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A microplate assay for the screening of ADAMTS-4 inhibitors
Matrix Biology 25 (2006) 261 – 267 www.elsevier.com/locate/matbio
A microplate assay for the screening of ADAMTS-4 inhibitors Marie Thomas a , Massimo Sabatini a,⁎, Fabrice Bensaude b , Benoit Mignard b , Jean-Claude Ortuno c , Isabelle Caron d , Jean A. Boutin b , Gilles Ferry b a Division of Rheumatology, Institut de Recherches Servier, IdRS, 11, rue des Moulineaux, 92150 Suresnes, France Division of Molecular and Cellular Pharmacology, Institut de Recherches Servier, 125, chemin de Ronde, 78290 Croissy-sur-Seine, France Division of Medicinal Chemistry, Institut de Recherches Servier, Institut de Recherches Servier, 125, chemin de Ronde, 78290 Croissy-sur-Seine, France d Beckman Coulter France S.A., Roissy, France b
c
Received 7 October 2005; received in revised form 13 December 2005; accepted 13 December 2005
Abstract Aggrecanase plays a major role in cartilage proteoglycan degradation in rheumatic diseases such as osteoarthritis and rheumatoid arthritis. The search of new inhibitors of aggrecanase activity necessitates a robust assays in order to be able to screen large numbers of compounds. We present in this paper an assay based on the cleavage of His-tagged aggrecan interglobular domain by N- and C- terminus truncated, active aggrecanase-1/ ADAMTS-4, with formation of the aggrecanase-specific ARGSV neoepitope. This is detected by anti-ARGSV antibody, in turn recognized by a fluorescent anti-IgG. Furthermore, the formation of the reaction products was confirmed by high-pressure capillary electrophoresis. This assay allows the rapid screening of aggrecanase inhibitors in a 96-well plate format, allowing an immediate transposition to high-throughput scale up. © 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved. Keywords: Aggrecanase; Screening assay; ADAMTS-4; Proteoglycan degradation; Anti-ARGSV antibody; HPCE
1. Introduction Osteoarthritis, the most common rheumatic disease, is characterized by progressive erosion of articular cartilage, finally leading to exposure of the underlying bone (MartelPelletier, 1999). Cartilage loss is an active process, due to an imbalance of tissue turn-over, consequent to an increase of degradation and decrease of synthesis of the extracellular matrix. Cartilage main components are collagen type II, which forms a tridimensional scaffold responsible for tensile strength, and aggrecan, a highly hydrated proteoglycan responsible for cartilage resilience and resistance to compression. Aggrecan degradation is carried out by metalloproteinases of the matrix metalloproteinase (MMP) and aggrecanase families (Caterson et al., 2000). Even if the relative contributions of the two groups of enzymes are not yet clearly defined, aggrecanases seem to play the main role in aggrecan catabolism, as it is suggested by the predominance of aggrecanase-generated neoepitopes in both the synovial fluid (Sandy et al., 1992; Lohmander et al., 1993) ⁎ Corresponding author. Tel.: +33 1 55 72 24 11; fax: +33 1 55 72 27 37. E-mail address: [email protected] (M. Sabatini).
and cartilage of patients with OA (Sandy and Verscharen, 2001). Aggrecanases belong to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin domain) family and cleave aggrecan at multiple sites, first in the G2–G3 chondroitin sulfate-rich domain, and then at the classical NITEGE373– A374RGSVIL position in the G1–G2 interglobular domain (IGD) (Sandy et al., 2000), corresponding to position E392–A393 of the full-length human sequence including the 19-residue leader sequence. Several aggrecanases have been so far identified in cartilage, and shown to be produced by chondrocytes: aggrecanase-1/ADAMTS-4 (Tortorella et al., 1999), aggrecanase-2/ADAMTS-5 (Abbaszade et al., 1999), ADAMTS-1 (Rodriguez-Manzaneque et al., 2002) and ADAMTS-8 (Collins-Racie et al., 2004). Synthesis of aggrecanases seems to be differentially regulated in chondrocytes. While aggrecanase-1 expression is stimulated by the inflammatory cytokines interleukin-1 (IL-1) β and tumor necrosis factor (TNF) α, and by transforming growth factor (TGF) β, aggrecanase-2 seems to be produced in a constitutive way (Moulharat et al., 2004). Aggrecanase-1/ADAMTS-4 is produced as 100 kDa proenzyme, which is N-terminally cleaved by furin (Gao et al., 2002), to produce a secreted 68 kDa form that
0945-053X/$ - see front matter © 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved. doi:10.1016/j.matbio.2005.12.003
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up producing a time-resolved fluorescent signal between antibodies linked to these beads and directed against the chondroitin and keratan sulfate regions of aggrecan. The region between these zones is the target of aggrecanase cleavage of aggrecan, leading to the decrease of the fluorescent signal between the beads, since the cleavage physically separates the zones against which the antibodies were raised. The test is based on the use of (a) C-terminally His-tagged aggrecan IGD immobilized on Ni-coated plates as a substrate and (b) N- and C-terminally truncated ADAMTS-4 as enzyme. Aggrecanase activity is measured by immunodetection of ARGSVIL neoepitope produced through cleavage of aggrecan IGD by ADAMTS-4.
