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.

Fig. 1.

Hydroxamate based-selective MMP inhibitors. IC₅₀, 50% inhibitory concentration.

Fig. 2.

Experimental setup and schematic outline for MMP inhibitor screening through cell surface display of biologically active MMP-2 and MMP-9 in P. pastoris. (a) Expression cassette containing an N-terminal signal sequence (α-MF) to ensure proMMP import into the secretory pathway and signal peptidase cleavage, releasing proMMP into the ER lumen. The C-terminal anchoring domain of Sed1p mediates covalent MMP immobilization on the yeast cell wall via a GPI anchor-mediated process. (b) Recombinant proteins are delivered to the P. pastoris cell surface through the secretory pathway and are subsequently activated by autocatalytic removal of the MMP prodomain. (c) Activated MMPs are supplied with the desired inhibitor (red triangles) and fluorescein-labeled gelatin as a substrate (spirals). Subsequent cleavage of the substrate liberates fluorescing fragments that are detectable in a microplate fluorescence reader. (d) Model kinetics of remaining MMP activity at a given MMP inhibitor concentration (slopes in different colors) and subsequent calculation of a dose-effect diagram allowing quantitative comparison of different MMPIs. RFU, relative fluorescence units.

Fig. 3.

P. pastoris cell surface display of human MMP-2 and MMP-9 verified by indirect immunofluorescence microscopy. Cells of P. pastoris KM71 expressing the indicated MMP were cultivated for 72 h under inducing conditions in the presence of methanol. The MMPs were labeled with anti-MMP antibodies and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG and analyzed by fluorescence microscopy (Keyence BZ-8000; λ_A = 480 nm; λ_E > 510 nm). Scale bars = 10 μm.

Fig. 4.

Synthesis and evaluation of MMP inhibitor 2. The reagents and conditions were as follows. (a) 1.2 eq ZnCl₂, 2.5 eq lithium diisopropylamide (LDA), tetrahydrofurane (THF), −78°C to room temperature (RT), 20 h. (b) 1.1 eq Cs₂CO₃, 3.5 eq benzyl bromide (BnBr), dimethylformamide (DMF), 0°C to RT, 20 h. (c) 1, O_3, CH₂Cl₂, −78°C, 5 min; 2, PPh₃, −78°C, 30 min. (d) 10 eq NaClO₂, 7 eq NaH₂PO₄ · 2H₂O, 20 eq 2-methyl-2-butene, t-butanol/H₂O, RT, 16 h. (e) 1.0 eq 2-(1H-benzotriazole-1-yl-1,1,3,3-tetramethylaminum tetrafluoroborate (TBTU), 1.0 eq NEt₃, 1.2 eq (S)-PhNHMe, CH₂Cl₂, 0°C to RT, 18 h. (f) Pd/C (10%), H₂, THF (90%). (g) 1.0 eq ClCOOiBu, 1.0 eq NEt₃, 2.0 eq O-benzylhydroxylamine, THF, −20°C to RT, 18 h (90%). (h) Pd/C (10%), H₂, DMF (83%). ee, enantiomeric excess; ds, diastereoselectivity.