophenylalanine (R)-92 [72] with HMDS followed by the addition of ethyl isobutylacrylate and saponification of the ethyl ester, gave the C-phosphinate 93 in 72% yield, which by coupling with (S)-TrpNH2, deprotection and HPLC separation, afforded the phosphinic tripeptide (R,S,S)-94 in 87% (Scheme 39) [78].…”

“…In a similar way, Stratikos et al [84] reported the synthesis of the phosphinic tripeptide (R,S,S)-94, which was tested in the inhibition of the endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), involved in regulation of cytotoxic cellular responses. Thus, the reaction of N-Boc H-phosphinic acid analogue of homophenylalanine (R)-92 [72] with HMDS followed by the addition of ethyl isobutylacrylate and saponification of the ethyl ester, gave the C-phosphinate 93 in 72% yield, which by coupling with (S)-TrpNH2, deprotection and HPLC separation, afforded the Considering that phosphinic peptides are well recognized as peptide isosters and powerful inhibitors of many classes of enzymes, mainly zinc proteases, Yiotakis et al [81,82] reported the synthesis of phosphinopeptidic building blocks incorporating a triple bond, which through 1,3-dipolar cycloaddition process, gives access to novel class of isoxazole-containing phosphinic peptides.…”

“…According to this strategy, the treatment of N-Cbz phenylalanine H-phosphinic analogue (S)-66a [72] with TMSCl/DIPEA followed by addition of diethyl 2-methylenemalonate and subsequent alkaline hydrolysis, afforded the phosphinic pseudodipeptide (S)-95 in 67% yield, which was subjected to a Knoevenagel-type condensation with several aldehydes, to give the dehydrophosphinic peptides 96a-i in good yields (Scheme 40). In a similar way, Stratikos et al [84] reported the synthesis of the phosphinic tripeptide (R,S,S)-94, which was tested in the inhibition of the endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), involved in regulation of cytotoxic cellular responses. Thus, the reaction of N-Boc H-phosphinic acid analogue of homophenylalanine (R)-92 [72] with HMDS followed by the addition of ethyl isobutylacrylate and saponification of the ethyl ester, gave the C-phosphinate 93 in 72% yield, which by coupling with (S)-TrpNH 2 , deprotection and HPLC separation, afforded the phosphinic tripeptide (R,S,S)-94 in 87% (Scheme 39) [78].…”