Screening for E3-ubiquitin ligase inhibitors: challenges and opportunities

Abstract

The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, and its deregulation has been implicated in a wide range of human pathologies that include cancer, neurodegenerative and immunological disorders and viral infections. Targeting the UPS by small molecular regulators thus provides an opportunity for the development of therapeutics for the treatment of several diseases. The proteasome inhibitor Bortezomib was approved for treatment of hematologic malignancies by the FDA in 2003, becoming the first drug targeting the ubiquitin proteasome system in the clinic. Development of drugs targeting specific components of the ubiquitin proteasome system, however, has lagged behind, mainly due to the complexity of the ubiquitination reaction and its outcomes. However, significant advances have been made in recent years in understanding the molecular nature of the ubiquitination system and the vast variety of cellular signals that it produces. Additionally, improvement of screening methods, both in vitro and in silico, have led to the discovery of a number of compounds targeting components of the ubiquitin proteasome system, and some of these have now entered clinical trials. Here, we discuss the current state of drug discovery targeting E3 ligases and the opportunities and challenges that it provides.

Figure 1. Potential drug targets in the Ubiquitin Proteasome System (UPS)

Drugging the UPS has become a major research interest in recent years and several drugs targeting various components of the machinery are currently in clinical and pre-clinical development. Small molecules and peptides are being developed that either affect the intrinsic activity of enzymes involved in the cascade (depicted as red dotted lines) or interfere with protein-protein interactions (depicted as blue dotted lines). E1= ubiquitin- activating enzyme; E2= ubiquitin conjugating enzyme, ub=ubiquitin; ATP= adenosine triphosphate; AMP= adenosine monophosphate.

Figure 2. p53-MDM2 negative feedback loop

In response to its activation the tumour suppressor p53 leads to expression of a number of target genes including its key regulator MDM2. The E3 ligase MDM2 then mediates p53 ubiquitination leading to its proteasomal degradation. This negative feedback ensures fine control of the duration of the p53 response and immediate termination upon loss of p53 stimulating signals. MDM2=Mouse double minute 2.

Figure 3. Regulation of the HECT E3 ligase Itch Itch is an important negative regulator of the transcription factors p63 and p73 leading to their polyubiquitination and subsequent proteasomal degradation

p73 ubiquitination by Itch activity is inhibited by AMPK mediated phosphorylation of p73. c-Jun induces YAP expression which binds to Itch and inhibits its activity. Cyclic peptides mimicking the Itch binding interface on p63 inhibit Itch. AMPK= AMP-activated protein kinase; YAP= Yes-Associated Protein.

Figure 4. Development of an Itch inhibitor

(A) Flowchart of a high throughput screen to identify Itch inhibitors. The screen was carried out using an ELISA based assay with Itch immobilised on the plate and all other components of the ubiquitination reaction in solution. Itch ubiquitination was determined using an antibody detecting poly-ubiquitin. The effect of ~21 000 compounds on Itch autoubiquitination was determined. Compounds that showed an inhibitory effect were further validated using dose response curves of the ELISA based assay and in vitro ubiquitination assays with the Itch substrate p73. In a further validation step an effect of the identified compound, clomipramine, on the E1 and E2 enzymes was ruled out and structural homologues of clomipramine were analysed for Itch inhibitory activity. ELISA= enzyme-linked immunosorbent assay. (B) Drugable surface of Itch and predicted Clomipramine binding sites. The active cysteine of the HECT ligase Itch is shown as blue spheres and the predicated drugable area, based on electrostatics and proximity to the active cysteine, is shown in yellow. The binding site of clomipramine was predicted using docking programs and the two binding sites that exhibit low-energy conformations are shown in green.