Screening of potential a disintegrin and metalloproteinase with thrombospondin motifs-4 inhibitors using a collagen model fluorescence resonance energy transfer substrate

Autor: Janelle L. Lauer-Fields, Gregg B. Fields, Hideaki Nagase, Peter Chase, Gayle D. Burstein, Mare Cudic, Timothy P. Spicer, Peter Hodder
Rok vydání: 2008
Předmět:
Zdroj: Analytical Biochemistry. 373:43-51
ISSN: 0003-2697
Popis: Osteoarthritis (OA)1 is an age-related debilitating disease affecting more than 80% of people over the age of 75, and is caused by the destruction of articular cartilage [1]. Extracellular matrix (ECM) proteins make up approximately 90% of the dry weight of human cartilage [2]. The major components of the cartilage ECM are type II collagen and aggrecan. Type II collagen provides cartilage with its tensile strength, while the water-binding capacity of aggrecan provides compressibility and elasticity [3]. Aggrecan breakdown leads to an increase in proteolytic susceptibility of articular collagen, hence aggrecan may also have a protective effect on type II collagen [4]. Cartilage destruction associated with OA has been shown to be due to increased catabolism rather than decreased synthesis [5]. Therefore, the study of enzymes associated with aggrecan proteolysis compliments those of collagenolytic matrix metalloproteinases (MMPs) in reference to OA. Several members of the a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family have been found to catalyze the hydrolysis of aggrecan. Although ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, and ADAMTS-15 are all “aggrecanases,” ADAMTS-4 and ADAMTS-5 are the most robust [6] and have been implicated in OA [7; 8; 9; 10]. The products of ADAMTS-4/ADAMTS-5 aggrecan breakdown have been discovered in the synovial fluid of patients with OA [7; 8]. The processing of aggrecan by ADAMTS-4 and ADAMTS-5 may be complimentary, as ADAMTS-5 is responsible for cleavage within the interglobular domain of aggrecan, while ablation of ADAMTS-5 still results in aggrecan processing in the chondroitin sulfate-rich region, presumably by ADAMTS-4 [11]. Given their role in aggrecan degradation and differing substrate specificity profiles, the pursuit of inhibitors for both ADAMTS-4 and ADAMTS-5 is desirable. However, few inhibitors have been described to date for the aggrecanase members of the ADAMTS family [12; 13; 14]. The discovery of aggrecanase inhibitors could be facilitated by high-throughput screening (HTS) methods. Previously described assays for aggrecanases are not particularly convenient for HTS, as all require antibodies and most are discontinuous [15; 16; 17; 18]. A continuous assay method, such as one that utilizes an increase in fluorescence upon substrate hydrolysis, would allow for rapid and convenient evaluation of aggrecanase inhibitors. To develop an improved HTS assay for aggrecanases, we examined fluorescence resonance energy transfer (FRET) collagen-model substrates recently described by our laboratory [19]. More precisely, ADAMTS-4/ADAMTS-5 FRET substrates had been designed to incorporate the aggrecan 1480–1481 cleavage site within a collagen-model structure [19]. The fluorophore/quencher pair was 7-methoxycoumarin (Mca)/2,4-dinitrophenyl (Dnp), where Mca fluorescence was efficiently quenched by the Dnp group in the intact substrate. Substrate conformation had a significant role in ADAMTS-4 specificity, and these substrates interacted with secondary binding sites (exosites) located outside the enzyme catalytic domain [19]. Thus, HTS with collagen-model aggrecanase substrates may allow for the identification of active site and/or exosite-binding inhibitors. One of the collagen-model aggrecanase substrates, fSSPa [C6-(Gly-Pro-Hyp-Pro-Hyp-Gly)2-Gly-Pro-Hyp-Gly-Thr-Lys(Mca)-Gly-Glu~Leu~Glu~Gly-Arg~Gly-Thr-Lys(Dnp)-Gly-Ile-Ser-(Gly-Pro-Hyp-Pro-Hyp-Gly)2-Gly-Pro-Hyp-NH2], has been utilized here for screening of a compound library (n = 960) against ADAMTS-4 in a 384-well format. Inhibitory compounds were (a) confirmed by dose-dependence analysis and (b) verified using an RP-HPLC based assay (a “secondary” screen). The overall quality of the screen was also examined.
Databáze: OpenAIRE
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