Molecular docking-aided identification of small molecule inhibitors targeting ß-catenin-TCF4 interaction.

Here we report a molecular docking-based approach to identify small molecules that can target the ß-catenin (ß-cat)-TCF4 protein-protein interaction (PPI), a key effector complex for nuclear Wnt signaling activity. Specifically, we developed and optimized a computational model of ß-cat using publicly available ß-cat protein crystal structures, and existing ß-cat-TCF4 interaction inhibitors as the training set. Using our computational model to an in silico screen predicted 27 compounds as good binders to ß-cat, of which 3 were identified to be effective against a Wnt-responsive luciferase reporter. In vitro functional validation experiments revealed GB1874 as an inhibitor of the Wnt pathway that targets the ß-cat-TCF4 PPI. GB1874 also affected the proliferation and stemness of Wnt-addicted colorectal cancer (CRC) cells in vitro. Encouragingly, GB1874 inhibited the growth of CRC tumor xenografts in vivo, thus demonstrating its potential for further development into therapeutics against Wnt-associated cancer indications.

Identification and characterization of a novel Sso7d scaffold-based binder against Notch1.

Notch signaling has important functions in regulating cell growth and development, misregulation of which has been implicated in various cancers. Monoclonal antibodies (mAbs) targeting Notch protein activity have already moved into clinical trials. However due to the limitations associated with cost and productivity of mAbs, there has been a surge in the development of complementary approaches that are based on non-antibody scaffolds. Non-antibody scaffolds are small proteins that are stable and can be engineered to develop high-affinity binders against specific targets of interest. Here we describe the isolation and characterization of a novel Notch1-binding protein, N9, obtained by screening of a combinatorial library based on the ultra-stable Sso7d scaffold. N9 targets the extracellular EGF-like repeats (ELR) 11-13 in Notch1, and therefore serves as a competitive inhibitor for Notch ligands to decrease expression of Notch target genes. We demonstrate that N9 recognizes surface expression of Notch1 on the plasma membrane and binds preferentially to cell lines misexpressing Notch1. Although N9 was selected against Notch1, we also observe cross-reactivity against other Notch receptors, including Notch2/3. Finally, we demonstrate that N9 inhibits proliferation and generation of tumorspheres in Notch expressing cancer cell lines, suggesting its potential as a therapeutic agent in Notch-associated malignancies.

Disrupting Interactions Between ß-Catenin and Activating TCFs Reconstitutes Ground State Pluripotency in Mouse Embryonic Stem Cells.

The 2i-media, composed of two small molecule inhibitors (PD0325901 and CHIR99021) against MEK and GSK3-kinases, respectively, is known to establish naïve ground state pluripotency in mouse embryonic stem cells (mESCs). These inhibitors block MEK-mediated differentiation, while driving ß-catenin dependent de-repression of pluripotency promoting targets. However, accumulating evidence suggest that ß-catenin's association with activating TCFs (TCF7 and TCF7L2) can induce expression of several lineage-specific prodifferentiation genes. We posited that CHIR-induced upregulation of ß-catenin levels could therefore compromise the stability of the naïve state in long-term cultures. Here, we investigated whether replacing CHIR with iCRT3, a small molecule that abrogates ß-catenin-TCF interaction, can still retain ground state pluripotency in mESCs. Our data suggests that iCRT3 + PD mediated coinhibition of MEK and ß-catenin/TCF-dependent transcriptional activity over multiple passages significantly reduces expression of differentiation markers, as compared to 2i. Furthermore, the ability to efficiently contribute toward chimera generation and germline transmission suggests that the inhibition of ß-catenin's TCF-dependent transcriptional activity, independent of its protein expression level, retains the naïve ground state pluripotency in mESCs. Additionally, growth medium containing iCRT3 + PD can provide an alternative to 2i as a stable culture method. Stem Cells 2017;35:1924-1933.

Inhibition of ß-catenin-TCF1 interaction delays differentiation of mouse embryonic stem cells.

The ability of mouse embryonic stem cells (mESCs) to self-renew or differentiate into various cell lineages is regulated by signaling pathways and a core pluripotency transcriptional network (PTN) comprising Nanog, Oct4, and Sox2. The Wnt/ß-catenin pathway promotes pluripotency by alleviating T cell factor TCF3-mediated repression of the PTN. However, it has remained unclear how ß-catenin's function as a transcriptional activator with TCF1 influences mESC fate. Here, we show that TCF1-mediated transcription is up-regulated in differentiating mESCs and that chemical inhibition of ß-catenin/TCF1 interaction improves long-term self-renewal and enhances functional pluripotency. Genetic loss of TCF1 inhibited differentiation by delaying exit from pluripotency and conferred a transcriptional profile strikingly reminiscent of self-renewing mESCs with high Nanog expression. Together, our data suggest that ß-catenin's function in regulating mESCs is highly context specific and that its interaction with TCF1 promotes differentiation, further highlighting the need for understanding how its individual protein-protein interactions drive stem cell fate.

PAPTi: a peptide aptamer interference toolkit for perturbation of protein-protein interaction networks.

Signaling proteins often form dynamic protein-protein interaction (PPI) complexes to achieve multi-functionality. Methods to abrogate a subset of PPI interfaces without depleting the full-length protein will be valuable for structure-function relationship annotations. Here, we describe the use of Peptide Aptamer Interference (PAPTi) approach for structure-function network studies. We identified peptide aptamers against Dishevelled (Dsh) and ß-catenin (ß-cat) to target the Wnt signaling pathway and demonstrate that these FN3-based MONOBODYs (FNDYs) can be used to perturb protein activities both in vitro and in vivo. Further, to investigate the crosstalk between the Wnt and Notch pathways, we isolated FNDYs against the Notch Ankyrin (ANK) region and demonstrate that perturbing the ANK domain of Notch increases the inhibitory activity of Notch towards Wnt signaling. Altogether, these studies demonstrate the power of the PAPTi approach to dissect specific PPI interactions within signaling networks.

Temporal coordination of gene networks by Zelda in the early Drosophila embryo.

In past years, much attention has focused on the gene networks that regulate early developmental processes, but less attention has been paid to how multiple networks and processes are temporally coordinated. Recently the discovery of the transcriptional activator Zelda (Zld), which binds to CAGGTAG and related sequences present in the enhancers of many early-activated genes in Drosophila, hinted at a mechanism for how batteries of genes could be simultaneously activated. Here we use genome-wide binding and expression assays to identify Zld target genes in the early embryo with the goal of unraveling the gene circuitry regulated by Zld. We found that Zld binds to genes involved in early developmental processes such as cellularization, sex determination, neurogenesis, and pattern formation. In the absence of Zld, many target genes failed to be activated, while others, particularly the patterning genes, exhibited delayed transcriptional activation, some of which also showed weak and/or sporadic expression. These effects disrupted the normal sequence of patterning-gene interactions and resulted in highly altered spatial expression patterns, demonstrating the significance of a timing mechanism in early development. In addition, we observed prevalent overlap between Zld-bound regions and genomic "hotspot" regions, which are bound by many developmental transcription factors, especially the patterning factors. This, along with the finding that the most over-represented motif in hotspots, CAGGTA, is the Zld binding site, implicates Zld in promoting hotspot formation. We propose that Zld promotes timely and robust transcriptional activation of early-gene networks so that developmental events are coordinated and cell fates are established properly in the cellular blastoderm embryo.