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. 2016 Jul 6;53(4):1499-516.
doi: 10.3233/JAD-151179.

High-Throughput Screening for Identification of Blood-Brain Barrier Integrity Enhancers: A Drug Repurposing Opportunity to Rectify Vascular Amyloid Toxicity

Hisham Qosa  1 , Loqman A Mohamed  1 , Sweilem B Al Rihani  1 , Yazan S Batarseh  1 , Quoc-Viet Duong  1 , Jeffrey N Keller  2 , Amal Kaddoumi  1
Affiliations

High-Throughput Screening for Identification of Blood-Brain Barrier Integrity Enhancers: A Drug Repurposing Opportunity to Rectify Vascular Amyloid Toxicity

Hisham Qosa et al. J Alzheimers Dis. .

Abstract

The blood-brain barrier (BBB) is a dynamic interface that maintains brain homeostasis and protects it from free entry of chemicals, toxins, and drugs. The barrier function of the BBB is maintained mainly by capillary endothelial cells that physically separate brain from blood. Several neurological diseases, such as Alzheimer's disease (AD), are known to disrupt BBB integrity. In this study, a high-throughput screening (HTS) was developed to identify drugs that rectify/protect BBB integrity from vascular amyloid toxicity associated with AD progression. Assessing Lucifer Yellow permeation across in-vitro BBB model composed from mouse brain endothelial cells (bEnd3) grown on 96-well plate inserts was used to screen 1280 compounds of Sigma LOPAC®1280 library for modulators of bEnd3 monolayer integrity. HTS identified 62 compounds as disruptors, and 50 compounds as enhancers of the endothelial barrier integrity. From these 50 enhancers, 7 FDA approved drugs were identified with EC50 values ranging from 0.76-4.56 μM. Of these 7 drugs, 5 were able to protect bEnd3-based BBB model integrity against amyloid toxicity. Furthermore, to test the translational potential to humans, the 7 drugs were tested for their ability to rectify the disruptive effect of Aβ in the human endothelial cell line hCMEC/D3. Only 3 (etodolac, granisetron, and beclomethasone) out of the 5 effective drugs in the bEnd3-based BBB model demonstrated a promising effect to protect the hCMEC/D3-based BBB model integrity. These drugs are compelling candidates for repurposing as therapeutic agents that could rectify dysfunctional BBB associated with AD.

Keywords: Amyloid-β; blood-brain barrier; high-throughput screening; permeability; repurposing.

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Figures

Figure 1
Figure 1
Model optimization. (a) Optimization of basement membrane substitute. LY permeation across bEnd3 monolayer grown on the top of polycarbonate transwell coated with 150 or 300 μg/ml rat-tail collagen type I, 30 or 60 μg/ml fibronectin, and a mixture of 150 μg/ml rat-tail collagen and 30 μg/ml fibronectin. LY permeation was represented as permeation coefficient (Pc). Compared to other tested coatings, in the presence of cells, fibronectin at 30 μg/ml offered the lowest Pc value indicative of restricted transport due to formation of a tight monolayer, and in the absence of cells fibronectin did not act as a barrier to LY transport. Data represented as mean±SEM for 9 replicates from 3 independent experiments. * P<0.05, ** P<0.01 values compared to control, # P<0.05 compared to collagen at 150 μg/ml.(b) Time-dependent changes in bEnd3 cells growth (open circles) and LY permeation across the bEnd3 cells-based BBB model (open triangles). Growth pattern of bEnd3 cells after seeding indicates formation of confluent monolayer on day 3 post-seeding. On the other hand, LY permeation started to decrease from day 1 and continue to decrease until day 4 where it remains contestant until day 6. LY permeation change indicates formation of tight junction between cells in confluent bEnd3 monolayer. Data represented as mean±SEM for 6 replicates from 2 independent experiments (* p < 0.05).
Figure 2
Figure 2
Assay validation. (a) Comparison of LY permeation coefficient across bEnd3 monolayer grown on the top of polycarbonate transwell of 3 μm pore size and polyester transwell of 0.45 pore size. The results showed no differences between LY permeation coefficients across two bEnd3 monolayer grown on either supporting membrane. (b) Comparison of LY permeation coefficient to that of 14C-inulin. Inulin showed significantly lower permeation coefficient compared to LY. Data represented as mean±SEM for 6 replicates from 2 independent experiments. * P<0.05, ** P<0.01 values compared to filter.
Figure 3
Figure 3
(a) Performance of bEnd3 cells-based BBB model in the HTS for disruptors and enhancers of the barrier function. (b) Screening of 1280 compounds of Sigma LOPAC1280 library for their effect on the integrity of bEnd3 cells-based BBB model. LY permeation coefficient of each compound was normalized to the plate median and MAD to obtain a standardized z-score value that was plotted for each compound. The upper dotted line represents the z-score cutoff corresponding to 3 MAD above the plate median. z-Score values above the upper dotted line represent compounds which disrupt the barrier function of the in vitro model. The lower dotted line represents z-score cutoff corresponding to 2 MADs below the plate median. z-Score values below the lower dotted line represent compounds enhancers of the barrier function of the BBB model. DMSO (negative control, 0.1%), hydrocortisone (positive control for enhancers, 10 μM) and Mannitol (positive control for disruptors, 1.4 M) were plotted as box plots to show the model performance.
Figure 4
Figure 4
Hits validation. 10-Point concentration-response curves of the seven FDA approved drugs that showed significant enhancement in the barrier function of the bEnd3 cells-based BBB model. The drugs were tested in the concentration range 0.065–20 μM to calculate compounds EC50s. The 10th point (20 μM) was eliminated from the curves due to toxicity. Data represent mean±SEM, n = 6 for each concentration.
Figure 5
Figure 5
(a) Representative images of claudin-5 in bEnd3 cells monolayers treated with hit FDA drugs showing localization and expression of claudin-5 in the membrane of bEnd3 cells (scale bar = 50 μm) (b) Representative blots and densitometry analysis for the effect of hit FDA drugs treatment on cluadin-5 expression determined by Western blot. Data represented as mean±SEM from 3 independent experiments. * P<0.05.
Figure 5
Figure 5
(a) Representative images of claudin-5 in bEnd3 cells monolayers treated with hit FDA drugs showing localization and expression of claudin-5 in the membrane of bEnd3 cells (scale bar = 50 μm) (b) Representative blots and densitometry analysis for the effect of hit FDA drugs treatment on cluadin-5 expression determined by Western blot. Data represented as mean±SEM from 3 independent experiments. * P<0.05.
Figure 6
Figure 6
Effect of the hit FDA drugs on rectifying the attenuated integrity of bEnd3 cells-based BBB model (a), and the human hCMEC/D3 cells-based BBB model (b) by Aβ mixture (100 nM Aβ40 monomers and 200 nM Aβ42 oligomers with bEnd3 cells; 50 nM Aβ40 monomers and 100 nM Aβ42 oligomers with hCMEC/D3 cells). Data represented as mean±SEM for 6 replicates from 2 independent experiments. * P<0.05, ** P<0.01 values compared to filter.
Scheme 1
Scheme 1
Schematic presentation of bEnd3 cells-based BBB model optimization and application.
See this image and copyright information in PMC

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