Thiol rearrangements in a viral A/B protein toxin are induced by an increase in pH that reflects compartmental pH changes during host cell intoxication. Under endoplasmic reticulum-mimicking conditions, such rearrangements are prevented by protein disulfide isomerase activity, whereas cytosolic conditions induce toxin-subunit dissociation and release of cytotoxic α monomers.

Diverse malfunctions in the expression and regulation of matrix metalloproteinases (MMPs) are often the cause of severe human diseases, bringing the identification of specific MMP inhibitors into major focus, particularly in anticancer treatment. Here, we describe a novel bioassay based on recombinant yeast cells (Pichia pastoris) that express, deliver, and incorporate biologically active human MMP-2 and MMP-9 at the yeast cell surface. Using Sed1p for cell wall targeting and covalent anchorage, a highly efficient bioassay was established that allows high-throughput screening and subsequent validation of novel MMP inhibitors as potential anticancer drugs. In addition, we developed a straightforward synthesis of a new aspartate-derived MMP inhibitor active in the nM range and bearing an amino functionality that should allow the introduction of a wide range of side chains to modify the properties of these compounds.Diverse malfunctions in the regulation of human matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, play a key role in the development of a wide range of diseases, including diabetes mellitus, arthritis, cardiovascular diseases, and, most of all, cancer (5, 16). Recent oncology research is therefore focusing on the specific inhibition of distinct MMPs as potential therapeutic targets (3). Most of these inhibitors are peptidic succinates with a hydroxamate functionality, binding to the zinc ion at the active site of the proteolytic enzyme (1, 7, 10). For example, hydroxamate 1 (Fig. 1, structure 1) shows significant selectivity toward MMP-2, -8, and -9 relative to other MMPs (17), which can be explained by the interaction of the nonpolar phenylpropyl side chain with the deep, tunnellike binding pockets of these enzymes (20). The introduction of polar substituents (e.g., R ϭ OH) onto the ␣-position of the hydroxamate group in general results in higher solubility and oral bioavailability (23,27).Nevertheless, early generations of MMP inhibitors (MMPIs) did not meet the high expectations for them in clinical trials, as poor inhibitor specificities caused massive side effects due to the inhibition of non-MMP targets (5,14,16,24). Thus, identification of more specific inhibitors is currently a major focus in MMPI development. Until now, the design and validation of novel inhibitors have often been hampered by costly and timeconsuming MMP purification from human tumor cell lines or primary fibroblasts (9), as well as by the lack of a suitable high-throughput bioassay for comprehensive MMP inhibitor screening. To bypass such a limitation, the major objective of the present study was to immobilize biologically active human MMPs on the surface of yeast (Pichia pastoris) and to establish a cell-based bioassay for MMP inhibitor screening. In addition, we developed a straightforward synthesis of a potential inhibitor of these MMPs based on structure 1 (Fig. 1), bearing an amino functionality at the ␣ position, which should allow the introduction of a wide range of side chains to modify the properties of th...