Michael R. Mattern

Strategies for the identification of ubiquitin ligase inhibitors

Dysregulation of the UPS (ubiquitin-proteasome system) has been implicated in a wide range of pathologies including cancer, neurodegeneration and viral infection. Inhibiting the proteasome has been shown to be an effective therapeutic strategy in humans; however, toxicity with this target remains high. E3s (Ub-protein ligases) represent an alternative attractive therapeutic target in the UPS. In this paper, we will discuss current platforms that report on E3 ligase activity and can detect E3 inhibitors, and underline the advantages and disadvantages of each approach.

New Cytotoxic Manzamine Alkaloids from a Palaun Sponge

A crude extract of a marine sponge showed initial inhibitory bioactivities in a yeast assay for inhibitors of methionine amino- peptidase-2 (Met AP-2). Bioassay-directed fractionation indicated that the activity was concentrated in the CH2Cl2-soluble fraction, and chromatography on silica gel led to the isolation of the two new bioactive alkaloids N-methyl-epi-manzamine D 1 and epi-manzamine D 2. The structures of the epi-manzamines were assigned by 1 H and 13 C NMR, DEPT, HMQC, and HMBC spectroscopy, and by comparison with the spectra of related compounds, and the structure of 1 was confirmed by X-ray structure analysis. Neither of the two isolated compounds showed selectivity in the yeast assay for inhibitors of Met AP-2, but both compounds were cytotoxic to HeLa and B16F10 mammalian cells, with compound 1 showing strong activity against the B16F10 cell line. q 2000 Elsevier Science Ltd. All rights reserved. Angiogenesis, or the development of new blood vessels, is an essential requirement of solid tumor growth, and it has been proposed to be the rate-limiting factor for tumor growth; angiogenesis inhibitors could thus play an impor- tant role in cancer chemotherapy.The antiangiogenic agent fumagillin has been shown to target methionine aminopep- tidase type 2 (Met AP-2); the related enzyme Met AP-1 is not affected by fumagillin. 1,2 Yeast strains are available with the genes for both Met AP-1 and Met AP-2 deleted; 3 compounds that act in the same way as fumagillin will be selective inhibitors of Met AP-2, and will thus show a greater growth inhibition for yeasts lacking Met AP-1 than for those lacking Met AP-2. 1 The differential sensitivity of Dmap1 and Dmap2 strains of yeast thus provides a tool for screening for angiogenesis inhibitors in plant extracts.

Detection and Characterization of SUMO Protease Activity Using a Sensitive Enzyme-Based Reporter Assay

In this chapter we describe a novel, sensitive, homogenous high throughput reporter-based in vitro assay for SUMO protease activity developed by Progenra, Inc. A reporter construct was created by fusing His(6)-tagged small ubiquitin-like modifier (SUMO) to the amino terminus of the reporter enzyme phospholipase A(2) (PLA(2)). Following cleavage by a member of the sentrin specific proteases (SENPs), free PLA(2) is able to turn over its substrate, resulting in the release of a fluorescent product which is readily quantifiable using a fluorimeter or a fluorescence plate reader. The utility of this SUMO-CHOP-Reporter assay platform is demonstrated by its ability to determine K(m) values and to characterize inhibitors of SUMO proteases.