Differential Inhibition of Signal Peptide Peptidase Family Members by Established γ-Secretase Inhibitors

PLoS One. 2015 Jun 5;10(6):e0128619. doi: 10.1371/journal.pone.0128619. eCollection 2015.

Abstract

The signal peptide peptidases (SPPs) are biomedically important proteases implicated as therapeutic targets for hepatitis C (human SPP, (hSPP)), plasmodium (Plasmodium SPP (pSPP)), and B-cell immunomodulation and neoplasia (signal peptide peptidase like 2a, (SPPL2a)). To date, no drug-like, selective inhibitors have been reported. We use a recombinant substrate based on the amino-terminus of BRI2 fused to amyloid β 1-25 (Aβ1-25) (FBA) to develop facile, cost-effective SPP/SPPL protease assays. Co-transfection of expression plasmids expressing the FBA substrate with SPP/SPPLs were conducted to evaluate cleavage, which was monitored by ELISA, Western Blot and immunoprecipitation/MALDI-TOF Mass spectrometry (IP/MS). No cleavage is detected in the absence of SPP/SPPL overexpression. Multiple γ-secretase inhibitors (GSIs) and (Z-LL)2 ketone differentially inhibited SPP/SPPL activity; for example, IC50 of LY-411,575 varied from 51±79 nM (on SPPL2a) to 5499±122 nM (on SPPL2b), while Compound E showed inhibition only on hSPP with IC50 of 1465±93 nM. Data generated were predictive of effects observed for endogenous SPPL2a cleavage of CD74 in a murine B-Cell line. Thus, it is possible to differentially inhibit SPP family members. These SPP/SPPL cleavage assays will expedite the search for selective inhibitors. The data also reinforce similarities between SPP family member cleavage and cleavage catalyzed by γ-secretase.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alanine / analogs & derivatives
  • Alanine / chemistry
  • Alanine / metabolism
  • Amino Acid Sequence
  • Amyloid Precursor Protein Secretases / antagonists & inhibitors*
  • Amyloid Precursor Protein Secretases / metabolism
  • Amyloid beta-Peptides / genetics
  • Amyloid beta-Peptides / metabolism
  • Animals
  • Aspartic Acid Endopeptidases / antagonists & inhibitors*
  • Aspartic Acid Endopeptidases / genetics
  • Aspartic Acid Endopeptidases / metabolism
  • Azepines / chemistry
  • Azepines / metabolism
  • Benzodiazepinones / chemistry
  • Benzodiazepinones / metabolism
  • Cell Line
  • Enzyme-Linked Immunosorbent Assay
  • HEK293 Cells
  • Humans
  • Immunoprecipitation
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mice
  • Molecular Sequence Data
  • Protease Inhibitors / chemistry*
  • Protease Inhibitors / metabolism
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Substrate Specificity

Substances

  • 2-(((3,5-difluorophenyl)acetyl)amino)-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)propanamide
  • Amyloid beta-Peptides
  • Azepines
  • Benzodiazepinones
  • Membrane Proteins
  • N2-((2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl)-N1-((7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo(b,d)azepin-7-yl)-L-alaninamide
  • Protease Inhibitors
  • Recombinant Fusion Proteins
  • Amyloid Precursor Protein Secretases
  • Aspartic Acid Endopeptidases
  • signal peptide peptidase
  • Alanine