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. 2016 May 31;7(7):681-5.
doi: 10.1021/acsmedchemlett.6b00084. eCollection 2016 Jul 14.

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

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

Athanasios Stamogiannos et al. ACS Med Chem Lett. .
Free PMC article

Abstract

We employed virtual screening followed by in vitro evaluation to discover novel inhibitors of ER aminopeptidase 1, an important enzyme for the human adaptive immune response that has emerged as an attractive target for cancer immunotherapy and the control of autoimmunity. Screening hits included three structurally related compounds carrying the (E)-N'-((1H-indol-3-yl)methylene)-1H-pyrazole-5-carbohydrazide scaffold and (2-carboxylatophenyl)sulfanyl-ethylmercury as novel ERAP1 inhibitors. The latter, also known as thimerosal, a common component in vaccines, was found to inhibit ERAP1 in the submicromolar range and to present strong selectivity versus the homologous aminopeptidases ERAP2 and IRAP. Cell-based analysis indicated that thimerosal can effectively reduce ERAP1-dependent cross-presentation by dendritic cells in a dose-dependent manner.

Keywords: ERAP1; ERAP2; IRAP; aminopeptidase; antigenic peptide; docking; immune system; inhibitor.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Chart 1
Chart 1. Chemical Structures of Known ERAP1 Inhibitors and Inhibitors Identified in This Studya
Figure 1
Figure 1
Predicted conformation of 4 bound to the active site of ERAP1. The inhibitor is shown with orange C, the interacting residues with cyan C, and the zinc-bound residues including the catalytic Tyr438 with green C; all N are blue, O are red, S are yellow, and Zn(II) is shown as a green sphere. Intermolecular hydrogen bonding distances between heavy atoms are given in Å.
Figure 2
Figure 2
Representative titrations showing the ability of selected compounds to inhibit the ability of ERAP1 to hydrolyze the model fluorigenic substrate l-leucine-7-amino-4-methylcoumarin. Calculated IC50 values and 95% confidence interval are indicated.
Figure 3
Figure 3
(A) Characteristic chromatograms showing the trimming of peptide YTAFTIPSI by ERAP1 in the presence or absence of 7. (B) Calculated specific activity based on the surface of the peptide peaks in the chromatograms (average of three separate experiments).
Figure 4
Figure 4
Kinetic analysis of inhibition of ERAP1 by 7 (thimerosal). (A) Michaelis–Menten analysis in the absence or presence of 500 nM 7. (B) Calculated enzymatic parameters by fitting the experimental data of panel A to the classical Michaelis–Menten equation. (C) Normalized ERAP1 activity following the addition of 7 (15 s of dead mixing time). (D) Recovery of ERAP1 activity after 100-fold dilution of the sample used in (C).
Figure 5
Figure 5
Putative binding mode of thimerosal at the catalytic zinc of ERAP1 (A) and ERAP2 (B). Hg(II) is shown as gray sphere, and all other atom colors are the same as those in Figure 1. Interactions between the ethylmercury moiety and the two nonconserved serine residues 316 and 869 are unique for ERAP1 and may be underlying the observed selectivity.
Figure 6
Figure 6
In vitro inhibitory effect of 7 (thimerosal) and inactive analogue thiosalicylic acid on cross-presentation by BMDCs. The IL-2 secretion of transgenic OT-I CD8+ T cells incubated with OVA loaded-ERAP+/+ and ERAP–/– BMDCs in the presence of 7 (thimerosal) (A) or thiosalicylic acid (B) was assessed by ELISA. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as calculated by a two-way ANOVA test, with correction for multiple comparisons by the Bonferroni method. Histograms represent the mean of triplicates obtained in four independent experiments.

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