Casitas B-Lineage Lymphoma RING Domain Inhibitors Protect Mice against High-Fat Diet-Induced Obesity and Insulin Resistance.

The casitas b-lineage lymphoma (c-Cbl) is an important adaptor protein with an intrinsic E3 ubiquitin ligase activity that interacts with E2 proteins such as UbCH7. c-Cbl plays a vital role in regulating receptor tyrosine kinase signaling. c-Cbl involves in whole-body energy homeostasis, which makes it a potential target for the treatment of type 2 diabetes and obesity. In the present study, we have designed two parental peptides and 55 modified peptides based on the structure of UbCH7 loop L1 and L2. Thirteen of the modified peptides showed increased inhibitory activity in a fluorescence polarization-based assay. In the in vivo proof of study principle, mice treated with peptides 10, 34, 49 and 51 were protected against high-fat diet-induced obesity and insulin resistant. These inhibitors may potentially lead to new therapeutic alternatives for obesity and type 2 diabetes

Development of a Fluorescence Polarization Based High-Throughput Assay to Identify Casitas B-Lineage Lymphoma RING Domain Regulators

<div><p>The E3 ubiquitin protein ligase Casitas B-lineage Lymphoma (Cbl) proteins and their binding partners play an important role in regulating signal transduction pathways. It is important to utilize regulators to study the protein-protein interactions (PPIs) between these proteins. However, finding specific small-molecule regulators of PPIs remains a significant challenge due to the fact that the interfaces involved in PPIs are not well suited for effective small molecule binding. We report the development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule regulators of Cbl (RING) domain. The FP assay was used to measure binding affinities and inhibition constants of UbCH7 peptides and small molecule regulators of Cbl (RING) domains, respectively. In order to rule out promiscuous, aggregation-based inhibition, two assay conditions were developed and compared side by side. Under optimized conditions, we screened a 10,000 natural compound library in detergent-free and detergent-present (0.01% Triton X-100) systems. The results indicate that the detergent-present system is more suitable for high-throughput screens. Three potential compounds, methylprotodioscin, leonuride and catalpol, have been identified that bind to Cbl (RING) domain and interfere with the Cbl (RING)-UbCH7 protein-protein interaction.</p></div

Sequences and inhibitory activity of Cbl RING domain inhibitors derived from UbCH7 L2.

<p>Letters in bold in the sequence refer to the structure in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135916#pone.0135916.g001" target="_blank">Fig 1</a>.</p><p>Sequences and inhibitory activity of Cbl RING domain inhibitors derived from UbCH7 L2.</p

<i>In vivo</i> studies of c-Cbl inhibitors.

<p><b>A-B</b>. Pharmacokinetic studies of parental peptides and modified peptides. Peptides were administered to mice by i.p. injection (4 mg/kg). Blood samples were collected from the tail tip at the indicated time points. Plasma samples were harvested and analyzed using RP-HPLC with EI-MS detection. <b>C-D</b>. Body weight during 12 weeks feeding with a high-fat diet. <b>E</b>. Food intake. <b>D</b>. Percentage of perigonadal fat mass. Mice were fed ad libitum with the high-fat diet before experiments and for another 12 weeks during the experiments. Mice were treated with vehicle or indicated peptides with a daily i.p. injection at 5 mg/kg. Body weight, food intake were measured manually on a daily basis. Perignonadal fat mass was measured using NMR technology. * p > 0.05. **p < 0.05.</p

Indirect energy expenditure and physical activity measurements result.

<p>Indirect energy expenditure and physical activity measurements result.</p

FP assay performance and post-screen analysis summary.

<p>FP assay performance and post-screen analysis summary.</p

Summary of hit compounds.

<p>Summary of hit compounds.</p

Assay development.

<p>A. The structure of PFKPP derived from UbCH7 L1 loop and c-Cbl RING domain complex. B. The structure of KPATK derived from UbcH7 L2 loop and c-Cbl RING domain complex (PDB ID: 1FBV). C. c-Cbl (RING) – Probe 1 peptide binding isotherms: an increasing concentration of protein (5 nmol/L to 11 µmol/L) was titrated into 100 nmol/L probe probe 1. K_d = 1.67±0.09 µmol/L; Δmp = 150 units. D. c-Cbl (RING) – Probe 2 peptide binding isotherms: an increasing concentration of protein (5 nmol/L to 11 µmol/L) was titrated into 100 nmol/L probe 2. K_d = 0.14±0.08 µmol/L; Δmp = 230 units.</p

<i>In vivo</i> study experimental groups.

<p><i>In vivo</i> study experimental groups.</p

A-B. Adipocyte volume.

<p>Mice were fed ad libitum with the high-fat diet before experiments and for another 12 weeks during the experiments. Mice were treated with vehicle or indicated peptides with a daily i.p. injection at 5 mg/kg. After the experiment, isolated adipocytes were obtained from excised epididymal fat pad.</p