9 research outputs found
Differential Inhibition of Signal Peptide Peptidase Family Members by Established γ-Secretase Inhibitors
<div><p>The signal peptide peptidases (SPPs) are biomedically important proteases implicated as therapeutic targets for hepatitis C (human SPP, (hSPP)), plasmodium (<i>Plasmodium</i> 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 (Aβ<sub>1-25</sub>) (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 (<i>Z-LL</i>)<sub>2</sub> ketone differentially inhibited SPP/SPPL activity; for example, IC<sub>50</sub> 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 IC<sub>50</sub> 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.</p></div
γ‐Secretase inhibitors in cancer clinical trials are pharmacologically and functionally distinct
Abstract γ‐Secretase inhibitors (GSIs) are being actively repurposed as cancer therapeutics based on the premise that inhibition of NOTCH1 signaling in select cancers is therapeutic. Using novel assays to probe effects of GSIs against a broader panel of substrates, we demonstrate that clinical GSIs are pharmacologically distinct. GSIs show differential profiles of inhibition of the various NOTCH substrates, with some enhancing cleavage of other NOTCH substrates at concentrations where NOTCH1 cleavage is inhibited. Several GSIs are also potent inhibitors of select signal peptide peptidase (SPP/SPPL) family members. Extending these findings to mammosphere inhibition assays in triple‐negative breast cancer lines, we establish that these GSIs have different functional effects. We also demonstrate that the processive γ‐secretase cleavage pattern established for amyloid precursor protein (APP) occurs in multiple substrates and that potentiation of γ‐secretase cleavage is attributable to a direct action of low concentrations of GSIs on γ‐secretase. Such data definitively demonstrate that the clinical GSIs are not biological equivalents, and provide an important framework to evaluate results from ongoing and completed human trials with these compounds
IC<sub>50</sub>s (nM) of select GSI on FBA/SPPL co-transfected cells.
<p>Each assay was repeated at least 3 times. The values in the table are the means ± standard errors. Numbers in parenthesis represent the rank order of inhibition of each SPP/SPPL by each compound.</p><p>IC<sub>50</sub>s (nM) of select GSI on FBA/SPPL co-transfected cells.</p
Molecular weight of FLAG-BRI2 peptide detected in FBA/SPP co-transfected cell lysates.
<p>*The number in the brackets is the difference between observed and calculated MW. Error between observed and calculated mass is less than ±0.1%.</p><p>Molecular weight of FLAG-BRI2 peptide detected in FBA/SPP co-transfected cell lysates.</p
The FBA substrate is efficiently cleaved by SPP/SPPLs.
<p>A. Design of SPP assay substrate based on BRI2 transmembrane domain. A FLAG tag fused to the NH<sub>2</sub>-terminus of BRI2<sub>1-81</sub>. Aβ<sub>1-25/K16A</sub> fused to the COOH-terminus of BRI2<sub>1-81</sub>. Potential cleavage in the transmembrane domain releases the ectodomain fragment xAβ<sub>1-25/K16A</sub>. B. Western blotting of FBA ±SPPL transfected cell lysates with and without 10 μM (Z-LL)<sub>2</sub> ketone. Blot detected with anti-FLAG M2 antibody. The intact FBA and ICDs are marked with arrows. C. SPP/SPPLs significantly increase xAβ<sub>1-25/K16A</sub> secretion from FBA transfected cells. xAβ<sub>1-25/K16A</sub> levels were determined with Aβ ELISA. D. The FBA cleavages conducted by SPPLs co-transfection are largely inhibited by 10 μM (Z-LL)<sub>2</sub> ketone. E. Western blotting of SPPLs transiently transfected HEK cell lysates. Blots detected with antisera against SPPL2a, SPPL2b, hSPP, and pSPP respectively. Monomer bands are marked with arrows. All experiments repeated 3 times. Statistical analysis performed by 1-way ANOVA ((*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).</p
GSIs inhibit SPP/SPPLs in a dose-dependent manner.
<p>FBA/SPPLs co-transfected HEK cells were treated with DMSO or GSIs as appropriate. Twenty-four hours later the medium was collected for assay by Aβ ELISA. xAβ<sub>1-25/K16A</sub> levels from DMSO-treated cells served as the control. All tests were repeated 3 times. Data were analyzed using GraphPad.</p
Molecular weight of x-Aβ<sub>1-25/K16A</sub> peptide detected in FBA/SPP co-transfected cell media.
<p><sup>a</sup> Error between observed and calculated mass is less than ±0.1%.</p><p>Molecular weight of x-Aβ<sub>1-25/K16A</sub> peptide detected in FBA/SPP co-transfected cell media.</p
Each SPP/SPPL has preferential cleavage site on FBA.
<p>A. Cell lysates of FBA-SPP/SPPL co-transfected cells are used for IP/MS with anti-FLAG M2 magnetic beads. Top and bottom panels are the spectra for cells with DMSO or 20 μM LY-411,575. Main peaks observed are labeled with molecular weight (Da). The peaks below 7000 Da and at ~8080 Da are non-specific. B. 10–20 ml of FBA/SPP co-transfected cell culture media is used for IP/MS with the anti-Aβ<sub>1–16</sub> antibody AB5 bound to magnetic beads. Main peaks observed in the xAβ<sub>1-25/K16A</sub> (ECDs) are labeled with molecular weights. The peaks at ~4190 Da are non-specific. C. Schematic representation of the cleavages of FBA. Putative TMD is underlined. Open and solid arrows indicate observed ICDs and xAβ<sub>1-25/K16A</sub> respectively. “Minor” peaks labeled with * are not shown in these spectra, but have been observed in others (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128619#pone.0128619.s001" target="_blank">S1 Fig</a>).</p