Aurintricarboxylic acid is a canonical disruptor of the TAZ-TEAD transcriptional complex

Abstract

Disrupting the formation of the oncogenic YAP/TAZ-TEAD transcriptional complex holds substantial therapeutic potential. However, the three protein interaction interfaces of this complex cannot be easily disrupted using small molecules. Here, we report that the pharmacologically active small molecule aurintricarboxylic acid (ATA) acts as a disruptor of the TAZ-TEAD complex. ATA was identified in a high-throughput screen using a TAZ-TEAD AlphaLISA assay that was tailored to identify disruptors of this transcriptional complex. We further used fluorescence polarization assays both to confirm disruption of the TAZ-TEAD complex and to demonstrate that ATA binds to interface 3. We have previously shown that cell-based models that express the oncogenic TAZ-CAMTA1 (TC) fusion protein display enhanced TEAD transcriptional activity because TC functions as an activated form of TAZ. Utilizing cell-based studies and our TC model system, we performed TC/TEAD reporter, RNA-Seq, and qPCR assays and found that ATA inhibits TC/TEAD transcriptional activity. Further, disruption of TC/TEAD and TAZ/TEAD interaction by ATA abrogated anchorage-independent growth, the phenotype most closely linked to dysregulated TAZ/TEAD activity. Therefore, this study demonstrates that ATA is a novel small molecule that has the ability to disrupt the undruggable TAZ-TEAD interface.

Fig 1. TAZ-TEAD AlphaLISA assay.

A, A schematic showing the TAZ-TEAD AlphaLISA assay design. The interaction between the tagged, full-length, TAZ and TEAD proteins brings the donor and acceptor beads close enough to allow singlet oxygen transfer, which generates an emission signal at 615 nm. B, Flowchart of the screening strategy, 56,115 compounds were screened using the TAZ-TEAD AlphaLISA screen. Filtering the hits via a counterscreen followed by IC₅₀ determination and assessment using a ThermoFluor assay identified aurintricarboxylic acid (ATA) as a disruptor of the TAZ-TEAD complex. C, The molecular structure of ATA. D, Dose-response curves obtained after titration of ATA either in the TAZ-TEAD AlphaLISA assay or in the counterscreen; a FLAG-His fusion peptide was used in the counterscreen. E and F, ThermoFluor assays measuring the melting temperatures (T_m) of full-length TAZ or TEAD in the presence of ATA. Data are representative of three independent experiments performed using technical duplicates.

Fig 2. ATA binds to TEAD and disrupts the TAZ-TEAD interaction.

A, Sensorgrams showing resonance responses that were recorded after passing the indicated concentrations of ATA over the YAP/TAZ-binding domain of TEAD. B, Affinity between TEAD and ATA was calculated by fitting a one-site binding curve to the steady-state response versus concentration. C, Cartoon showing the superposition of VGLL1-TEAD (PDB ID: 5Z2Q) and TAZ-TEAD structures (PDB ID: 5GN0). TAZ forms interfaces 2 and 3, whereas VGLL1 forms interfaces 1 and 2. The central pocket that houses the lipid is shown as a red mesh. D, Acylated and deacylated forms of TEAD were used in a TAZ-TEAD FP assay to characterize whether ATA binds to the surface or the central pocket of TEAD, the IC₅₀ values were obtained through a four-parameter curve fit. E, YAP-TEAD FP assay using a YAP peptide probe to evaluate whether ATA disrupts the formation of the YAP-TEAD complex. F, Vgll1-TEAD FP assay using a mouse Vgll1 peptide probe to monitor whether ATA binds at interfaces 1 and 2. Peptide 7 was used as a positive control and peptide 17 was used as a negative control. All FP experiments were repeated three times using technical duplicates with similar outcome. The data is shown as mean (n = 2) and error bars represent standard deviation of the mean.

Fig 3. ATA inhibits TC/TEAD transcriptional activity.

A, TC/TEAD reporter assay to measure TC/TEAD activity in cells transfected with empty vector (EV), cells stably expressing TC treated with vehicle control (TC), and TC-expressing cells treated with ATA (TC + ATA). The experiment was repeated three times and similar results were obtained. Data are presented as mean ± SD. B, Volcano plot showing the distribution of differentially expressed genes in TC-transfected compared to EV-transfected cells, blue points: FDR < 0.05 and either Log₂FC > 2 or Log₂FC<-2, red points: FDR>0.05 or 2 < Log₂FC > -2, FDR: False discovery rate. C, Gene set enrichment analysis of ATA-treated cells stably expressing TC versus TC-expressing, vehicle control treated cells utilizing the TC up Genes from Fig 3B, NES: Normalized enrichment score D, qPCR to probe the levels of indicated target genes in empty EV, TC and TC+ATA NIH3T3 cells. Error bars represent the standard deviation of the mean (n = 3) normalized to EV, P values were obtained through two-tailed t tests (***P < 0.0008, **P < 0.008, *P < 0.03).

Fig 4. ATA inhibits soft agar colony growth that is dependent on TC/TAZ-TEAD activity.

Bright-field images and the corresponding quantification of soft agar colonies determined as percent normalized to vehicle controls (VC). Cells were treated with the indicated doses of ATA in (A) NIH3T3 cells expressing TAZ-CAMTA1 (B) NIH3T3 expressing TAZ ^S89A and (C) NIH3T3 cells expressing NRAS ^G12V. Data represent results of two independent experiments. Data are presented as mean (n = 3) and error bars represent the standard deviation of the mean.