The ubiquitin proteasome system is an essential intracellular degradation pathway intrinsic to cellular growth control and therapeutics targeting the proteosome, the endpoint in this pathway, are now used in clinical practice for the treatment of cancers. E6AP is an E3 ubiquitin ligase, targeting proteins for proteasomal degradation. E6AP is a HECT domain E3 ligase, critical for the suppression of p53 in HPV-related cancers, and in certain contexts also in non-HPV infected cells. More recently, we have revealed a role for E6AP as a key regulator of PML. p53 and PML are known tumour suppressors. A partial loss of E6AP is sufficient to attenuate Myc-induced B-cell lymphomagenesis
We developed a high throughput functional in vitro ubiquitination assay, utilizing E1 and E2 enzymes in conjunction with E6AP and protein substrates. The ubiquitination of E6AP or E6AP substrate was measured with Alphascreen technology.
The AlphasScreen assay was optimized, validated and subsequently miniaturized to 1536-well plate format and used to screen a library of 250,000 lead-like molecules for inhibitors of E6AP activity. Additional in vitro E3 ligase assays were also developed and used in both selectivity and counter screens to deconvolute E6AP inhibitors from those that blocked E1 and E2 ligases. We identified a number of compounds with selective inhibitory activity against E6AP.
E6AP is an emerging target for the development of new drugs for the treatment of HPV-related cancers and malignancies where PML expression is suppressed.
A versatile and robust HTS platform has been designed and validated to identify small molecule inhibitors of E3 ligases.
In one of the first examples of an E3 ligase high-throughput screen, we have been able to identify potent inhibitors of E6AP, an E3 ligase capable of marking tumour suppressor proteins for proteasomal degradation.