Regulating the ARNT/TACC3 axis: multiple approaches to manipulating protein/protein interactions with small molecules

Abstract

For several well-documented reasons, it has been challenging to develop artificial small molecule inhibitors of protein/protein complexes. Such reagents are of particular interest for transcription factor complexes given links between their misregulation and disease. Here we report parallel approaches to identify regulators of a hypoxia signaling transcription factor complex, involving the ARNT subunit of the HIF (Hypoxia Inducible Factor) activator and the TACC3 (Transforming Acidic Coiled Coil Containing Protein 3) coactivator. In one route, we used in vitro NMR and biochemical screening to identify small molecules that selectively bind within the ARNT PAS (Per-ARNT-Sim) domain that recruits TACC3, identifying KG-548 as an ARNT/TACC3 disruptor. A parallel, cell-based screening approach previously implicated the small molecule KHS101 as an inhibitor of TACC3 signaling. Here, we show that KHS101 works indirectly on HIF complex formation by destabilizing both TACC3 and the HIF component HIF-1α. Overall, our data identify small molecule regulators for this important complex and highlight the utility of pursuing parallel strategies to develop protein/protein inhibitors.

Figure 1. Overview of the ARNT/TACC3 complex

a. Schematic of HIF complexes, which are bHLH-PAS heterodimers that include an O₂-sensitive HIF-α subunit and a constitutive ARNT subunit. Under normoxia, O₂-dependent hydroxylation of HIF-α decreases its abundance and activity. Hypoxia stops these modifications, allowing HIF-α to accumulate in the nucleus and dimerize with ARNT. This heterodimer binds to hypoxia responsive enhancer (HRE) sites, controlling target gene transcription. In addition to binding HIF-α, ARNT PAS-B directly recruits CCC proteins.^, b. Structural model of an ARNT/CCC complex showing how the TACC3 coiled coil interacts with the helical surface of ARNT PAS-B, opposite from where HIF-α PAS-B binds.

Figure 2. Screening ARNT PAS-B for small molecule effectors of the ARNT/TACC3 interaction

a. Diagram of the ARNT PAS-B crystal structure, shown inset with secondary structure designations and internal cavities (grey mesh). The larger cavity is adjacent to the TACC3 binding site and contains three waters; the.smaller cavity is primarily hydrophobic. b. Schematic of the NMR-based screen for compounds that bind ARNT PAS-B. Initial ¹⁵N/¹H HSQC spectra were acquired with 250 µM ¹⁵N-labeled ARNT PAS-B with a mixture of five compounds (1 mM each); mixtures producing large chemical shift perturbations (compared to DMSO) were deconvoluted as shown. c. Lead compounds (500 µM each) from NMR-based screen were tested for their ability to disrupt complexes of ARNT PAS-B with CCC fragments of TRIP230 (1583–1716) and TACC3 (561–631 = TACC3-CT). d. Summary of ARNT PAS-B binding and ARNT/CCC disruption of tested compounds. All ten compounds generated chemical shift perturbations when titrated into ARNT PAS-B; KG-548 and KG-655 (red box) also disrupted TRIP230 and TACC3 binding to ARNT PAS-B in vitro.

Figure 3. KG-548 appears to bind within the ARNT PAS-B cavities

a. ¹⁵N/¹H HSQC spectra of a KG-548 titration (0–1 mM from light to dark crosspeaks) into 320 µM ¹⁵N ARNT PAS-B. Slow exchange behavior was observed, indicated by the disappearance of apo-crosspeaks and concomitant appearance of new peaks. b. Minimum chemical shift analysis of KG-548 titration into ARNT PAS-B, mapped onto the sequence and secondary structure. c. Chemical shift mapping suggests that KG-548 binds the ARNT PAS-B cavities, as shown by a heat map of KG-548-induced chemical shift changes on the ARNT PAS-B crystal structure (Δδ_mcs colored from low (blue) to high (red)) with the largest changes near the internal cavities (mesh).

Figure 4. KG-548 disrupts in vitro ARNT/TACC3 interactions

a. Titration of KG-548 into an in vitro pulldown assay of minimal ARNT PAS-B and TACC3-CT interacting fragments shows a dose-dependent reduction in ARNT/TACC3 complex formation. b. Quantification of KG-548 potency for disrupting the ARNT PAS-B/TACC3-CT interaction as provided by AlphaScreen, showing an apparent IC₅₀ of 25 µM. c. Co-immunoprecipitation assays of full length ARNT and TACC3 proteins in HEK293T cell lysates show that KG-548 weakens the ARNT/TACC3 interaction as demonstrated by the dose-dependent decrease in the intensity of the ARNT protein band (quantitated with % remaining compared to DMSO control) associated with immunoprecipitated TACC3 protein.

Figure 5. KHS101 decreases TACC3 levels in cells and regulates HIF gene expression

a. KHS101 facilitates TACC3 degradation in a proteasome-dependent manner. HEK293T cells were treated with 5 µM KHS101, 100 µg/ml cycloheximide (CHX) (upper panel); additional cells were similarly treated with KHS101 and CHX plus 20 µM MG132 (lower panel). Cells were harvested 0µ8 hr post-treatment and prepared for TACC3 immunoblot analyses. b. Quantification of TACC3 protein levels from data shown in panel a. Without MG132, TACC3 protein levels decreased, with greater drops observed in KHS101-treated cells (compared to DMSO) after 6 hr incubation. A statistically-significant difference was observed 6 hr post-treatment (p < 0.01 by Student’s t-test); the 8 hr timepoint also shows a substantial decrease in TACC3 levels, but this is not statistically significant due to large variations in data values. In the presence of MG132, we observed little decrease in TACC3 levels with no KHS101-dependent effects, implicating a proteasomal-dependent degradation pathway. c. Steady state treatment with KHS101 reduces TACC3 levels. HEK293T cells were treated with KHS101 (0 – 15 µM) and immunostained with TACC3 AB1 after 14 hr. TACC3 intensity was negatively affected by KHS101. d. KHS101 induces cell differentiation with a minimum of 6 hr exposure. Adult rat NPCs were exposed to 5 µM KHS101 (or DMSO) for the indicated times, after which media were replaced with compound-free versions and incubated for a total of 100 hr. Neuronal differentiation was assessed by expression of the TuJ1 marker, showing upregulation after times consistent with TACC3 levels falling in CHX-treated cells (panels a, b).

Figure 6. KHS101 inhibits HIF target gene expression and decreases HIF-1α protein levels

a. KHS101 treatment potently reduces HIF target gene expression. Hep3B cells were treated with KHS101 (0–25 µM) and incubated under hypoxia (1% O₂) for 16 hr. The expression of three HIF-driven genes (EPO, PGK1, GLUT1) were assessed by qPCR, demonstrating KHS101 inhibiting transcription with IC₅₀ < 5 µM. b. HIF-1α mRNA level was measured by qPCR, showing no significant change with increasing KHS101 concentration. c. HIF-1α protein levels are reduced in a KHS101 dose-dependent manner, using anti HIF-1α Western blot.