associates to membrane-type (MT)-4 MMP in chondrosarcoma cells (Gao et al., 2004). MT-4 MMP is in turn linked to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor, and cleaves ADAMTS-4 C-terminally to produce 53 and 40 kDa forms, in which substrate specificity progressively switches from the G2–G3 chondroitin-rich domain to the G1–G2 IGD (Gao et al., 2004; Kashiwagi et al., 2004). Inhibition of aggrecanases is a promising approach for the treatment of arthritic diseases (Arner et al., 1999; Malfait et al., 2002). Screening of large numbers of chemicals in search of inhibitors depends on the availability of simple and reproducible assays of aggrecanase activity. Miller et al. (2003) recently described a microplate assay based on the immunodetection of the aggrecan neoepitope ARGSVIL, generated by the cleavage of a synthetic, biotinylated 41-mer peptide substrate, immobilized to streptavidin-coated plates and degraded by aggrecanase partially purified from cultures of bovine cartilage. We used a partially different approach, mainly dictated by the choice to use commercially available reagents. This paper presents a protocol for testing inhibitors of aggrecanase activity. Previous HTS assays (Peppard et al., 2003) used a proprietary technology (AlphaScreen™) requiring the development of beads that end
2. Results It was first verified that truncated, constitutively active ADAMTS-4 (p40 form) could cleave aggrecan IGD, with formation of the aggrecan neoepitope ARGSVIL, as expected for aggrecanase activity. Aggrecan IGD, at concentrations between 0.3 and 3 μM, was incubated with 10 and 100 nM ADAMTS-4 for 2 h. In the presence of 100 nM enzyme, the
A: Gel Ruby
B: Western Blot
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Fig. 1. Degradation of aggrecan IGD by ADAMTS-4 (A and B), and effects of an aggrecanase inhibitor (C). Panels A and B: aggrecan IGD at concentrations of 0.3, 0.6 and 3 μM, was exposed to 100 nM ADAMTS-4 for 2 h at 37 °C. The reaction was stopped by adding SDS sample buffer with reducing agent and heating at 90 °C for 5 min. Samples were separated by SDS-PAGE and either stained with sypro ruby (A) or subjected to Western blot and probed by anti-ARGSVIL antibody (B). Panel C: aggrecan IGD (3 μM) was incubated without or with 100 nM ADAMTS-4 in the absence or presence of aggrecanase inhibitor at concentrations in the range 10− 9 to 10− 5 M, for 2 h at 37 °C. The reaction was stopped by adding SDS sample buffer with reducing agent and heating at 90 °C for 5 min, then the samples were separated by SDS-PAGE in parallel with Mark 12 MW standards and stained by colloidal blue.
M. Thomas et al. / Matrix Biology 25 (2006) 261–267
concentrations between 0.6 and 3 μM (0.12 to 0.6 nmoles/well, respectively) was added to the plates and incubated for 24 h at 4 °C, or 2 h at 37 °C. After repeated washes to remove unbound material, bound IGD was desorbed from the plates, and a fraction separated by SDS-PAGE and visualized by protein staining (not shown). The bands of recovered aggrecan IGD were compared with a scale of aggrecan IGD standards similarly separated and stained. It was determined by densitometric analysis that IGD binding reached a maximum of around 30 pmol/well for peptide solutions between 1.8 and 3 μM left in the wells for 24 h at 4 °C. Comparable binding was obtained by incubating 3 μM IGD for 2 h at 37 °C. It was then determined if plate-bound aggrecan IGD could be degraded by ADAMTS-4. Ni-coated plates were coated with 1.8 μM aggrecan IGD (0.36 nmol/well) for 24 h at 4 °C, washed to remove the unbound protein, then exposed to 100 nM ADAMTS-4 for 2 h at 37 °C. After repeated washes to remove N-terminal cleaved fragments, plate-bound IGD, intact or
20 kDa aggrecan IGD was partially converted to a 12 kDa form (Fig. 1A), which presented the ARGSVIL neoepitope (Fig. 1B) and therefore represented the C-terminal fragment of aggrecanase-cleaved IGD. A very weak signal was obtained using 10 nM ADAMTS-4 (not shown). Aggrecan IGD degradation was inhibited by a selective aggrecanase inhibitor (compound 8 in Yao et al., 2001), which added at concentrations between 10− 9 and 10− 5 M, dose-dependently inhibited the appearance of the 12 kDa fragment (Fig. 1C). No band could be observed, corresponding to the smaller N-terminal fragment, with a calculated MW of 4.8 kDa. In a similar experiment, aggrecan IGD degradation in the absence (Fig. 2A) and presence of the inhibitor (Fig. 2B) was quantified using high performance capillary electrophoresis. An IC50 value of 3.5 × 10− 7 M was calculated (Fig. 2B). Knowing that aggrecan IGD could be correctly cleaved by ADAMTS-4, it was then examined if aggrecan IGD could be adsorbed onto Ni-coated multiwell plates by its C-terminal poly His-tag tail. Aggrecan IGD at
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Time (minutes) Fig. 2. A: Analysis of aggrecanase activity by HPCE and PAGE. 100 nM ADAMTS-4 was incubated with 3 μM aggrecan IGD for 2 h at 37 °C. The reaction was stopped by adding SDS sample buffer with reducing agent and heating at 90 °C for 5 min, then the samples were analysed in parallel by HPCE (elution profile at 220 nm) and by SDS-PAGE (insert: colloidal blue staining of gel). Lane 1: IGD alone; lane 2: IGD plus ADAMTS-4. Letters a (in gel) and b, c (in both gel and profile) indicate ADAMTS-4 (p40), IGD and IGD degradation product, respectively. B: Effect of the aggrecanase inhibitor on ADAMTS-4 activity. Reaction was carried out as in Fig. 1C, in the absence or presence of the inhibitor at concentrations between 10− 8 and 10− 5. Values are means of triplicates. In both A and B panels, absorbance profiles of depicted runs were shifted along the y axes in order to set apart the different profiles. Depicted run actually showed the same baseline absorbance values.
