Development of high throughput screening assays and pilot screen for inhibitors of metalloproteases meprin α and β

Abstract

Zinc metalloproteinases meprin α and meprin β are implicated in a variety of diseases, such as fibrosis, inflammation and neurodegeneration, however, there are no selective small molecule inhibitors that would allow to study their role in these processes. To address this lack of molecular tools we have developed high throughput screening (HTS) assays to enable discovery of inhibitors of both meprin α and meprin β and screened a collection of well characterized pharmaceutical agents (LOPAC, n = 1,280 compounds). Two compounds (PPNDS, NF449) confirmed their activity and selectivity for meprin β. Kinetic studies revealed competitive (PPNDS) and mixed competitive/non-competitive (NF449) inhibition mechanisms suggesting that binding occurs in meprin β active site. Both PPNDS and NF449 exhibited low nanomolar IC50 and Ki values making them the most potent and selective inhibitors of meprin β reported to the date. These results demonstrate the ability of meprin α and β assays to identify selective compounds and discard artifacts of primary screening.

Metalloproteinase meprin α regulates migration and invasion of human hepatocarcinoma cells and is a mediator of the oncoprotein Reptin

“…While this work was in progress, however, OuYang et al showed that it could induce HCC cell migration and invasion [ 14 ]. Interestingly, there are already available good meprin α inhibitors like actinonin [ 15 ] that can be used in vivo [ 16 – 18 ], and more specific inhibitors are currently under development [ 19 ], raising the hope that meprin α may become a clinically useful target.…”

Discovery and Optimization of Selective Inhibitors of Meprin α (Part I)

“…Assay miniaturization and optimization in 1,536 well plate format. The meprin α and meprin β assays, which utilize the substrates (Mca)-YVADAPK-(K-ε-Dnp) and (Mca)-EDEDED-(K-ε-Dnp), respectively, have been described previously 7 . To enable an ultra-high-throughput screening (uHTS) campaign, we proceeded to miniaturize both assays to 1,536 well plate format (wpf).…”

“…All data generated during this effort were uploaded to the SRMSC's institutional screening database (Assay Explorer, Symyx). Sample to background (S/B) ratios, as well as Z and Z' values, were calculated on a per-plate basis as described 7 . Curve fitting and resulting IC50 determinations were performed as previously reported 19 .…”

“…and PPNDS ( Fig. 1, Ki = 22 nM and 8 nM, respectively), with ~100-fold selectivity against meprin α and good selectivity against adamalysins and matrixins 7 . Ramsbeck et al, (2017) reported the low nanomolar selective meprin β inhibitor, 11g, with 46-fold selectivity against meprin α ( Fig.…”

Discovery and Optimization of Selective Inhibitors of Meprin α (Part II)

“…Understanding meprin 's precise role, alone or in combination with other metzincins, including its close relative meprin , has been difficult to establish due to the lack of known selective inhibitors. While many preliminary reports for meprin inhibitors have emerged [4][5][6][7][8] , no compounds with suitable potency, selectivity, and druglike attributes for in vivo use are known. We recently reported an HTS campaign to identify lead meprin  and meprin  inhibitors 9 .…”