Structural and Inhibition Analysis Reveals the Mechanism of Selectivity of a Series of Aggrecanase Inhibitors

Abstract

Several inhibitors of a series of cis-1(S)2(R)-amino-2-indanolbased compounds were reported to be selective for the aggrecanases, ADAMTS-4 and -5 over other metalloproteases. To understand the nature of this selectivity for aggrecanases, the inhibitors, along with the broad spectrum metalloprotease inhibitor marimastat, were independently bound to the catalytic domain of ADAMTS-5, and the corresponding crystal structures were determined. By comparing the structures, it was determined that the specificity of the relative inhibitors for ADAMTS-5 was not driven by a specific interaction, such as zinc chelation, hydrogen bonding, or charge interactions, but rather by subtle and indirect factors, such as water bridging, ring rigidity, pocket size, and shape, as well as protein conformation flexibility. Osteoarthritis (OA)3 pathology includes degradation of articular cartilage, along with subchondral bone sclerosis and osteophyte formation, all contributing to impaired joint function. Pain, restricted movement, and joint instability accompany these structural changes and often result in the need for total joint replacement. Current therapies alleviate the mild to moderate pain and inflammation associated with OA, but do not protect the cartilage from further damage and have not demonstrated an effect on disease progression (1). Therefore, therapeutics that prevent or slow the alteration of joint structure and function will address a major unmet medical need.Loss of aggrecan, a macromolecular proteoglycan providing cartilage with its properties of compressibility and resilience, is a major phenotype associated with OA and is believed to be a critical event in driving disease progression (2, 3). Both ex vivo and in vivo proof of concept studies support ADAMTS-4 and ADAMTS-5, commonly referred to as aggrecanase-1 and -2, respectively, as the two major enzymes responsible for the proteolytic breakdown of cartilage aggrecan (reviewed in Ref. 4). Blocking their activity may be an attractive strategy to stop or slow down the progression of the disease, as suggested by studies in knock-out mice (5). Given the chronic nature of the disease, long term treatment will be likely, demanding very safe therapeutic interventions only achievable with ADAMTS-4-and ADAMTS-5-specific inhibitors lacking off-target side effects. Designing selectivity has been very challenging and a major source of difficulty is that at least 57 metalloproteases (MP) divided in three major families, 1) matrix metalloproteases (MMP); 2) a disintegrin and metalloproteinase (ADAM); and 3) a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), are present in humans. ADAMTS-4 and -5 belong to the ADAMTS family and share common catalytic and structural features with the other MP members. These features include the highly conserved amino acid sequence, HEXXHXXGXXH, harboring a catalytic zinc cation, required for activation of the peptide bond toward hydrolysis. In addition, many MPs share significant structural topology in the active site, such as...

The Role of Synovial Macrophages and Macrophage-Produced Mediators in Driving Inflammatory and Destructive Responses in Osteoarthritis

“…Several pharmaceutical companies (Wyeth/Pfizer, Schering-Plough, Rottapharm SpA, Alantos Pharm, Japan Tobacco) have patented small-molecule inhibitors of ADAMTS4 and ADAMTS5, developed mainly as potential DMOADs. Some of these compounds are claimed to be specific, whereas others have effect against both enzymes, against other ADAMTS members, or even against MMPs (Wittwer et al, 2007;Tortorella et al, 2009). The Wyeth/Pfizer compound was recently used in a phase I clinical trial in osteoarthritis (Hellio le Graverand-Gastineau, 2010;Gilbert et al, 2011).…”

Structure analysis reveals the flexibility of the ADAMTS‐5 active site

“…The alterations seen with Compound 12 are specific to this inhibitor. We believe the reasons for the compound's ability to change the cataTS5 S1 0 loop are due to its strong anchoring groups located on the indanolsuccinamide moiety that interacts with cataTS5, as described by Tortorella et al 12 as well as to the rigidity and hydrophobicity of the P1 0 phenylbenzodioxole moiety. The changes can be described as a two-step movement, as shown in Figure 6.…”

“…16,17 Inhibition of ADAMTS-4 or ADAMTS-5 mediated cleavage of bovine aggrecan at the Glu 373 /Ala 374 site was determined as reported in previous work. 12 …”

“…12 This new discovery reveals that the P1 0 moieties of these inhibitors bear important factors that recognize or exclude binding to MPs. The uniqueness of this newly discovered S1 0 pocket in cataTS5 generated by Compound 12 and its likely formation in the two-domain structure, cataTS5- disintegrin-like domain, upon binding Compound 12, is important for drug design.…”

“…Recently our group reported the mechanism of selectivity of a series of cis-1(S)2(R)-amino-2-indanol based compounds by generating the crystal structures of these molecules with the catalytic domain of ADAMTS-5. 12 These structures reveal that subtle or secondary interactions, such as water bridging and polarity distribution, are responsible for selectivity, whereas the reaction pocket of cataTS5 in these structures is essentially unchanged. The S1 0 pockets in MPs have been shown to be flexible and readily changeable.…”