Exosite inhibition of ADAMTS-5 by a glycoconjugated arylsulfonamide

Salvatore Santamaria,Francesca Liva,Doretta Cuffaro,Rens De Groot,Felicia D’Andrea,Tiziano Tuccinardi,Lidia Ciccone,Armando Rossello,Elisa Nuti,Josefin Ahnström

doi:10.1038/s41598-020-80294-1

Salvatore Santamaria, Francesca Liva + Show 8 more

Open Access

https://doi.org/10.1038/s41598-020-80294-1

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Abstract

ADAMTS-5 is a major protease involved in the turnover of proteoglycans such as aggrecan and versican. Dysregulated aggrecanase activity of ADAMTS-5 has been directly linked to the etiology of osteoarthritis (OA). For this reason, ADAMTS-5 is a pharmaceutical target for the treatment of OA. ADAMTS-5 shares high structural and functional similarities with ADAMTS-4, which makes the design of selective inhibitors particularly challenging. Here we exploited the ADAMTS-5 binding capacity of β-N-acetyl-d-glucosamine to design a new class of sugar-based arylsulfonamides. Our most promising compound, 4b, is a non-zinc binding ADAMTS-5 inhibitor which showed high selectivity over ADAMTS-4. Docking calculations combined with molecular dynamics simulations demonstrated that 4b is a cross-domain inhibitor that targets the interface of the metalloproteinase and disintegrin-like domains. Furthermore, the interaction between 4b and the ADAMTS-5 Dis domain is mediated by hydrogen bonds between the sugar moiety and two lysine residues (K532 and K533). Targeted mutagenesis of these two residues confirmed their importance both for versicanase activity and inhibitor binding. This positively-charged cluster of ADAMTS-5 represents a previously unknown substrate-binding site (exosite) which is critical for substrate recognition and can therefore be targeted for the development of selective ADAMTS-5 inhibitors.

Highlights

A Disintegrin And Metalloproteinase with Thrombospondin motif (ADAMTS)-5 is a major protease involved in the turnover of proteoglycans such as aggrecan and versican
These regions are essential for proteolysis of both aggrecan and versican and lie in loops which are hypervariable within the ADAMTS family[5], offering a target for the development of selective inhibitors
The most common disaccharide unit is composed of a 2-O-sulfated iduronic acid and 6-O-sulfated, N-sulfated β-N-acetyl-d-glucosamine (GlcNAc), which we here have exploited for the development of an ADAMTS-5 inhibitor

Summary

Introduction

ADAMTS-5 is a major protease involved in the turnover of proteoglycans such as aggrecan and versican. The interaction between 4b and the ADAMTS-5 Dis domain is mediated by hydrogen bonds between the sugar moiety and two lysine residues (K532 and K533) Targeted mutagenesis of these two residues confirmed their importance both for versicanase activity and inhibitor binding. We have recently identified two such exosites in the ADAMTS-5 Sp domain (residues 739–744 and 837–844)[5] These regions are essential for proteolysis of both aggrecan and versican and lie in loops which are hypervariable within the ADAMTS family[5], offering a target for the development of selective inhibitors. The most common disaccharide unit is composed of a 2-O-sulfated iduronic acid and 6-O-sulfated, N-sulfated β-N-acetyl-d-glucosamine (GlcNAc), which we here have exploited for the development of an ADAMTS-5 inhibitor For this purpose, we probed ADAMTS-5 with a small library of GAG-mimetic molecules, containing the GlcNAc moiety (Fig. 1). Through mutagenesis and biochemical characterization, we show that these residues compose a novel exosite which is amenable to selective inhibition

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