Novel Yeast Bioassay for High-Throughput Screening of Matrix Metalloproteinase Inhibitors

Abstract

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...

Characterization and regulation of MT1‐MMP cell surface‐associated activity

“…The inhibition of MMP cell surface activity has been quantified previously using fluoresceinconjugated gelatin (Diehl et al, 2011). However, gelatin is a substrate for multiple proteases, and MMPs were expressed on the surface of yeast cells (Diehl et al, 2011), which are quite different from mammalian cell surfaces. As several selective and/or secondary binding site (exosite) MT1-MMP inhibitors have been described (Levin, Udi, Solomonov, & Sagi, 2017;Ling et al, 2017;Pahwa et al, 2014;Santamaria & de Groot, 2018), the evaluation of activity at the cell surface will allow for examination of such inhibitors in a more native-like environment.…”

“…Both small molecule and mini‐protein inhibitors were found to be active toward cell surface triple‐helical peptidase activity, albeit with reduced efficiency compared with inhibition in solution. The inhibition of MMP cell surface activity has been quantified previously using fluorescein‐conjugated gelatin (Diehl et al., ). However, gelatin is a substrate for multiple proteases, and MMPs were expressed on the surface of yeast cells (Diehl et al., ), which are quite different from mammalian cell surfaces.…”

Yeast-Based Biosensors: Current Applications and New Developments

“…Inhibitors of specific matrix metalloproteinases are therefore potential therapeutic targets and their search is one of the leads in oncology research. Diehl and colleagues developed a recombinant Pichia pastoris yeast strain that expresses biologically active human MMPs at the cell surface allowing to screen for MMPs [39]. Another screen based on the model yeast S. cerevisiae allows the identification of inhibitors of phosphatidylinositol 3-kinase (PI3K) [38].…”

Overview of Recent Progress in Protein-Expression Technologies for Small-Molecule Screening

“…80 The P. pastoris expression system has been recently used to screen for inhibitors of human matrix metalloproteases (MMPs) that are very difficult to express as active enzymes in prokaryotic systems due to their mechanism of activation. 81 MMP2 and MMP9 were both expressed on the cell surface of P. pastoris by fusing the target constructs to a cell wall anchor sequence, and the intact cells were used as the enzyme source for a screen with a fluorescent substrate that resulted in the discovery of potent inhibitors. 81…”

“…81 MMP2 and MMP9 were both expressed on the cell surface of P. pastoris by fusing the target constructs to a cell wall anchor sequence, and the intact cells were used as the enzyme source for a screen with a fluorescent substrate that resulted in the discovery of potent inhibitors. 81 Interestingly, descriptions of expression of human proteins in mammalian systems, and human cell-based platforms, are somewhat rare in the screening literature and in the online database of crystallized proteins. Even though such systems have been commercially available for some time, their use has often been limited to the production of antibodies.…”