Identification and development of novel cyclic peptide inhibitors of IDOL mediated LDLR degradation

Cholesterol is an essential component of the plasma membrane however if present in excess, free cholesterol is toxic to cells and can lead to complications such as atherosclerosis, and ultimately cardiovascular disease. The low density lipoprotein receptor binds and internalises circulating plasma cholesterol, present in low density lipoprotein molecules. The low density lipoprotein receptor has two main transcriptional regulators: liver X receptors and sterol regulatory element binding proteins and post-transcriptional regulators, namely the E3-ubiquitin ligase IDOL, which binds and ubiquitinates the low density lipoprotein receptor intracellular tail, targeting it for lysosomal degradation. Several protein-protein interactions are required for the ubiquitination activity of IDOL, serving as potential therapeutic modulation sites for the treatment of hypercholesterolemia via an upregulation of low density lipoprotein receptor protein levels.Bacterial reverse-two hybrid systems were designed for the IDOL-LDLR heterodimerisation and the IDOL homodimerisation interactions. The latter system was used to screen a library of 3.2 million cyclic peptides, identifying a series of peptides capable of inhibiting the IDOL homodimerisation event. These peptides were synthesised and their activity assessed using a selection of in vitro assays, providing a lead candidate.The efficacy of the lead candidate peptide was improved through the development of a small library of non-natural derivatives, improving binding activity by 7-fold. The activity of the new generation peptide was assessed both in vitro for ability to inhibit autoubiquitination and in hepatic cells, elucidating biological implications of the inhibition of IDOL mediated LDLR degradation

Methods for the creation of cyclic peptide libraries for use in lead discovery

The identification of initial hits is a crucial stage in the drug discovery process. Although many projects adopt high-throughput screening of small-molecule libraries at this stage, there is significant potential for screening libraries of macromolecules created using chemical biology approaches. Not only can the production of the library be directly interfaced with a cell-based assay, but these libraries also require significantly fewer resources to generate and maintain. In this context, cyclic peptides are increasingly viewed as ideal scaffolds and have proven capability against challenging targets such as protein-protein interactions. Here we discuss a range of methods used for the creation of cyclic peptide libraries and detail examples of their successful implementation

Methods for the Creation of Cyclic Peptide Libraries for Use in Lead Discovery

The identification of initial hits is a crucial stage in the drug discovery process. Although many projects adopt high-throughput screening of small-molecule libraries at this stage, there is significant potential for screening libraries of macromolecules created using chemical biology approaches. Not only can the production of the library be directly interfaced with a cell-based assay, but these libraries also require significantly fewer resources to generate and maintain. In this context, cyclic peptides are increasingly viewed as ideal scaffolds and have proven capability against challenging targets such as protein-protein interactions. Here we discuss a range of methods used for the creation of cyclic peptide libraries and detail examples of their successful implementation

Inhibition of low-density lipoprotein receptor degradation with a cyclic peptide that disrupts the homodimerization of IDOL E3 ubiquitin ligase

Cellular uptake of circulating cholesterol occurs via the low density lipoprotein receptor (LDLR). The E3 ubiquitin ligase IDOL is a mediator of LDLR degradation, with IDOL homodimerization thought to be required for its activity. To probe the possibility of modulating LDLR levels with an inhibitor of IDOL homodimerization, we screened a SICLOPPS library of 3.2 million cyclic peptides for compounds that disrupt this protein-protein interaction. We identified cyclo-CFFLYT as the lead inhibitor, and improved its activity through the incorporation of non-natural amino acids. The activity of the optimized cyclic peptide was assessed in hepatic cells, with a dose-dependent increase in LDLR levels observed in the presence of our IDOL homodimerization inhibitor

Dataset for Inhibition of low-density lipoprotein receptor degradation with a cyclic peptide that disrupts the homodimerization of IDOL E3 ubiquitin ligase

Raw data supporting: Leitch, E. et al (2018). Inhibition of low-density lipoprotein receptor degradation with a cyclic peptide that disrupts the homodimerization of IDOL E3 ubiquitin ligase. Chemical Science. </span