Identification of small-molecule antagonists that inhibit an activator: coactivator interaction

Abstract

Phosphorylation of the cAMP response element binding protein (CREB) at Ser-133 in response to hormonal stimuli triggers cellular gene expression via the recruitment of the histone acetylase coactivator paralogs CREB binding protein (CBP) and p300 to the promoter. The NMR structure of the CREB:CBP complex, using relevant interaction domains called KID and KIX, respectively, reveals a shallow hydrophobic groove on the surface of KIX that accommodates an amphipathic helix in phospho (Ser-133) KID. Using an NMR-based screening approach on a preselected small-molecule library, we identified several compounds that bind to different surfaces on KIX. One of these, KG-501 (2-naphthol-AS-E-phosphate), targeted a surface distal to the CREB binding groove that includes Arg-600, a residue that is required for the CREB:CBP interaction. When added to live cells, KG-501 disrupted the CREB: CBP complex and attenuated target gene induction in response to cAMP agonist. These results demonstrate the ability of small molecules to interfere with second-messenger signaling cascades by inhibiting specific protein-protein interactions in the nucleus.

Fig. 1.

Analysis of KIX residues affected by addition of KG-122, pamoic acid (a), and KG-501, naphthol-AS-E-phosphate (b). (Top) The ¹⁵N-¹H heteronuclear single quantum coherence spectra show KIX alone (black) and with compound added (red) at [protein] = 250 μM and [compound] = 500 μM. (Middle) The bar charts represent the combined ¹H and ¹⁵N chemical shift changes (Δδ = [Δδ(¹H)² + 0.1*Δδ(¹⁵N)²]^1/2) per residue. Helical regions of the KIX domain are represented as rectangles. Gray bars in a indicate amide residues whose peaks have broadened beyond detection because of addition of KG-122 and for which an arbitrary value of 0.35 ppm was assigned. The black line represents that average shift, and the red line indicates +1 SD. (Bottom) Ribbon representations of KIX, yellow (Protein Data Bank ID code 1KDX), highlighting backbone amides exhibiting significant chemical shift changes upon the addition of KG-122 or KG-501. For reference, the pKID polypeptide has been included and is shown in green. For KG-122, the average shift was 0.078 ppm with a SD of 0.064 ppm. Magenta spheres, Δδ > 0.142 ppm (1 SD more than average); pink spheres, 0.11 <Δδ < 0.142 ppm (0.5 SD). For KG-501 the average shift was 0.063 ppm with a SD of 0.048 ppm. Magenta spheres, Δδ > 0.111 ppm (1 SD); pink spheres, 0.087 <Δδ < 0.111 ppm (0.5 SD). KG-122 and KG-501 are shown on the same scale as the KIX structure (stick model) to compare their relative sizes.

Fig. 2.

KG-501 inhibits the in vitro interaction between the CBP KIX domain and phospho (Ser-133) CREB. (a) Comparison of KG-501 (2-naphthol-AS-E-phosphate) and KG-122 (pamoic acid) effects on CREB:CBP interaction relative to 2-naphthol. Purified GST-KIX protein was preincubated with 100 μM of each compound. ³²P-labeled Ser-133 phosphorylated CREB protein (0.2 pmol) was added to the binding reaction, and the bound proteins were precipitated with glutathione-agarose. Samples were analyzed by gel electrophoresis followed by autoradiography. In, 20% input ³²P-CREB. (b) Purified GST-KIX protein was preincubated with increasing micromolar concentrations of KG-501 or 2-naphthol. In, 10% input ³²P-CREB. (c) KG-501 inhibits binding of CREB to full-length p300. Purified flag-tagged full-length p300 protein was preincubated with the indicated micromolar concentrations of KG-501. ³²P-labeled Ser-133 phosphorylated CREB protein was added to the binding reaction, and the bound proteins were precipitated with anti-Flag antibody-conjugated agarose. Samples were analyzed as in a. In, 20% input ³²P-CREB. (d) KG-501 does not inhibit CREB dimer formation or complex formation between CREB and the coactivator TORC1. GST, GST-KIX, GST-CREB bZIP (amino acids 271–341) (Upper) or GST-TORC1 (Lower) fusion proteins were preincubated with DMSO vehicle (–) or increasing amounts of KG-501 compound [100 and 250 μM(Upper) and 100, 250, and 500 μM(Lower)]. Complexes of CREB bound to KIX were precipitated and analyzed as in a. In, 10% input ³²P-CREB. (e) Effect of KG-501 on p300 HAT activity. Purified p300 was incubated with DMSO (–), KG-501, or the p300 HAT inhibitor lysyl CoA (28). Acetylase activity was measured by incubation with histones and ¹⁴C-acetyl CoA followed by gel electrophoresis (15% gel) and autoradiography.

Fig. 3.

KG-501 inhibits CREB signaling in vivo. (a) Effect of KG-501 on induction of the CREB-responsive promoter evenskipped (EVX-1) by cAMP agonist. EVX-1 luciferase reporter plasmid was transfected into HEK293T cells. At 24 h posttransfection, cells were pretreated with the indicated amount of KG-501 or DMSO vehicle for 20 min followed by coincubation with forskolin or DMSO. Cells were harvested at 4 h, and luciferase activity was measured. Luciferase activity was normalized to β-gal activity from cotransfected Rous sarcoma virus-β-gal expression plasmid. (b) Quantitative PCR analysis of mRNA levels for CREB target gene NR4A2. Relative mRNA levels were normalized to GAPDH expression. Effect of KG-501 (10 and 25 μM) on target gene expression in HEK293T cells under basal and forskolin (10 μM, 45 min) treated conditions are shown. (c) KG-501 does not interfere with CREB Ser-133 phosphorylation in response to forskolin. HEK293T cells were pretreated with 10 μM KG-501 or DMSO vehicle for 30 min and then coincubated with forskolin. (Upper) Cells were harvested at the indicated time points, and whole-cell lysates were analyzed by Western blot analysis using a specific anti-phospho-CREB antibody (5322). (Lower) Effect of pretreatment with DMSO, KG-501, or the PKA inhibitor H89 on CREB phosphorylation after addition of forskolin for 30 min. (d) Effect of KG-501 on GAL4 CREB (amino acids 1–283) activity in HEK293T cells cotransfected with GAL4-luciferase reporter plasmid and treated with forskolin as in a. (e) KG-501 inhibits the mammalian two-hybrid interaction between Gal4-KID with KIX-VP16. HEK293T cells were transfected with GAL4-KID, VP16-KIX, and GAL4-luciferase reporter plasmids and treated as in a with 10 μM KG-501. (f) Effect of KG-501 on the CREB:CBP interaction in living cells. FRET analysis of YFP-KID (YKIDN) with CFP-KIX (KIXCN) polypeptides. HEK293T cells were transfected with the YKIDN and KIXCN plasmids and preincubated with KG-501 (50 μM). The cells were then stimulated with forskolin, and FRET efficiency was measured as described (18).