Identification of inhibitors for MDM2 ubiquitin ligase activity from natural product extracts by a novel high-throughput electrochemiluminescent screen.

High-throughput screening technologies have revolutionized the manner in which potential therapeutics are identified. Although they are the source of lead compounds for ~65% of anticancer and antimicrobial drugs approved by the Food and Drug Administration between 1981 and 2002, natural products have largely been excluded from modern screening programs. This is due, at least in part, to the inherent difficulties in testing complex extract mixtures, which often contain nuisance compounds, in modern bioassay systems.

Assays for high-throughput screening of E2 AND E3 ubiquitin ligases.

We developed a series of assays for biochemical activities involving ubiquitin. These assays use electrochemiluminescence detection to measure the ubiquitylation of target proteins. To enable electrochemiluminescence detection, the target proteins were prepared as bacterially expressed fusion proteins and captured on the surface of specially designed microtiter plates having integrated electrodes.

In vitro screening for substrates of the N-end rule-dependent ubiquitylation.

We describe a systematic, high-throughput approach to the discovery of protein substrates of ubiquitylation. This method uses a library of cDNAs in combination with a reticulocyte lysate-based, transcription-translation system that acts as both an excellent means for high-throughput protein expression and a source of ubiquitylation enzymes. Ubiquitylation of newly expressed proteins occurs in this milieu from the action of any one of a number of E3 ligases that are present in the lysate.

Small molecule inhibitors of HDM2 ubiquitin ligase activity stabilize and activate p53 in cells.

The p53 tumor suppressor protein is regulated by its interaction with HDM2, which serves as a ubiquitin ligase (E3) to target p53 for degradation. We have identified a family of small molecules (HLI98) that inhibits HDM2's E3 activity. These compounds show some specificity for HDM2 in vitro, although at higher concentrations effects on unrelated RING and HECT domain E3s are detectable, which could be due, at least in part, to effects on E2-ubiquitin thiol-ester levels.

Assay for ubiquitin ligase activity: high-throughput screen for inhibitors of HDM2.

An assay for the autoubiquitination activity of the E3 ligase HDM2 (Mdm2) was developed and adapted to a high-throughput format to identify inhibitors of this activity. The assay can also be used to measure the activity of other E3s and may be useful in finding both inhibitors and activators of a wide range of different ubiquitin ligases.

Hodgkin disease: pharmacologic intervention of the CD40-NF kappa B pathway by a protease inhibitor.

The malignant Reed-Sternberg cell of Hodgkin disease is an aberrant B cell that persists in an immunolgically mediated inflammatory infiltrate. Despite its nonproductive immunoglobulin genes, the Reed-Sternberg cell avoids the usual apoptotic fate of defective immune cells through an unknown mechanism. A likely candidate is the surface receptor, CD40, consistently expressed by Reed-Sternberg cells, and the first link in the pathway to NF-kappa B activation, the central regulator of cytokine production and apoptosis.