title: Differential Inhibition of Signal Peptide Peptidase Family Members by Established γ-Secretase Inhibitors.
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.
authors.affiliation: Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, and McKnight Brain Institute, College of Medicine University of Florida, Gainesville, Florida, United States of America.
authors.forename: Yong
authors.initials: Y
authors.lastname: Ran
authors.affiliation: College of Pharmacy, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Gabriela Z
authors.initials: GZ
authors.lastname: Ladd
authors.affiliation: Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, and McKnight Brain Institute, College of Medicine University of Florida, Gainesville, Florida, United States of America.
authors.forename: Carolina
authors.initials: C
authors.lastname: Ceballos-Diaz
authors.affiliation: Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, and McKnight Brain Institute, College of Medicine University of Florida, Gainesville, Florida, United States of America.
authors.forename: Joo In
authors.initials: JI
authors.lastname: Jung
authors.affiliation: Pennsylvania Drug Discovery Institute, Philadelphia, Pennsylvania, United States of America.
authors.forename: Doron
authors.initials: D
authors.lastname: Greenbaum
authors.affiliation: Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, and McKnight Brain Institute, College of Medicine University of Florida, Gainesville, Florida, United States of America.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Felsenstein
authors.affiliation: Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, and McKnight Brain Institute, College of Medicine University of Florida, Gainesville, Florida, United States of America.
authors.forename: Todd E
authors.initials: TE
authors.lastname: Golde
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D001381:Azepines / Q000737:chemistry / Q000378:metabolism; D001570:Benzodiazepinones / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: C529132:2-(((3,5-difluorophenyl)acetyl)amino)-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl)propanamide; D016229:Amyloid beta-Peptides; D001381:Azepines; D001570:Benzodiazepinones; D008565:Membrane Proteins; C484278: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; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C460085:signal peptide peptidase; D000409:Alanine