Discovery of inhibitors of SIAH function
Sue Eccles, Swen Hoelder, Will Court, Lisa Pickard, Nadia Kadi, Lewis Vidler, Rosemary Burke, Rob van Montfort
The SIAH1 and 2 (Seven in Absentia Homologue) proteins are RING finger E3 ligases responsible for regulating levels of a number of important intracellular proteins via polyubiquitination and proteasomal degradation. This is a 3-step enzymatic process involving ubiquitin activation (E1) conjugation (E2) and ligation (E3). The latter step controls the specificity of the process, since E3 ligases recognise selective degradation signals (degrons) within their target substrates.
SIAH proteins have been identified as key proximal regulators of cellular responses to hypoxia and also have additional functions in RAS signalling. Hypoxia inducible factor-1alpha (HIF-1alpha) is normally rapidly degraded by the ubiquitin-proteasomal pathway, but under hypoxic conditions (common in many solid tumours) it is stabilised and dimerises with HIF-1beta.
The resulting transcription factor complex upregulates not only angiogenic factors, but also proteins promoting invasion and glycolytic metabolism, favouring progression towards a more malignant and therapy-resistant phenotype. Targeting SIAH proteins, which operate at the apex of the hypoxic response, could potentially overcome the issues of targeting individual downstream angiogenic factors which may lead to escape or enhanced malignant potential.
To increase our chances for success in discovering SIAH inhibitors, we are simultaneously pursuing several complementary screening approaches within an international consortium of collaborators (including Dr David Bowtell at PeterMac Cancer Centre, Dr Michael Parker at St Vincent’s Institute, Dr Ian Street, CTx (Melbourne) and Dr Ali Tavassoli (University of Southampton). At ICR we have initially focused on inhibiting the interaction between SIAH and peptides containing the VxP degron recognition motif e.g. the Drosophila protein phyllopod (PHYL) which binds with high affinity to the SIAH substrate binding domain (SBD).
This interface is well characterised in crystal structures (see Figure) and inhibitory peptides have provided proof of principle for this approach in vitro and in vivo. Secondly we are undertaking a fragment screen using a recently acquired Biacore SPR system. To support the drug discovery effort, we are engineering cells to express tagged SIAH and PHD3 (substrate) proteins in order to follow binding (for example using Duolink proximity ligation assays) and degradation steps. We have in place functional assays in hypoxia and appropriate tumour and angiogenesis models for downstream evaluation of promising compounds.