Table 1.
IC50 (nM) for APP 5′UTR blockers in pIRES-APP-5′UTR transfectants (top row) and pIRES-PrP-5′UTR transfectants (bottom row).
Figure 1.
Alignment of human and mouse APP 5′UTRs with human PrP 5′UTR sequences relative to the L- and H-ferritin Iron-responsive elements (IREs).
Panel A: The human and mouse APP 5′UTR specific IRE-like RNA stem loops, the human PrP 5′UTR, and the human and mouse SNCA specific IRE –like stem loops each aligned adjacent to the ferritin L- and H IRE RNA stem loops. Shown, the L- and H-mRNAs encode canonical IRE RNA stem loops whereas the APP IRE in non canonical although fully iron responsive [6]. The α-synuclein IRE (SNCA IRE) represents a non canonical IRE traversing the central splice junction of exon-1 and exon-2 (the CAGUGN loop/splice site sequences) of SNCA mRNA [49]. Typical IRE stem loops fold to exhibit an apical AGU pseudotriloop which is depicted in red lettering at the apex of the H-ferritin and SNCA IREs [28] relative to an analogous AGA from the IRE–like stem loop encoded by APP mRNA [6]. Panel B: Maps of the 5′UTRs encoding by the human and mouse APP mRNAs, PrP mRNA, SNCA mRNA, and the mRNAs for L- and H-ferritin (IRE stem loops are displayed as lollipops). Panel C: Relative alignment of the sequences that encode the 5′UTR specific IRE-like stem loops in human APP mRNA, PrP mRNA, SNCA mRNA, and the L- and H-ferritin mRNAs. Panel D: Screen and counter-screening Constructs [21]: The human APP 5′UTR cassette was subcloned in front of the luciferase reporter gene in the dicistronic pCD(APP) reporter construct. The same-sized and related PrP 5′UTR was subcloned in an identical format into the pCD(PrP) reporter construct for the purpose of counter-screening, as described in the materials and methods section.
Figure 2.
Relative capacity of thirteen APP 5′UTR translation blockers to reduce Aβ levels in the conditioned medium of SH-SY5Y cells.
Following 48 h treatment (1 µM) for each inhibitor, the histogram shows reduction of total Aβ levels confirmed after averaging five independent samplings from the following:- JTR-009 treated<control, p<0.01). Total Aβ levels were also documented for the APP blockers JTR-004, JTR-10, JTR-0011 JTR-0013 (N = 5). Data are means ± SEM, N = 5, * = p<0.01, ** = p<0.01, *** = p<0.0013, p<0.01, **** p<0.011, *****, where each treatment was analyzed by ANOVA+Dunnett's post hoc test compared to untreated samples. JTR-009 was the ninth and JTR-005 was fifth in the series of 13 APP translation blockers.
Figure 3.
The effect of JTR-009 to reduce the steady state levels of APP in SH-SY5Y cells with a high degree of selectivity in the absence of changes to the levels of β-actin and α-synuclein (SNCA).
Panels A and B: Dose-responsive (0, 10 µM, 20 µM, 30 µM) treatment of SH-SY5Y cells for 48 h to measure the capacity of JTR-009 and PFT-α to limit APP expression relative to β-actin and SNCA levels. The representative western blot experiment in Panel A contributed to densitometry for the histogram shown in Panel B (N = 3). Right Panel: Chemical structure of JTR-009, 4-(5-methyl-1H-benzimidazol-2yl) aniline, compared to the anti-apoptotic stroke agent PFTα, (275 Da), a tricyclic benzothiazole. Panel C: Dose-responsive measurement of total amyloid Aβ levels in response to the APP 5′UTR inhibitors JTR-005 and JTR-009, measured by benchmarked ELISA in conditioned medium of 72-hour treated SH-SY5Y cells. Shown are the mean values for the reduction of levels of Aβ ± SEM (N = 4) after treatment of the cells with JTR-009 and JTR-005 at 0.01 µM (* = p<0.01), 0.1 µM (** = p<0.015), and 1 µM (*** = p<0.01) analyzed by ANOVA (N = 5). Dotted line: Representative LDH assay parallel to Aβ determination for SH-SY5Y cells treated for 72 h at concentrations up to 100 µM of JTR-009 (N = 4). Panel D: MTS assay for cellular mitochondrial viability after treatment of SH-SY5Y cells with JTR-005 and JTR-009 at the concentrations shown. Y axis: Percent of maximal viability ± SEM after treatment of the cells with JTR-009 and JTR-005 (N = 3)). Shown are the relative trend-lines for the dose-responsive viability of JTR-005 and JTR-009 compared to untreated cells (‘poly’ = non linear polynomial regression of the data).
Figure 4.
Evaluation of the potency and selectivity of APP blocker-9.
