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. 2015 Aug 13;58(15):5808-24.
doi: 10.1021/acs.jmedchem.5b00354. Epub 2015 Aug 4.

SAR Studies of Exosite-Binding Substrate-Selective Inhibitors of A Disintegrin And Metalloprotease 17 (ADAM17) and Application as Selective in Vitro Probes

Anna M Knapinska  1 , Daniela Dreymuller  2 , Andreas Ludwig  2 , Lyndsay Smith  1 , Vladislav Golubkov  3 , Anjum Sohail  4 , Rafael Fridman  4 , Marc Giulianotti  5   6 , Travis M LaVoi  5 , Richard A Houghten  5 , Gregg B Fields  1   7 , Dmitriy Minond  5
Affiliations

SAR Studies of Exosite-Binding Substrate-Selective Inhibitors of A Disintegrin And Metalloprotease 17 (ADAM17) and Application as Selective in Vitro Probes

Anna M Knapinska et al. J Med Chem. .

Abstract

ADAM17 is implicated in several debilitating diseases. However, drug discovery efforts targeting ADAM17 have failed due to the utilization of zinc-binding inhibitors. We previously reported discovery of highly selective nonzinc-binding exosite-targeting inhibitors of ADAM17 that exhibited not only enzyme isoform selectivity but synthetic substrate selectivity as well ( J. Biol. Chem. 2013, 288, 22871). As a result of SAR studies presented herein, we obtained several highly selective ADAM17 inhibitors, six of which were further characterized in biochemical and cell-based assays. Lead compounds exhibited low cellular toxicity and high potency and selectivity for ADAM17. In addition, several of the leads inhibited ADAM17 in a substrate-selective manner, which has not been previously documented for inhibitors of the ADAM family. These findings suggest that targeting exosites of ADAM17 can be used to obtain highly desirable substrate-selective inhibitors. Additionally, current inhibitors can be used as probes of biological activity of ADAM17 in various in vitro and, potentially, in vivo systems.

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Figures

Figure 1
Figure 1
Results of cell viability assay with ADAM17 inhibitors. (A) Viability assay with CHO-K1 cells. (B) Viability assay with A549 cells.
Figure 2
Figure 2
Results of TNFα shedding assay in THP-1 cells with ADAM17 inhibitors. (A) Single point assay. One-way analysis of variance (ANOVA) was used followed by Dunnett posthoc test. The data shown are the mean ± SEM, n = 5. *****p < 0.0001; ***p < 0.001, **p < 0.01, *p < 0.05. (B) Dose–response assay with compounds 17 and 19.
Figure 3
Figure 3
Effect of ADAM17 inhibitors on cytokine shedding in HTMS cells. (A) LPS-stimulated IL-8 shedding inhibition assay. (B) IL-1-stimulated IL-8 shedding inhibition assay. (C) Fractalkine constitutive shedding inhibition assay. One-way analysis of variance (ANOVA) was used followed by Dunnett posthoc test. The data shown are the mean ± SEM, n = 3 from three independent experiments. *****p value <0.0001, ***p value <0.001, **p value <0.01, *p value <0.05. GI: control ADAM10 selective inhibitor GI254023X.
Figure 4
Figure 4
Results of DDR1 release assay in HCC1806 breast cancer cells with compound 17. One, untreated control; 2, 10 μM compound 17; 3, 20 μM compound 17, 4–40 μM compound 17.
Figure 5
Figure 5
Effect of compound 17 on PTK7 shedding and invasion. (A) PTK7 shedding assay in HT1080 (fibrosarcoma) and SW480 (colorectal cancer) cells. (B) Invasion assay in HT1080 cells. sPTK7: soluble PTK7.
Figure 6
Figure 6
Effect of ADAM17 inhibitors on growth factors shedding in A549 cells. (A) Effect on shedding of heregulin. M-stat: marimastat. (B) Effect on shedding of betacellulin. One-way analysis of variance (ANOVA) was used followed by Dunnett posthoc test. The data shown are the mean ± SEM, n = 3. ***p <0.001. (C) Effect on shedding of TGFα. One-way analysis of variance (ANOVA) was used followed by Dunnett posthoc test. The data shown are the mean ± SEM, n = 3. ***p <0.001.
Figure 7
Figure 7
Results of synergy studies of compound 17 with FDA-approved lung cancer drugs. (A) Synergy study with gefitinib. (B) Synergy study with etoposide. (C) Synergy study with doxorubicin.
Scheme 1
Scheme 1. General Synthesis Procedure for Leads of Pyrrollidine-bis-diketopiperazine Series Used in Present Studies
See this image and copyright information in PMC

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