Wnt signaling: chemical probes and drug discovery
Background and importance: The canonical Wnt/β-catenin signalling pathway plays a pivotal role in animal development and tissue homeostasis, and in human cancer. general Wnt… The signal transducer protein β-catenin lies at the centre of the pathway. Under normal conditions, the cellular concentration of β-catenin is tightly regulated and maintained to a low level. This regulatory mechanism is mediated by the action of a cytoplasmic multiprotein complex that induces phosphorylation and ubiquitination of β-catenin, leading to its proteasomal degradation (left). The scaffolding proteins Axin and Adenomatous Polyposis Coli (APC) form the core of this so-called “destruction complex” and can recruit the casein kinases 1α, β and δ (CK1) and glycogen synthase kinase 3 (GSK3). A sequential process involves binding of β-catenin to axin, followed by phosphorylation of β-catenin at Ser45 by CK1, and further phosphorylation at Ser33, Ser37 and Ser41 by GSK3. This series of phosphorylations promotes the transfer of β-catenin from axin to APC and allows axin to bind a new molecule of β-catenin. The APC:β-catenin complex exposes the N-terminally phosphorylated part of β-catenin to the ubiquitin ligase β-transducin-repeat-containing protein (β-TrCP). The latter is responsible for the ubiquitination of β-catenin that results in its degradation by the proteasome. Upon binding of a Wnt protein ligand to the extracellular domain of Fz transmembrane receptors, a signalling cascade induces the disruption of this complex, leading to β-catenin accumulation in the cytoplasm. β-catenin further translocates to the nucleus and associates with transcriptional regulators, resulting in activation of Wnt target genes (e.g. myc) and cell proliferation (right). In some ways, the degradative mode of regulation of β-catenin is analogous to that of p53 by its negative regulators MDM2/4, and offers a fast cellular response mechanism at the post-translational level upon extracellular stimuli.
A relationship between misregulation of Wnt signalling and cancer is now well established, notably in colorectal cancer (CRC). However, current therapies targeting CRCs remain largely toxic and ineffective. Approximately 1.4 million new CRC cases and 700,000 deaths by CRC are recorded every year, making CRC one of the leading causes of cancer related death in developed countries. In particular, hyperactivity of β-catenin (encoded by the CTNNB1 gene) is a hallmark of CRC, and down-regulation of its activity has been proposed as a high profile approach for developing novel classes of anticancer drugs. For example, disruption of PPIs between β-catenin and its nuclear partners has been proposed to inhibit Wnt signalling and cancer cell proliferation. A small number of compounds targeting the interface between β-catenin and Tcf4, Bcl9, B9L, LEF1 and CBP have been claimed and are currently under scrutiny. However, the majority of these compounds originate from functional screening assays and unambiguous evidence of target engagement through biophysical binding assessment and structural methods has not been reported. The high affinity of β-catenin PPIs (e.g. nanomolar Kd), large interacting surfaces (e.g. > 4000 Å2) and the high degree of overlap of the binding surfaces makes the development of small molecule inhibitors a daunting task. In particular, the lack of biophysical and structural data in this area has generally hampered further hit to lead optimisation, and perhaps provides an explanation for why none of the reported hits from high throughput screening campaigns have reached advanced clinical evaluation. As a result, the direct targeting of β-catenin with small molecules has remained unsuccessful, contributing to the common view that β-catenin is an “undruggable” target.
Research: Our aim is to challenge this dogma and to demonstrate that the “soluble pool” of β-catenin remains a prime target for small molecule modulation and anti-cancer drug development, and that its claimed low druggability in fact reflects the need for new approaches to assemble small molecules modulators. We are using a combination of fragment screening, bioprospecting and HTVS combined with molecular/cell/structural biology to identify new β-catenin signalling inhibitors as proof-of-principle chemical probes towards first-in-class therapeutics in CRC.