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A
B MW
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IGD Cleaved IGD 6
IGD ADAMTS-4
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Fig. 3. Aggrecan IGD (1.8 μM, 200 μl/well) was adsorbed to Ni-coated plates for 24 h at 4 °C. After 3 washes to remove unbound material, bound IGD was exposed to 100 nM ADAMTS-4 for 2 h at 37 °C. After 3 more washes to remove N-terminal cleaved fragments, plate-bound IGD, intact or degraded, was desorbed from the wells, and a fraction separated by SDS-PAGE, in parallel with Mark 12 MW standards, and either stained by sypro ruby (A) or subjected to Western blot and probed by anti-ARGSV antibody (B).
0.25 μM without major loss of signal (data not shown). Linear neoepitope formation occurred during the first 30 min of reaction, using 0.25 μM aggrecan IGD for coating and ADAMTS-4 at 0.125 or 0.25 nM for substrate degradation (Fig. 4A). The signal obtained at 30 min was proportional to enzyme concentration in the range from 0.031 to 0.25 nM (Fig. 4B). Therefore, conditions selected for the screening test provided for coating with 0.25 μM aggrecan, and substrate degradation by 0.25 nM ADAMTS-4 for 30 min. Similar signals were obtained using as a primary antibody either antiARGSV (diluted 1 / 500) by HDM or anti-ARGSVIL (diluted 1 / 100) by Agro-bio. Using the conditions finally selected (see Section 4.2 for details), the aggrecanase inhibitor antagonized ADAMTS-4 with an IC50 value of 1.6 × 10− 7 (Fig. 5).
degraded, was desorbed from the wells and separated by SDSPAGE (Fig. 3A). The 20 kDa band of aggrecan IGD was converted to the 12 kDa form, which was confirmed to possess the ARGSV neoepitope by Western blot (Fig. 3B). It was then verified that degraded aggrecan IGD still attached to the plate by its poly-His tail could be detected in situ by the anti-ARGSV antibody. Bound primary antibody was detected using a Cy™5conjugated secondary antibody. Aggrecan IGD (0.75 μM) was adsorbed to the plate, then exposed to ADAMTS-4. It was found that enzyme concentration of 100 nM, well adapted to the degradation of solubilised aggrecan IGD, was in large excess when used against the immobilized substrate. The signal detected after 1 h of reaction was strongly decreased after 2 h and had almost disappeared by 4 h (data not shown). This could be due to further degradation of the substrate and shedding of the neoepitope, as suggested by the fact that further dilutions of ADAMTS-4 in the range from 0.25 to 0.031 nM allowed a time-dependent increase of neoepitope formation. It was also found that within this range of enzyme concentrations, aggrecan-IGD coating solution could be diluted from 0.75 to
3. Discussion We describe in this paper the development of a rapid and simple immunoassay for screening of ADAMTS-4 inhibitors. When examining the conditions of aggrecan IGD cleavage by
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Fig. 4. A: Time course of neoepitope signal generation. Aggrecan IGD (0.250 μM) was adsorbed to 96-well Ni-coated plates, then exposed to ADAMTS-4 at concentrations of 0.031 (squares), 0.063 (triangles), 0.125 (circles) and 0.250 (diamonds) nM, for 15, 30, 45 and 60 min at 37 °C. B: Data in A were replotted to show fluorescence vs. ADAMTS-4 concentration at 15 (squares), 30 (circles), 45 (triangles), and 60 min (triangles).
M. Thomas et al. / Matrix Biology 25 (2006) 261–267
30000 IC50=1.6x10-7 M
Fluorescence unit
25000 20000 15000 10000 5000 0 0
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Inhibitor concentration (Log M) Fig. 5. Inhibition of ADAMTS-4 activity by the aggrecanase inhibitor. Aggrecan (0.25 μM) was preadsorbed to Ni-Coated plates. The inhibitor was added at final concentrations between 10− 9 and 10− 3 M, followed by ADAMTS-4 at the final concentration of 0.25 nM for 30 min at 37 °C (see Section 4.2 for details). Data are means ± ESM; n = 3 points at each concentration.
ADAMTS-4 (p40 form) in solution, reaction kinetics could not be determined, for lack of an analytical system of the necessary sensitivity for the measurement of the substrate and the reaction products. It was however verified that IGD, which showed an apparent MW of 20 kDa in SDS-PAGE, could be cleaved by truncated ADAMTS-4 and form a fragment of 12 kDa, exhibiting the ARGSVIL neoepitope, which is a specific marker of aggrecanase activity. Formation of the smaller, N-terminal fragment, with a calculated MW of 4.8 kDa, could not be monitored by SDS-PAGE on a 6–12% gel, possibly also because of secondary cleavage at the MMP site N341–F342 (corresponding to position N360–F361 of the full-length human sequence) (Westling et al., 2002), further decreasing MW to 3.7 kDa. Cleavage of IGD and production of the 12 kDa fragment could be inhibited by a specific aggrecanase inhibitor. Results obtained by SDS-PAGE and Western blot were supported by studies with high performance capillary electrophoresis, which confirmed both IGD degradation by ADAMTS-4 and cleavage inhibition by the anti-aggrecanase compound. That aggrecan IGD, consisting of G1–G2 region only, could be cleaved by truncated ADAMTS-4 was not obvious, in light of the paper by Tortorella et al. (2000) showing that the G2–G3 region of aggrecan, rich in glycosaminoglycan, is important for substrate recognition by ADAMTS-4, via the C-terminal thrombospondin-1 region (TSP-1) of the enzyme. However, the lack of the G2–G3 region in the substrate was probably offset by less stringent substrate recognition by the enzyme, due to the absence of the C-terminal cys-rich and spacer domains in the p 40 form of ADAMTS-4 (Gao et al., 2002, 2004; Kashiwagi et al., 2004; Flannery et al., 2002). After verification of substrate cleavage in solution, the conditions were defined under which aggrecan IGD could be adsorbed to the Ni-coated plates via its poly-His tail, and degraded by ADAMTS-4 with generation of ARGSVIL neoepitope. Compared with the conditions used for substrate degradation in solution, it was necessary to decrease
