Target-Activated Prodrugs (TAPs) for the Autoregulated Inhibition of MMP12

Abstract

We describe a prodrug concept in which the target enzyme MMP12 produces its own inhibitor in a two-step activation procedure. By using an MMP12-specific peptide sequence and a known sulfonamide drug integrated in the backbone, the active inhibitor is released upon enzyme cleavage. In in vitro experiments, we present proof of concept that the activation proceeds with useful kinetics. The approach is highly selective over the closely related MMP8. If applied in vivo in the future, these prodrugs might release the active entity in a highly specific manner only at such sites where enzyme activity resides.

Keywords: MMP; inflammation; inhibitors; prodrug; proteases.

Figure 1

HPLC traces showing hMMP12-mediated drug release from TAP. (a) Prodrug 3 (10 μM) was incubated with hMMP12 (12 nM) in TCN buffer at 37 °C for different times. After 3 h, the prodrug had been significantly cleaved by hMMP12 to generate predrug 5. Over time, 5 hydrolyzed spontaneously to the potent inhibitor 1. Uncleaved prodrug 3 hydrolyzed to compound 7. (b) Compound 5 (10 μM) was incubated without enzyme to study the spontaneous hydrolysis of the molecule. After 20 h, a substantial amount of 5 had converted to the free inhibitor (1). (c) In the absence of hMMP12, prodrug 3 degraded slowly to the noninhibitory compound 7. (d) When prodrug 3 (10 μM) was incubated with hMMP8 (20 nM), the enzyme did not cleave the prodrug substantially, and thus, inhibitor release was poor. Instead, most of the prodrug degraded to compound 7 over time.

Scheme 1. Mechanism of TAP Activation by hMMP12 and Inhibitor Release

An MMP12 inhibitor is inserted into a peptidic sequence (3), which is specifically cleaved by hMMP12. Upon proteolytic cleavage, the released molecule (5) shows a moderate IC₅₀ towards hMMP12 but degrades spontaneously, generating the more potent hMMP12 inhibitor 1. In the absence of enzyme, the prodrug eventually degrades to the poor hMMP12 inhibitor (7).

Figure 2

Activity-based assay showing the selective inhibition of hMMP12 by TAP. (a) Prodrug 3 (10 μM) was preincubated with hMMP12 (12 nM) in TCN buffer at 37 °C for different times. After that, the activity of the enzyme was measured with the fluorogenic substrate LaRee5. With longer preincubation times, the enzyme was significantly inhibited due to the presence of compound 1. (b) Compound 5 (10 μM) was preincubated in buffer without hMMP12. After that, we added hMMP12 (12 nM) and measured its activity. Strong inhibition occurred after long preincubation times due to the spontaneous conversion of 5 into 1. (c) Incubation of prodrug 3 in the absence of hMMP12 did not generate any inhibitory molecule over time; thus, hMMP12 activity remained high. (d) When prodrug 3 was incubated with hMMP8 (20 nM), the enzyme activity also remained high over time, as measured with a commercial fluorogenic substrate, since compound 1, which also inhibits hMMP8, is not significantly formed.