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

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.

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

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

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

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

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

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

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. General Synthesis Procedure for Leads of Pyrrollidine-bis-diketopiperazine Series Used in Present Studies