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ADAMTS-4 concentration by at least 400 times in order to assure linear generation of neoepitope signal. Moreover, aggrecan IGD concentration for substrate adsorption could be limited to 250 nM. Exposure of plate-adsorbed aggrecan IGD to 100 nM ADAMTS-4 produced a transient signal, probably because of further degradation of the plate-bound substrate. It is known that the G1-carrying fragment deriving from cleavage of aggrecan IGD at E373–A374 can be secondarily attacked by ADAMTS-4 at the N341–F342 region, which is normally recognized by MMPs (Westling et al., 2002). However, no secondary cleavage has been so far reported for the G2 carrying fragment. Further degradation at the A374 N-extremity could be due to altered substrate conformation due to the presence of the poly-His tail linked to the well surface. Another possibility could be that the enzyme competed the substrate out of the Ni-coated suface, since they both possess a C-terminal His-tag. However, addition of the aggrecanase inhibitor blocked the disappearance of the signal (not shown), suggesting that the enzyme's poly-His tail does not interfere with substrate binding to the plate. The conditions selected for the test allowed signal generation that was linear over 30 min, and proportional to enzyme concentration in the 0.031 to 0.25 nM range. When a specific aggrecanase inhibitor (Yao et al., 2001) was tried on the plate test, the IC50 found (1.6 × 10− 7 M) was of the same order as that determined when the reaction was carried out in a test tube with the substrate in solution, and the analysis carried out by capillary electrophoresis (3.5 × 10− 7 M). This value was however higher than that (6.4 × 10− 8 M) (Yao et al., 2001) found when the inhibitor was tested according to a protocol using full-length human ADAMTS-4 and purified bovine aggrecan (Tortorella et al., 2000). Most of this difference could be due to the chiral nature of the compound we used, if only one of the two forms present were able, as it is often the case, to inhibit enzyme activity. This difference could be also explained by changes of affinity between ADAMTS-4 and aggrecan, when truncated instead of full-length forms are used (Gao et al., 2002; 2004; Kashiwagi et al., 2004). Moreover, different concentrations of substrate and enzyme were used in the two assays, which would also contribute to IC50 changes. It can also be added that the absence of the cysrich and spacer domain in ADAMTS-4 p40 does not allow interactions between the inhibitor and regions that are otherwise important for substrate recognition in p53 and p68 forms of the enzyme. Compared with already published protocols (Miller et al., 2003; Tortorella et al., 2000) this assay has the advantage of being based on commercially available reagents and therefore offers the possibility of a rapid set-up. A possibly limiting factor in the routine use of this protocol is the primary antibody, due to the limited dilution (1 / 100 for Agro-bio; 1 / 500 for HDM antibodies) that has to be used for the detection of the aggrecanase-generated neoepitope. However, we easily produced different batches of rabbit affinity-purified anti-ARGSVIL of consistent affinity for the epitope. Furthermore, it also has the advantage of not being based on a proprietary technology, as is the already described HTS assay (Peppard et al., 2003).
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In conclusion, we present in this paper an immunoassay well suited for rapid screening of aggrecanase inhibitors in chemical libraries. Initially set up for manual procedure, it has been easily automatized, thanks to its limited number of steps, opening the route towards a large scale screen for the discovery of aggrecanase inhibitors. 4. Experimental procedures 4.1. Reagents Human recombinant aggrecan IGD and ADAMTS-4 were from Invitek (Berlin, Germany). Aggrecan IGD consisted of aminoacids T331–G458, which correspond to residues T350–G477 of the full-length human sequence including the leader sequence, and a C-terminal poly-His tail, with a calculated molecular weight (MW) of 15.5 kDa. According to the manufacturer, this peptide has an apparent MW of 20 kDa in sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE). ADAMTS-4 consisted of aminoacids F213–A579 and a C-terminal poly-His tail, with a calculated MW of 40.4 kDa. According to the manufacturer, this peptide has an apparent MW in SDS-PAGE of 44 kDa. This fragment is the equivalent of the p40 form described by others, which consists of the catalytic, disintegrin and thrombospondin domains, and cleaves aggrecan in both the CS-rich domain and the IGD (Gao et al., 2004; Kashiwagi et al., 2004; Flannery et al., 2002). This form has wider substrate competence than the p68 form, due to the absence of the cysteine-rich and spacer domains, which play an important role in substrate recognition. The aggrecanase inhibitor is the molecule identified as compound 8 in the paper by Yao et al. (2001), and was synthesized, as a mixture of two chiral isomers, at Institut de Recherches Servier. Its formula is: N4-hydroxy-2-(3-hydroxybenzyl) N1-[(1S,2R) 2-hydroxy-2,3dihydro-1H-inden-1-yl] succinamide. This compound inhibited aggrecanase activity with an IC50 of 6.4 × 10− 8 M, when tested according to the protocol by Tortorella et al. (2000). Bovine serum albumin (BSA) of RIA grade, Brij, Tris-buffered saline (TBS), phosphate-buffered saline (PBS), Tween 20, and HISselect™ nickel-coated plates of high binding capacity were from Sigma (St. Louis, MO, USA). Two different antibodies were used to detect the aggrecanase-generated neoepitope in aggrecan IGD. Rabbit anti-ARGSV serum (antibody 2171) was from HDM (Montreal, Canada). Rabbit anti-ARGSVIL was produced by Agro-bio (La Ferté St Aubin, France), using as antigen the peptide consisting of ARGSVIL plus a C-terminal TC dimer conjugated to keyhole limpet hemocyanin (KLH). The anti-ARGSVIL antiserum was affinity-purified by antigenconjugated beads. Cy™5-conjugated donkey anti-rabbit IgG was from Jackson Immunoresearch (Baltimore, MD, USA). The solution used to detach aggrecan IGD from Ni-coated plates was made of NuPage LDS sample buffer and NuPage sample reducing agent (both from Invitrogen) diluted 4 and 10 times, respectively, into TBS (Sigma). 50 μl/well were added to the plates, incubated for 30 min on an orbital shaker at room temperature, then reduced and separated by SDS-PAGE as described below.