Panel A: Dose responsive measurement of the capacity of JTR-009 to limit APP 5′UTR-luciferase expression relative to posiphen, a known APP translation blocker (JTR-009: IC50 = 0.1 µM; posiphen: IC50 = 5 µM, N = 4). Panel B: Dose-responsive reduction APP levels in SH-SY5Y cells treated 48 hours at 0.1 µM, 0.5 µM and 1 µM JTR-009. Western blot for APP levels using N- terminal 22C11 antibody (standardization with β-actin as loading control). Bottom Panel: Histogram quantitation of the relative expression of APP/β-actin in SH-SY5Y cells. Panel C: Lysates from the experiment in Panel B was analyzed by Western blotting using APP the C-terminal specific (A8717) antibody and β-actin antibody. Bottom Panel: histogram quantitation of the relative expression of APP/β-actin in SH-SY5Y cells from autoradiographic film subjected to densitometry (N = 3). Panel D: Dose-responsive capacity of JTR-009 to limit APP expression in primary E-18 mouse neurons (1 nM). The relative α-synuclein (SNCA) expression was calculated. Shown, the combined data was graphed into a histogram where mean values from separate assays were calculated from densitometry of Western blots (N = 5). Panel E: Real-time qPCR measurement of the dose-responsive measurement of the levels of APP mRNA in SH-SY5Y cells treated with escalating concentrations of JTR-009 for 48 hours. Panel F: Equivalent real-time qRT-PCR analysis to measure APP mRNA and TfR mRNA levels in SH-SY5Y cells after 48 h treatment with 25 µM desferrioxamine (DFO) (Positive control for qRT-PCR analysis shown in Panel E).
Table 2.
Comparative IC50 of JTR-009 relative to posiphen to inhibit APP 5′UTR driven luciferase expression relative to suppression of APP and Aβ levels in SH-SY5Y cells and primary neurons.
Figure 5.
RNA pulldown assay to measure the dose-dependent capacity of the cyclic benzimidazole JTR-009 to substitute for IRP1 binding to APP 5′UTR sequences in SH-SY5Y cells: correlated repression of APP translation.
RNA pulldown assays were conducted as ilustrated in Figure 6 and as described by Cho et al., 2010 [6] Panel A and B: Representative RNA binding assays in which recovered beads measured the dose-responsive capacity of JTR-009 (0 µM 0.3 µM, 3 µM and 30 µM) to inhibit IRP1 binding to 30 base biotinylated probes encoding the APP 5′UTR. In Panel B Western blots measured relative levels of IRP1 and IRP2 bound to biotinylated RNA probes for APP IRE sequences after recovery in steptavidin bead fractions. Densitomteric quantitation of bead-specific IRP1 is shown in Panel A. Panel C: Measurement of the dose-dependent off-target action of JTR-009 to suppress H-ferritin IRE binding to SH-SY5Y specific IRP1 and IRP2 (bead fraction). Panel D and E: Dose-dependent decrease of APP levels in response to JTR-009 measured in the supernatants of bead fractions (experimental<control set (p<0.00 1). Panel E: Western blots of lysate supernatants showing APP as measured using the N terminal specific 22C11 and C-terminal specific A8717 anitibodies. Panel D: Densitometric quantitation of the data in Panel E to measure the extent to which JTR-009 dose dependently repressed APP expression in SH-SY5Y cells (Dunnetts test, p = 0.03). Data from 5 separate trials, each in triplicate.
Figure 6.
RNA binding assay to measure the capacity of JTR-009 to replace IRP1 binding to biotinylated probes encoding core APP IRE sequences compared to IRP1 binding to the H-ferritin IRE sequences (N = 4).
Panel A: Top Panel: Cartoon representation of the protocol employed to detect RNA binding between IRE probes and IRP1 in SH-SY5Y cell lysates. Bottom Panel: Effect of JTR-009 treatment of SH-SY5Y cells (24 h, 10 µM) to alter ferritin-H IRE binding to IRP1 compared to that of the APP IRE. Panel B: The calcein assay for iron levels in SH-SY5Y cells in response to treatment with JTR-009. Cells were treated with either DMSO (negative control), extracellular iron chelator (DPTA), or JTR-009 at 10 µM for 48 hours. Panel C: The anti-amyloid-Aβ-42 efficacy of the APP 5′UTR inhibitors JTR-005 and JTR-009, as measured in conditioned medium from SH-SY5Y neuronal cells (Chemiluminescent BetaMark x-42 ELISA assay from Covance, inc). Data shows the mean values for the reduction of levels of Aβ-42 ± SEM (N = 3) after 72 h treatment of the cells with JTR-009 compared to JTR-005 at 10 µM (p<0.01), analyzed by ANOVA.
Figure 7.
Model for the action of the bezimidazole JTR-009 to act as an inhibitor of APP translation by irreversibly replacing the iron dependent translation repressor IRP1 from interacting with APP 5′UTR sequences.
Binding of JTR-009 selectively targeted APP 5′UTR sequences and then was found to repress APP levels leading to reduced amyloid levels without perturbing cellular iron uptake.