Optimization and Structure–Activity Relationships of Phosphinic Pseudotripeptide Inhibitors of Aminopeptidases That Generate Antigenic Peptides

Abstract

The oxytocinase subfamily of M1 aminopeptidases, consisting of ER aminopeptidase 1 (ERAP1), ER aminopeptidase 2 (ERAP2), and insulin-regulated aminopeptidase (IRAP), plays critical roles in the generation of antigenic peptides and indirectly regulates human adaptive immune responses. We have previously demonstrated that phosphinic pseudotripeptides can constitute potent inhibitors of this group of enzymes. In this study, we used synthetic methodologies able to furnish a series of stereochemically defined phosphinic pseudotripeptides and demonstrate that side chains at P' and P' positions are critical determinants in driving potency and selectivity. We identified low nanomolar inhibitors of ERAP2 and IRAP that display selectivity of more than 2 and 3 orders of magnitude, respectively. Cellular analysis demonstrated that one of the compounds that is a selective IRAP inhibitor can reduce IRAP-dependent but not ERAP1-dependent cross-presentation by dendritic cells with nanomolar efficacy. Our results encourage further preclinical development of phosphinic pseudotripeptides as regulators of adaptive immune responses.

A systematic re-examination of processing of MHCI-bound antigenic peptide precursors by endoplasmic reticulum aminopeptidase 1

“…To probe this putative interaction, we designed a phosphinic pseudopeptide based on the sequence of AI12. The peptide H-AΨ[P(O)(OH)CH2]KAALRSRYWAI-OH, named DG078, carries the phosphinic group in place of the N-terminal peptide bond which is non-hydrolysable and is expected to bind to the active site of ERAP1 with high affinity as previously shown for other similar peptides (40,44). DG078 was successfully folded with B08 at a similar yield as AI12, and the presence of the peptide in the purified complex was confirmed by MALDI-MS (Supporting Figure 14).…”

“…Iodide 2 (1.8 g, 6.0 mmol) was dissolved in 28 mL of 1:1 mixture DMF/toluene, HC(CO2Et)3 (1.53 g 6.6 mmol), K2CO3 (1.08 g, 7.82 mmol) was added and the mixture was refluxed for 1 h, according to our previously published procedure (44). After aqueous workup, the crude product was saponified with KOH (10 equiv) in EtOH and the resulting diacid was subjected to Knoevenagel condensation, affording acrylic acid analogue of 3 (887 mg, 61% yield for 3 steps) (44). The resulting acid (787 mg, 3.23 mmol) was dissolved in CH2Cl2 (5 mL) and benzyl alcohol (350 mg, 3.24 mmol), DCC (801 mg, 3.89 mmol) and DMAP (40 mg, 0.32 mmol) were successively added.…”

The Discovery of Insulin-Regulated Aminopeptidase (IRAP) Inhibitors: A Literature Review

“…The mechanism of inhibition by this rationally designed inhibitor was further investigated by solving the crystal structure of ERAP2/ DG013A complex which revealed that the hydroxyphosphinyl moiety interacts with Zn 2+ through the two oxygen atoms of tetrahedral phosphorous, forming also hydrogen bonds with Glu371 and Tyr455, two residues critical for catalysis. Based on the analogy of DG013A binding pose with the conformation of a substrate during the transition state of hydrolytic reaction, a typical property of phosphinic pseudopeptide protease inhibitors (Georgiadis and Dive, 2015), a subsequent SAR study was performed aiming to unveil those structural determinants that confer selectivity between ERAPs and IRAP (Kokkala et al, 2016). Two inhibitors derived from this study displayed distinct properties in terms of IRAP inhibition: DG026 and DG046 (Figure 2).…”

Is There an Interplay Between the Functional Domains of IRAP?

“…These include their limited stability in the circulation and lack of specificity for IRAP over other aminopeptidases and receptors such as the AT1R for Ang IV and μ-opioid receptor for LVV-H7 ( Lew et al, 2003 ). Thus, a range of novel peptidomimetic ( Axen et al, 2007 ; Lukaszuk et al, 2008 ; Andersson et al, 2010 ; Kokkala et al, 2016 ) and small molecule inhibitors ( Albiston et al, 2008 ; Borhade et al, 2014 ) have been developed to address these limitations with some of these compounds showing promise as potential therapeutic agents.…”