4.2. Test of aggrecanase inhibition Nickel-coated 96-well plates (His Select™, Sigma) were coated with 200 μl/well of 0.250 μM recombinant His-tagged aggrecan IGD in TBS for 24 h at 4 °C or 2 h at 37 °C. The wells were washed 3 times with 300 μl/well of 0.05% Tween TBS, then filled with 10 μl of inhibitor or vehicle plus 190 μl ADAMTS-4 (0.25 nM final concentration) in 10 mM CaCl2, 0.05% Brij TBS, and incubated at 37 °C for 30 min, at the end of which the supernatants were discarded. After saturation with 200 μl of 1% BSA, 0.1% Tween PBS for 1 h at room temperature, and 3 washes with 300 μl of 0.05% Tween TBS, the plates were incubated overnight at 4 °C with 200 μl/well of 1 / 100 rabbit anti-ARGSVIL antibody (Agro-bio) or 1 / 500 anti-ARGSV (HDM) in 1% BSA PBS. After 3 more washes, 100 μl of 1/125 Cy™5-conjugated donkey anti rabbit IgG was added. After 2-h incubation at room temperature, and 3 washes with 300 μl of 0.05% Tween TBS, wells were emptied and fluorescence was read at excitation and emission wavelengths of 650 and 680 nm, respectively using a Synergy HT microplate reader (Bio-Tek, Winooski, VT, USA). 4.3. High performance capillary electrophoresis (HPCE) Experiments were carried out on a ProteomeLab TM PA-800 system with photodiode array detection (Beckman Coulter Inc. Fullerton, CA, USA), using a Fused Silica Capillary 50 μm ID × 30.2 cm (20 cm to detector). Samples were electrokinetically introduced by applying voltage at − 5 kV for 60 s. Electrophoresis was performed at constant voltage, with an applied field strength of (−) 497 V/cm with a capillary thermostated to 25 °C using recirculating liquid coolant. UV detection was performed at 220 nm. 32 Karat software version 7.0 was used for instrument control and for data collecting and processing. Between two consecutive injections, the capillary was rinsed with 0.1 N NaOH (70 psi × 3 min), 0.1 N HCl (70 psi × 1 min), deionized water (70 psi × 1 min) and SDS gel buffer (70 psi × 6 min). Reagents : SDS-MW Gel buffer, SDS sample buffer (100 mM Tris HCl pH = 9–1% SDS) were all manufactured at Beckman Coulter Inc., Fullerton, CA, USA. 4.4. SDS-PAGE and Western blots Samples were reduced at 90 °C for 5 min by NuPAGE reducing agent (Invitrogen), and separated by SDS-PAGE on 4– 12% gels (Invitrogen) in parallel with either Magic Mark or Mark 12 MW markers from Invitrogen. Separated samples were then either stained (by sypro ruby protein gel stain, or by colloidal blue staining kit, both from Invitrogen) or transferred onto Hybond ECL membranes (Amersham Pharmacia), using an Xcell system from Invitrogen. Membranes were saturated with 1% BSA, 0.1% Tween PBS for 1 h, then probed with anti ARGSV antiserum from HDM (1 / 1000) in 1% BSA PBS for 1 h at room temperature. After 3 washes, membranes were probed with 1 / 2000 peroxidase-labeled anti rabbit IgG in 1% BSA PBS for 1 h at room temperature. After 4 washes, signals were visualized using an ECL detection kit from Amersham
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Pharmacia. Blot images were captured by an Agfa Arcus II scanner (Ista, Paris, France) controlled by a Kodak 1D imageanalysis software (Rochester, NY, USA). References Abbaszade, I., Liu, R.Q., Yang, F., Rosenfeld, S.A., Ross, O.H., Link, J.R., Ellis, D.M., Tortorella, M.D., Pratta, M.A., Hollis, J.M., Wynn, R., Duke, J.L., George, H.J., Hillman, M.C., Murphy, K., Wiswall, B.H., Copeland, R.A., Decicco, C.P., Bruckner, R., Nagase, H., Itoh, Y., Newton, R.C., Magolda, R.L., Trzaskos, J.M., Hollis, G.F., Arner, E.C., Burn, T.C., 1999. Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family. J. Biol. Chem. 274, 23443–23450. Arner, E.C., Pratta, M.A., Decicco, C.P., Xue, C.-B., Newton, R.C., Trzaskos, J.M., Magolda, R.L., Tortorella, M.D., 1999. Aggrecanase. A target for the design of inhibitors of cartilage degradation. Ann. N. Y. Acad. Sci. 878, 92–107. Caterson, B., Flannery, C.R., Hughes, C.E., Little, C.B., 2000. Mechanisms involved in cartilage proteoglycan catabolism. Matrix Biol. 19, 333–344. Collins-Racie, L.A., Flannery, C.R., Zeng, W., Corcoran, C., Annis-Freeman, B., Agostino, M.J., Arai, M., DiBlasio-Smith, E., Dorner, A.J., Georgiadis, K.E., Jin, M., Tan, X.Y., Morris, E.A., LaVallie, E.R., 2004. ADAMTS8 exhibits aggrecanase activity and is expressed in human articular cartilage. Matrix Biol. 23, 