Discovery of Small-Molecule Inhibitors of the PTK7/β-Catenin Interaction Targeting the Wnt Signaling Pathway in Colorectal Cancer

Abstract

Colorectal cancer (CRC), the second cause of death due to cancer worldwide, is a major public health issue. The discovery of new therapeutic targets is thus essential. Pseudokinase PTK7 intervenes in the regulation of the Wnt/β-catenin pathway signaling, in part, through a kinase domain-dependent interaction with the β-catenin protein. PTK7 is overexpressed in CRC, an event associated with metastatic development and reduced survival of nonmetastatic patients. In addition, numerous alterations have been identified in CRC inducing constitutive activation of the Wnt/β-catenin pathway signaling through β-catenin accumulation. Thus, targeting the PTK7/β-catenin interaction could be of interest for future drug development. We have developed a NanoBRET screening assay recapitulating the interaction between PTK7 and β-catenin to identify compounds able to disrupt this protein−protein interaction. A high-throughput screening allowed us to identify small-molecule inhibitors targeting the Wnt pathway signaling and inducing antiproliferative and antitumor effects in vitro in CRC cells harboring β-catenin or adenomatous polyposis coli (APC) mutations. Thus, inhibition of the PTK7/β-catenin interaction could represent a new therapeutic strategy to inhibit cell growth dependent on the Wnt signaling pathway. Moreover, despite a lack of enzymatic activity of its tyrosine kinase domain, targeting the PTK7 kinase domain-dependent functions appears to be of interest for further therapeutic development.

PTK7 is a positive allosteric modulator of GPR133 signaling in glioblastoma

“…Third, we have so far analyzed the agonistic influence of the PTK7-GPR133 interaction on GPR133 signaling, but its effects on PTK7 function are unknown. PTK7 is a known component of Wnt signaling complexes, 23 – 25 and the Wnt system, in turn, is an important tumorigenic mechanism in GBM. 26 Our signaling data suggested a trend of Wnt5a administration interfering with PTK7’s positive allosteric effect on GPR133 signaling, which could suggest that Wnt5a and GPR133 are competing for PTK7 binding.…”

PTK7 is a positive allosteric modulator of GPR133 (ADGRD1) signaling in GBM

“…Third, we have so far analyzed the agonistic influence of the PTK7-GPR133 interaction on GPR133 signaling, but its effects on PTK7 function are unknown. PTK7 is a known component of Wnt signaling complexes (Dunn & Tolwinski, 2016; Ganier et al , 2022; Peradziryi et al , 2012), and the Wnt system, in turn, is an important tumorigenic mechanism in GBM (Barzegar Behrooz et al , 2022). We, therefore, intend to dissect bidirectional effects of this novel interaction on both PTK7 and GPR133 function in the future.…”

Recent insights into the therapeutic strategies targeting the pseudokinase PTK7 in cancer

“…Despite having a crystal structure of the PTK7 kinase domain, we do not yet understand the precise molecular mechanism behind these interactions and how these transmit cellular signaling as done with CASK and its partners [ 24 ]. Nevertheless, the binding of the PTK7 pseudokinase domain to the armadillo repeats of β-catenin is direct, required for a signaling cascade from the membrane to the nucleus, and amenable to pharmacological inhibition [ 32 , 35 ].

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“…5B ) [ 32 ]. We recently reconstituted the PTK7-β-catenin interaction using an in cellulo NanoBRET (Bioluminescence Resonance Energy Transfer) format and identified small-molecule inhibitors able to disrupt this protein-protein interaction and downregulate WNT signaling similarly to PTK7 knockdown [ 32 , 35 ]. Our observation in CRC cells was not confirmed in normal cells, probably due to the different landscape of WNT co-receptors and ligands associated to PTK7 [ 44 ].…”

“…We and others have accumulated evidence that the PTK7 kinase domain is endowed with signaling functions linked to cancer despite its deficient catalytic activity, probably through signalling docking property [ 32 – 34 , 51 , 93 ]. We identified β-catenin as a direct partner of PTK7 and demonstrated the requirement of this interaction in WNT/β-catenin signaling regardless of the mutational status of APC and CTNN1 genes [ 32 , 35 ]. Through a multi-screening strategy based on a newly developed NanoBRET assay recapitulating the interaction between PTK7 and β-catenin that combined virtual screening, high throughput screening, and repurposing of drugs in development, we demonstrated that inhibition of this protein-protein interaction is achievable with small molecules.…”