Discovery of novel inhibitors of a disintegrin and metalloprotease 17 (ADAM17) using glycosylated and non-glycosylated substrates

Abstract

A disintegrin and metalloprotease (ADAM) proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off-target toxicity. We hypothesized that secondary binding site (exosite) inhibitors should provide a viable alternative to active site inhibitors. Potential exosites in ADAM structures have been reported, but no studies describing substrate features necessary for exosite interactions exist. Analysis of ADAM cognate substrates revealed that glycosylation is often present in the vicinity of the scissile bond. To study whether glycosylation plays a role in modulating ADAM activity, a tumor necrosis factor α (TNFα) substrate with and without a glycan moiety attached was synthesized and characterized. Glycosylation enhanced ADAM8 and -17 activities and decreased ADAM10 activity. Metalloprotease (MMP) activity was unaffected by TNFα substrate glycosylation. High throughput screening assays were developed using glycosylated and non-glycosylated substrate, and positional scanning was conducted. A novel chemotype of ADAM17-selective probes was discovered from the TPIMS library (Houghten, R. A., Pinilla, C., Giulianotti, M. A., Appel, J. R., Dooley, C. T., Nefzi, A., Ostresh, J. M., Yu, Y., Maggiora, G. M., Medina-Franco, J. L., Brunner, D., and Schneider, J. (2008) Strategies for the use of mixture-based synthetic combinatorial libraries. Scaffold ranking, direct testing in vivo, and enhanced deconvolution by computational methods. J. Comb. Chem. 10, 3-19; Pinilla, C., Appel, J. R., Borràs, E., and Houghten, R. A. (2003) Advances in the use of synthetic combinatorial chemistry. Mixture-based libraries. Nat. Med. 9, 118-122) that preferentially inhibited glycosylated substrate hydrolysis and spared ADAM10, MMP-8, and MMP-14. Kinetic studies revealed that ADAM17 inhibition occurred via a non-zinc-binding mechanism. Thus, modulation of proteolysis via glycosylation may be used for identifying novel, potentially exosite binding compounds. The newly described ADAM17 inhibitors represent research tools to investigate the role of ADAM17 in the progression of various diseases.

FIGURE 1.

Glycosylated substrate and substrate characterization by circular dichroism spectroscopy. A, structure of glycosylated substrate (Glu(EDANS)-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser(TF)-Ser-Lys(DABCYL)). Shown are circular dichroism spectra of non-glycosylated (B) and glycosylated substrates (C). D, molar ellipticity of non-glycosylated and glycosylated substrates at λ = 222 nm as a function of TFE concentration. TF, Thomsen-Freidenreich.

FIGURE 2.

Summary of screening of TPIMS library for selective inhibitors of ADAM17.

FIGURE 3.

Results of the pilot “scaffold ranking” screen of TPIMS drug-like library against ADAM10 and 17. Shown is an ADAM10 (A) and ADAM17 (B) screen using glycosylated (checked bars) and non-glycosylated substrate (small checked bars). The arrows indicate libraries containing potential exosite inhibitors of ADAM10 and 17. All assays were performed in triplicate. Activity and selectivity of all libraries were confirmed in reversed-phase HPLC-based assays. (C), basic scaffold of library 1344.

FIGURE 4.

Results of the positional scan analysis of library 1344 against ADAM10 and -17. Positional scan of R₁ (A), R₂ (B), R₃ (C), and R₄ (D and E) defined moieties against ADAM10 (black bars) and ADAM17 (checked bars) using glycosylated substrate.

FIGURE 5.

Results of dose-response study of most ADAM17-selective and potent mixture samples for each defined R position against metalloprotease panel. Shown are test libraries (A–E) and pharmacological assay controls (F) against ADAM10 (□), ADAM17 (♦), MMP-8 (♢), and MMP-14 (○). Structures of functional groups present in the defined R position for each sublibrary are shown as insets. Structures of MMP-9/-13 inhibitor (G) and actinonin (H) are shown. Note that sample R₂-35 (see Fig. 5B) exhibits inhibition of MMP-14 only at the highest dose tested (1 μm).

FIGURE 6.

Results of dose response study of most ADAM17 selective and potent individual compounds. ADAM10 (□), ADAM17 (♦), MMP-8 (♢), and MMP-14 (○). Structures of individual compounds (compounds 9 (A), 27 (B), 15 (C), 34 (D), 17 (E), and 36 (F)) are shown as insets.

FIGURE 7.

Lineweaver-Burk plots of glycosylated substrate hydrolysis by ADAM17 in the presence of representative members of library 1344. A, compound 24; B, compound 35; C, compound 61; D, compound 119.

FIGURE 8.

Characterization of mechanism of inhibition of ADAM17 catalytic domain and ectodomain by compound 15. A, structure of AHA; B, Yonetani-Theorell plot of glycosylated substrate hydrolysis by ADAM17 in the presence of AHA and compound 15. Note the non-parallel lines of best fit, indicating mutually non-exclusive binding by two inhibitors. C, Lineweaver-Burk plot of glycosylated substrate hydrolysis by ADAM17 in the presence of compound 15. Shown is a dose-response study of inhibition of ADAM17 catalytic domain and ectodomain by AHA (D), MMP-9/-13 inhibitor (E), and compound 15 (F). CD, catalytic domain-only.