Abstract
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membrane-type 1 MMP (MMP-14) are important targets for inhibition, since their roles in various diseases, including cancer, have been well established. We describe herein a set of mechanism-based inhibitors that show high selectivity to gelatinases and MMP-14 (inhibitor 3) and to only MMP-2 (inhibitors 5 and 7). These molecules bind to the active sites of these enzymes, initiating a slow binding profile for the onset of inhibition, which leads to covalent enzyme modification. The full kinetic analysis for the inhibitors is reported. These are nanomolar inhibitors (Ki) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (k(on) of 10(2) to 10(4) M(-1) s(-1) and the reversal of the process is slow (k(off) of 10(-3) to 10(-4) s(-1)). However, with the onset of covalent chemistry with the best of these inhibitors (e.g. inhibitor 3), very little recovery of activity (<10%) was seen over 48 h of dialysis. We previously reported that broad spectrum MMP inhibitors like GM6001 enhance MT1-MMP-dependent activation of pro-MMP-2 in the presence of tissue inhibitor of metalloproteinases-2. Herein, we show that inhibitor 3, in contrast to GM6001, had no effect on pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 3 reduced tumor cell migration and invasion in vitro. These results show that these new inhibitors are promising candidates for selective inhibition of MMPs in animal models of relevant human diseases.
Highlights
Progression and gelatinase expression and activity has been well established in many studies [2]
We previously described the design and properties of inhibitor 1 (Fig. 1), which is a selective mechanism-based inhibitor for gelatinases. This compound binds to the active sites of Matrix metalloproteinases (MMPs)-2 and MMP-9, with the thiirane moiety coordinating with the zinc ion
The possibility for specific electrostatic interactions near the terminal phenyl group in inhibitor 1 bound to the active site of MMP-2 was anticipated for judiciously designed chemical functionalities into the molecular template of compound 1
Summary
Synthesis—1H and 13C NMR spectra were recorded on either a Varian UnityPlus 300-MHz or a Varian INOVA 500-MHz spectrometer. The combined organic layer was washed with 1 M aqueous HCl, saturated NaHCO3 solution, and brine, dried over Na2SO4, and concentrated under reduced pressure. 4-(4-Acetamidophenoxy)-1-allylthiobenzene—To a stirred solution of 12 (794 mg), which was prepared from 11 (830 mg, 2.89 mmol) in the same manner as described for compound 13, in CH2Cl2 (15 ml) were added pyridine (500 l, 6.18 mmol) and acetic anhydride (292 l, 3.09 mmol) at ice-water temperature, and the mixture was stirred at the same temperature for 1 h. For analysis of simple linear competitive inhibition, reaction mixtures containing the enzyme (ϳ1 nM) and increasing concentrations of the inhibitor, in buffer R (final volume 1 ml), were incubated for ϳ16 h at 25 °C in acrylic semimicrocuvettes. Equilibrium Dialysis—Mixtures of enzyme (10 nM) in the presence and absence of inhibitor (1 mM) were incubated at room temperature
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