GDC-0879, a BRAFV600E Inhibitor, Protects Kidney Podocytes from Death

Abstract

Progressive kidney diseases affect approximately 500 million people worldwide. Podocytes are terminally differentiated cells of the kidney filter, the loss of which leads to disease progression and kidney failure. To date, there are no therapies to promote podocyte survival. Drug repurposing may therefore help accelerate the development of cures in an area of tremendous unmet need. In a newly developed high-throughput screening assay of podocyte viability, we identified the BRAF^V600E inhibitor GDC-0879 and the adenylate cyclase agonist forskolin as podocyte-survival-promoting compounds. GDC-0879 protects podocytes from injury through paradoxical activation of the MEK/ERK pathway. Forskolin promotes podocyte survival by attenuating protein biosynthesis. Importantly, GDC-0879 and forskolin are shown to promote podocyte survival against an array of cellular stressors. This work reveals new therapeutic targets for much needed podocyte-protective therapies and provides insights into the use of GDC-0879-like molecules for the treatment of progressive kidney diseases.

Keywords: BRAF; ER stress; ERK; MAPK; SB-682330; cell death; forskolin; glomerular disease; kidney disease; melanoma; post-mitotic cells.

Figure 1. GDC-0879 and forskolin identified in HTS of clinically active compounds

A, B) High throughput assay development and reproducibility. A favorable Z′ ≥0.5 was obtained at 96 hours of thapsigargin–treatment. Fully differentiated podocytes were re–plated into 384–well plates at 2000 (A), 4000 (A) and 8000 (B) cells per well and thapsigargin was applied for the indicated times, respectively. Scatterplot between several replicates as percent change of cell viability relative to DMSO control–treated cells determined by assessing ATP levels. Z′ indicates screen potency of the different conditions. C) Scheme of HTS workflow. D) 1649 compounds were screened in duplicates for their effect on thapsigargin–mediated cell death. Scatterplot between two replicates as percent change of cell viability relative to thapsigargin–treated cells determined by assessing ATP levels. Protective hits were defined as compounds eliciting responses for both replicates individually that are larger than 40% (red dots). The chemical structures of GDC-0879 and forskolin are shown below. E) Dose responsive effect of GDC-0879 and forskolin on thapsigargin–treated podocytes at 96 hours. Bar graph represents relative cell viability (ATP levels) ± SD, data normalized to thapsigargin treatment (n=32). F) Annexin V in podocytes treated with thapsigargin in the presence or absence of GDC and forskolin at 48 hours. Bar graph shows mean % ± SD of annexin V positive cells (n=3). * p < 0.05, ** p < 0.01, ## p < 0.0001 (vs. thapsigargin, Bonferroni–corrected).

Figure 2. GDC-0879 promotes podocyte survival through activation of MEK/ERK signaling

A) Decreased thermal stability assessed by cellular thermal shift assay indicated GDC-0879 as a target of BRAF. B) Knockdown of BRAF, ARAF and CRAF did not affect thapsigargin-mediated podocyte death. In the presence of GDC-0879, BRAF– and ARAF–depletion was sufficient to partially reverse the protective effect of GDC-0879. Bar graph represents relative annexin V positive cells ± SD, data normalized to thapsigargin (n=3), # p < 0.001 (vs. scrambled thapsi+GDC, Bonferroni-corrected). D) GDC-0879 restored ERK activity as assessed by phosphorylated p42/44 levels. Forskolin had no effect on p44/42 phosphorylation. CHOP abundance was not affected by compound treatment. D) Detection of simultaneous MEK1/2 and p44/42 phosphorylation by GDC-0879 at 4-dose points. E) MEK inhibitors Selumetinib and UO216-EtOH blocked GDC-0879-mediated p42/44 phosphorylation. F) MEK inhibitors Selumetinib and UO216-EtOH reversed the podocyte protective effect of GDC-0879. Bar graph represents relative cell viability (ATP levels) ± SD. Thapsigargin treatment was set to 1 (n=16); # p < 0.001, ## p < 0.0001 (vs thapsi + GDC-0879, Bonferroni-corrected).

Figure 3. Forskolin promotes podocyte survival by attenuating protein biosynthesis

A) GSEA of microarray and proteomics data of podocytes treated with thapsigargin revealed enrichment for genes/proteins associated with protein biosynthesis (bold). Top 20 GO term enriched gene sets are indicated (FDR, false discovery rate). Gene sets shared between thapsigargin ± forskolin are highlighted in green. B) Forskolin, but not GDC-0879, attenuated protein biosynthesis after sustained podocyte injury (thapsi) as measured by fluorescently labeled incorporated methionine analog L-azidohomoalanine. 15 hour de novo protein biosynthesis was evaluated at baseline (control) or after 9 hour of thapsigargin-treatment (9–24 hour). See methods for details.

Figure 4. GDC-0879 and forskolin protect podocytes from diverse cellular stressors

A, B) Cell survival was measured after cells were treated with palmitic acid, tunicamycin, brefeldin A, adriamycin or staurosporine in the presence of forskolin (A) and GDC-0879 (B). C) A matrix of the combination of GDC-0879 and forskolin at 8-dose points. D) Predicted values based on curve fits of the compound combinations, Bliss independence synergy model predicted activity, and difference between curve fits and model prediction (excess volume). No synergy was observed between the two compounds. In combination, observed rescue was modestly less than predicted by synergy model for higher concentrations of forskolin, suggesting mild antagonism that may be due to cytotoxicity. Poor fit for two values at 10 and 20 μM of GDC-0879 alone results in likely artifact in corresponding calculated excess volume. Similar results were seen with a Loewe additivity model.

Figure 5. New mechanistic insights into podocyte-protective therapeutic targets

A variety of agents (palmitic acid, tunicamycin, brefeldin A, adriamycin, staurosporine and thapsigargin) promote podocyte injury. This results in a reduction in MEK/ERK signaling, which leads to cell death. GDC-0879 promotes activation of MAPK signaling to rescue podocytes. In a parallel pathway, podocyte injury results in a compensatory induction of protein biosynthesis. Forskolin confers a protective advantage by attenuating protein biosynthesis.