219–230. Flannery, C.R., Zeng, W., Corcoran, C., Collins-Racie, L.A., Chockalingam, P.S., Hebert, T., Mackie, S.A., McDonagh, T., Crawford, T.K., Tomkinson, K.N., LaVallie, E.R., Morris, E.A., 2002. Autocatalytic cleavage of ADAMTS-4 (aggrecanase-1) reveals multiple glycosaminoglycan-binding sites. J. Biol. Chem. 277, 42775–42780. Gao, G., Westling, J., Thompson, V.P., Howell, T.D., Gottschall, P.E., Sandy, J.D., 2002. Activation of the proteolytic activity of ADAMTS4 (aggrecanase-1) by C-terminal truncation. J. Biol. Chem. 277, 11034–11041. Gao, G., Plaas, A., Thompson, V.P., Jin, S., Zuo, F., Sandy, J.D., 2004. ADAMTS-4 (aggrecanase-1) activation on the cell surface involves Cterminal cleavage by glycosylphosphatidyl inositol-anchored membrane type 4-matrix metalloproteinase and binding of the activated proteinase to chondroitin sulfate and heparan sulfate on syndecan. J. Biol. Chem. 279, 10042–10051. Kashiwagi, M., Enghild, J.J., Gendron, C., Hughes, C., Caterson, B., Itoh, Y., Nagase, H., 2004. Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing. J. Biol. Chem. 279, 10109–10119. Lohmander, L.S., Neame, P.J., Sandy, J.D., 1993. The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis. Arthritis Rheum. 36, 1214–1222. Malfait, A.M., Liu, R.Q., Ijiri, K., Komiya, S., Tortorella, M.D., 2002. Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage. J. Biol. Chem. 277, 22201–22208. Martel-Pelletier, J., 1999. Pathophysiology of osteoarthritis. Osteoarthr. Cartil. 7, 371–373.
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Miller, J.A., Liu, R.Q., Davis, G.L., Pratta, M.A., Trzaskos, J.M., Copeland, R. A., 2003. A microplate assay specific for the enzyme aggrecanase. Anal. Biochem. 314, 260–265. Moulharat, N., Lesur, C., Thomas, M., Rolland-Valognes, G., Pastoureau, P., Anract, P., De Ceuninck, F., Sabatini, M., 2004. Effects of transforming growth factor-β on aggrecanase production and proteoglycan degradation by human chondrocytes in vitro. Osteoarthr. Cartil. 12, 296–305. Peppard, J., Glickman, F., He, Y., Hu, S.I., Doughty, J., Goldberg, R., 2003. Development of a high-throughput screening assay for inhibitors of aggrecan cleavage using luminescent oxygen channelling (AlphaScreen™). J. Biomol. Screen. 8, 149–156. Rodriguez-Manzaneque, J.C., Westling, J., Thai, S.N.M., Luque, A., Knauper, V., Murphy, G., Sandy, J.D., Iruela-Arispe, M.L., 2002. ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitor. Biochem. Biophys. Res. Commun. 293, 501–508. Sandy, J.D., Flannery, C.R., Neame, P.J., Lohmander, L.S., 1992. The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373–Ala 374 bond of the interglobular domain. J. Clin. Invest. 89, 1512–1516. Sandy, J.D., Thompson, V., Doege, K., Verscharen, C., 2000. The intermediates of aggrecanase-dependent cleavage of aggrecan in rat chondrosarcoma cells treated with interleukin-1. Biochem. J. 351, 161–166. Sandy, J.D., Verscharen, C., 2001. Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo. Biochem. J. 358, 615–626. Tortorella, M.D., Burn, T.C., Pratta, M.A., Abbaszade, I., Hollis, J.M., Liu, R., Rosenfeld, S.A., Copeland, R.A., Decicco, C.P., Wynn, R., Rockwell, A., Yang, F., Duke, J.L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D.M., Ross, H., Wiswall, B.H., Murphy, K., Hillman, M.C., Hollis, G.F., Newton, R.C., Magolda, R.L., Trzaskos, J.M., Arner, E.C., 1999. Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins. Science 284, 1664–1666. Tortorella, M., Pratta, M., Liu, R.-Q., Abbaszade, I., Ross, H., Burn, T., Arner, E., 2000. The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage. J. Biol. Chem. 275, 25791–25797. Westling, J., Fosang, A.J., Last, K., Thompson, V.P., Tomkinson, K.N., Hebert, T., McDonagh, T., Collins-Racie, L.A., LaVallie, E.R., Morris, E.A., Sandy, J.D., 2002. ADAMTS4 cleaves at the aggrecanse site (Glu373–Ala374) and secondarily at the matrix metalloproteinase site (Asn341–Phe342) in the aggrecan interglobular domain. J. Biol. Chem. 277, 16059–16066. Yao, W., Wasserman, Z.R., Chao, M., Reddy, G., Shi, E., Liu, R.-Q., Covington, M.B., Arner, E.C., Pratta, M.A., Tortorella, M., Magolda, R.L., Newton, R., Qian, M.X., Ribadeneira, M.D., Christ, D., Wexler, R.R., Decicco, C.P., 2001. Design and synthesis of a series of (2R)-N4-hydroxy-2-(3-hydroxybenzyl)-N1-[(1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]buta-nediamide derivatives as potent, selective, and orally bioavailable aggrecanase inhibitors. J. Med. Chem. 44, 3347–3350.
