Isolation and characterization of β-transducin repeat-containing protein ligands screened using a high-throughput screening system

Abstract

β-transducin repeat-containing protein (β-TrCP) is an F-box protein subunit of the E3 Skp1-Cullin-F box (SCF) type ubiquitin-ligase complex, and provides the substrate specificity for the ligase. To find potent ligands of β-TrCP useful for the proteolysis targeting chimera (PROTAC) system using β-TrCP in the future, we developed a high-throughput screening system for small molecule β-TrCP ligands. We screened the chemical library utilizing the system and obtained several hit compounds. The effects of the hit compounds on in vitro ubiquitination activity of SCF^β-TrCP1 and on downstream signaling pathways were examined. Hit compounds NPD5943, NPL62020-01, and NPL42040-01 inhibited the TNFα-induced degradation of IκBα and its phosphorylated form. Hence, they inhibited the activation of the transcription activity of NF-κB, indicating the effective inhibition of β-TrCP by the hit compounds in cells. Next, we performed an in silico analysis of the hit compounds to determine the important moieties of the hit compounds. Carboxyl groups of NPL62020-01 and NPL42040-01 and hydroxyl groups of NPD5943 created hydrogen bonds with β-TrCP similar to those created by intrinsic target phosphopeptides of β-TrCP. Our findings enhance our knowledge of useful small molecule ligands of β-TrCP and the importance of residues that can be ligands of β-TrCP.

Keywords: Chemical biology; F-box protein; Ubiquitin.

Figure 1. Screening system and high-throughput screening for ligands of β-TrCP. (A) Insect cell lysates expressing mAG-β-TrCP1 were added to 96-well plates that are covalently attached with IκB phosphopeptides. In the presence of the small molecules competing with the phosphopeptides for mAG-β-TrCP binding, the fluorescence of the plate decreases. (B) The phosphopeptide of β-TrCP binding sequence (C-IκB-PP15) and unphosphorylated version (C-IκB-s15) were bound to 96-well plates. The binding of mAG-β-TrCP1 to C-IκB-PP15 (closed circle) and C-IκB-s15 (open circle) was determined using spectrofluorometry. A concentration-dependent binding decrease was observed in the presence of the free IκB phosphopeptides (C-IκB-PP15). (The data are presented as the average ± S.D. The statistical differences among the groups were evaluated using one-way ANOVA. p-value: **** < 0.0001) (C) Results of initial screening of ~5,000 compounds. The effect of each compound (at 0.2 mg/mL) on mAG-β-TrCP1 binding was standardized and is represented as a percentage value of control (100% indicating no inhibition). The compounds that inhibited the binding by >20% were the initial hits.

Figure 2. Dose-dependent inhibition of ubiquitination by hit compounds of Wee1KR. In the presence of these seven compounds, in vitro ubiquitination of Wee1KR is inhibited in a dose-dependent manner (IC 50 of each compound obtained in (B) is shown) (B) The results in (A) were quantitated by ImageJ and plotted.

Figure 3. NPL72038-01 and NPL62039-01 accumulated phosphorylated β-catenin; however, they did not significantly affect the β-catenin signaling pathway. (A) Effect of MG132, GS143, and two positive compounds on the levels of β-catenin and phosphorylated β-catenin in HeLa cells. The levels are quantitated and shown under the panel as % of DMSO control. Increased values (>115) are shown in red. (B) Wnt3a (100 ng/mL) increased the level of TCF transcription. (C) Effect of GS143 and two compounds on TCF transcription in HeLa cells. The data are presented as the average (n = 3) ± S.D. The statistical differences among the groups were evaluated using one-way ANOVA. (p-value: ** < 0.01).

Figure 4. NPD5943, NPL42040-01, and NPL 62020-01 show an inhibitory effect in the NF-κB signaling pathway in a dose-dependent manner. (A) Effect of MG132, GS143, and three compounds on TNF-α induced IκBα degradation in HeLa cells. The levels of IκBα and phosphorylated IκBα in HeLa cells after the treatment are shown. The levels are quantitated and shown under the panel as % of DMSO control. (B) Effect of three positive compounds on TNF α induced NF-κB activation of transcription in HeLa cells: Samples were examined in triplicate. The data are presented as the average ± S.D. The statistical differences among the groups were evaluated using one-way ANOVA (p-value: **** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05).

Figure 5. In silico analysis of the hit compounds. (A) The interface of phosphorylated β-catenin (pS33, pS37) and β-TrCP: β-TrCP is shown in red, with its side chains in pink, and the phosphodegron peptides of β-catenin are in yellow (phosphate groups on serines are shown in green and red). White dotted lines represent H bonds. Amino acid residues linked to pS33 of β-catenin and those to pS37 are marked in yellow and white circles, respectively. These phosphoamino acids of β-catenin, pS33 and pS37 are marked in yellow and white boxes, respectively. The original figure from [7] was modified. (B) The interface of hit compounds and β-TrCP: β-TrCP is shown in light blue. When amino acids shown in (A) create an H bond with the compound, they are shown in the same color as in (A). (C) The interface of negative compound (NPL72029-01) and β-TrCP: β-TrCP is shown in light blue. The structure of NPL72029-01 is shown on the right.