A microplate assay for the screening of ADAMTS-4 inhibitors Marie Thomas a , Massimo Sabatini a,⁎, Fabrice Bensaude b , Benoit Mignard b , Jean-Claude Ortuno c , Isabelle Caron d , Jean A. Boutin b , Gilles Ferry b a Division of Rheumatology, Institut de Recherches Servier, IdRS, 11, rue des Moulineaux, 92150 Suresnes, France Division of Molecular and Cellular Pharmacology, Institut de Recherches Servier, 125, chemin de Ronde, 78290 Croissy-sur-Seine, France Division of Medicinal Chemistry, Institut de Recherches Servier, Institut de Recherches Servier, 125, chemin de Ronde, 78290 Croissy-sur-Seine, France d Beckman Coulter France S.A., Roissy, France b
c
Received 7 October 2005; received in revised form 13 December 2005; accepted 13 December 2005
Abstract Aggrecanase plays a major role in cartilage proteoglycan degradation in rheumatic diseases such as osteoarthritis and rheumatoid arthritis. The search of new inhibitors of aggrecanase activity necessitates a robust assays in order to be able to screen large numbers of compounds. We present in this paper an assay based on the cleavage of His-tagged aggrecan interglobular domain by N- and C- terminus truncated, active aggrecanase-1/ ADAMTS-4, with formation of the aggrecanase-specific ARGSV neoepitope. This is detected by anti-ARGSV antibody, in turn recognized by a fluorescent anti-IgG. Furthermore, the formation of the reaction products was confirmed by high-pressure capillary electrophoresis. This assay allows the rapid screening of aggrecanase inhibitors in a 96-well plate format, allowing an immediate transposition to high-throughput scale up. © 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved. Keywords: Aggrecanase; Screening assay; ADAMTS-4; Proteoglycan degradation; Anti-ARGSV antibody; HPCE
1. Introduction Osteoarthritis, the most common rheumatic disease, is characterized by progressive erosion of articular cartilage, finally leading to exposure of the underlying bone (MartelPelletier, 1999). Cartilage loss is an active process, due to an imbalance of tissue turn-over, consequent to an increase of degradation and decrease of synthesis of the extracellular matrix. Cartilage main components are collagen type II, which forms a tridimensional scaffold responsible for tensile strength, and aggrecan, a highly hydrated proteoglycan responsible for cartilage resilience and resistance to compression. Aggrecan degradation is carried out by metalloproteinases of the matrix metalloproteinase (MMP) and aggrecanase families (Caterson et al., 2000). Even if the relative contributions of the two groups of enzymes are not yet clearly defined, aggrecanases seem to play the main role in aggrecan catabolism, as it is suggested by the predominance of aggrecanase-generated neoepitopes in both the synovial fluid (Sandy et al., 1992; Lohmander et al., 1993) ⁎ Corresponding author. Tel.: +33 1 55 72 24 11; fax: +33 1 55 72 27 37. E-mail address: [email protected] (M. Sabatini).
and cartilage of patients with OA (Sandy and Verscharen, 2001). Aggrecanases belong to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin domain) family and cleave aggrecan at multiple sites, first in the G2–G3 chondroitin sulfate-rich domain, and then at the classical NITEGE373– A374RGSVIL position in the G1–G2 interglobular domain (IGD) (Sandy et al., 2000), corresponding to position E392–A393 of the full-length human sequence including the 19-residue leader sequence. Several aggrecanases have been so far identified in cartilage, and shown to be produced by chondrocytes: aggrecanase-1/ADAMTS-4 (Tortorella et al., 1999), aggrecanase-2/ADAMTS-5 (Abbaszade et al., 1999), ADAMTS-1 (Rodriguez-Manzaneque et al., 2002) and ADAMTS-8 (Collins-Racie et al., 2004). Synthesis of aggrecanases seems to be differentially regulated in chondrocytes. While aggrecanase-1 expression is stimulated by the inflammatory cytokines interleukin-1 (IL-1) β and tumor necrosis factor (TNF) α, and by transforming growth factor (TGF) β, aggrecanase-2 seems to be produced in a constitutive way (Moulharat et al., 2004). Aggrecanase-1/ADAMTS-4 is produced as 100 kDa proenzyme, which is N-terminally cleaved by furin (Gao et al., 2002), to produce a secreted 68 kDa form that
0945-053X/$ - see front matter © 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved. doi:10.1016/j.matbio.2005.12.003
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up producing a time-resolved fluorescent signal between antibodies linked to these beads and directed against the chondroitin and keratan sulfate regions of aggrecan. The region between these zones is the target of aggrecanase cleavage of aggrecan, leading to the decrease of the fluorescent signal between the beads, since the cleavage physically separates the zones against which the antibodies were raised. The test is based on the use of (a) C-terminally His-tagged aggrecan IGD immobilized on Ni-coated plates as a substrate and (b) N- and C-terminally truncated ADAMTS-4 as enzyme. Aggrecanase activity is measured by immunodetection of ARGSVIL neoepitope produced through cleavage of aggrecan IGD by ADAMTS-4.
associates to membrane-type (MT)-4 MMP in chondrosarcoma cells (Gao et al., 2004). MT-4 MMP is in turn linked to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor, and cleaves ADAMTS-4 C-terminally to produce 53 and 40 kDa forms, in which substrate specificity progressively switches from the G2–G3 chondroitin-rich domain to the G1–G2 IGD (Gao et al., 2004; Kashiwagi et al., 2004). Inhibition of aggrecanases is a promising approach for the treatment of arthritic diseases (Arner et al., 1999; Malfait et al., 2002). Screening of large numbers of chemicals in search of inhibitors depends on the availability of simple and reproducible assays of aggrecanase activity. Miller et al. (2003) recently described a microplate assay based on the immunodetection of the aggrecan neoepitope ARGSVIL, generated by the cleavage of a synthetic, biotinylated 41-mer peptide substrate, immobilized to streptavidin-coated plates and degraded by aggrecanase partially purified from cultures of bovine cartilage. We used a partially different approach, mainly dictated by the choice to use commercially available reagents. This paper presents a protocol for testing inhibitors of aggrecanase activity. Previous HTS assays (Peppard et al., 2003) used a proprietary technology (AlphaScreen™) requiring the development of beads that end
2. Results It was first verified that truncated, constitutively active ADAMTS-4 (p40 form) could cleave aggrecan IGD, with formation of the aggrecan neoepitope ARGSVIL, as expected for aggrecanase activity. Aggrecan IGD, at concentrations between 0.3 and 3 μM, was incubated with 10 and 100 nM ADAMTS-4 for 2 h. In the presence of 100 nM enzyme, the
A: Gel Ruby
B: Western Blot
MW (kDa)
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IGD (µM) ADAMTS-4 (100 nM)
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Fig. 1. Degradation of aggrecan IGD by ADAMTS-4 (A and B), and effects of an aggrecanase inhibitor (C). Panels A and B: aggrecan IGD at concentrations of 0.3, 0.6 and 3 μM, was exposed to 100 nM ADAMTS-4 for 2 h at 37 °C. The reaction was stopped by adding SDS sample buffer with reducing agent and heating at 90 °C for 5 min. Samples were separated by SDS-PAGE and either stained with sypro ruby (A) or subjected to Western blot and probed by anti-ARGSVIL antibody (B). Panel C: aggrecan IGD (3 μM) was incubated without or with 100 nM ADAMTS-4 in the absence or presence of aggrecanase inhibitor at concentrations in the range 10− 9 to 10− 5 M, for 2 h at 37 °C. The reaction was stopped by adding SDS sample buffer with reducing agent and heating at 90 °C for 5 min, then the samples were separated by SDS-PAGE in parallel with Mark 12 MW standards and stained by colloidal blue.
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concentrations between 0.6 and 3 μM (0.12 to 0.6 nmoles/well, respectively) was added to the plates and incubated for 24 h at 4 °C, or 2 h at 37 °C. After repeated washes to remove unbound material, bound IGD was desorbed from the plates, and a fraction separated by SDS-PAGE and visualized by protein staining (not shown). The bands of recovered aggrecan IGD were compared with a scale of aggrecan IGD standards similarly separated and stained. It was determined by densitometric analysis that IGD binding reached a maximum of around 30 pmol/well for peptide solutions between 1.8 and 3 μM left in the wells for 24 h at 4 °C. Comparable binding was obtained by incubating 3 μM IGD for 2 h at 37 °C. It was then determined if plate-bound aggrecan IGD could be degraded by ADAMTS-4. Ni-coated plates were coated with 1.8 μM aggrecan IGD (0.36 nmol/well) for 24 h at 4 °C, washed to remove the unbound protein, then exposed to 100 nM ADAMTS-4 for 2 h at 37 °C. After repeated washes to remove N-terminal cleaved fragments, plate-bound IGD, intact or
20 kDa aggrecan IGD was partially converted to a 12 kDa form (Fig. 1A), which presented the ARGSVIL neoepitope (Fig. 1B) and therefore represented the C-terminal fragment of aggrecanase-cleaved IGD. A very weak signal was obtained using 10 nM ADAMTS-4 (not shown). Aggrecan IGD degradation was inhibited by a selective aggrecanase inhibitor (compound 8 in Yao et al., 2001), which added at concentrations between 10− 9 and 10− 5 M, dose-dependently inhibited the appearance of the 12 kDa fragment (Fig. 1C). No band could be observed, corresponding to the smaller N-terminal fragment, with a calculated MW of 4.8 kDa. In a similar experiment, aggrecan IGD degradation in the absence (Fig. 2A) and presence of the inhibitor (Fig. 2B) was quantified using high performance capillary electrophoresis. An IC50 value of 3.5 × 10− 7 M was calculated (Fig. 2B). Knowing that aggrecan IGD could be correctly cleaved by ADAMTS-4, it was then examined if aggrecan IGD could be adsorbed onto Ni-coated multiwell plates by its C-terminal poly His-tag tail. Aggrecan IGD at
b
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MW (kDa) 55
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Time (minutes) Fig. 2. A: Analysis of aggrecanase activity by HPCE and PAGE. 100 nM ADAMTS-4 was incubated with 3 μM aggrecan IGD for 2 h at 37 °C. The reaction was stopped by adding SDS sample buffer with reducing agent and heating at 90 °C for 5 min, then the samples were analysed in parallel by HPCE (elution profile at 220 nm) and by SDS-PAGE (insert: colloidal blue staining of gel). Lane 1: IGD alone; lane 2: IGD plus ADAMTS-4. Letters a (in gel) and b, c (in both gel and profile) indicate ADAMTS-4 (p40), IGD and IGD degradation product, respectively. B: Effect of the aggrecanase inhibitor on ADAMTS-4 activity. Reaction was carried out as in Fig. 1C, in the absence or presence of the inhibitor at concentrations between 10− 8 and 10− 5. Values are means of triplicates. In both A and B panels, absorbance profiles of depicted runs were shifted along the y axes in order to set apart the different profiles. Depicted run actually showed the same baseline absorbance values.
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A
B MW
kDa
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IGD Cleaved IGD 6
IGD ADAMTS-4
+ -
+
+ +
+ +
+ +
+
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-
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Fig. 3. Aggrecan IGD (1.8 μM, 200 μl/well) was adsorbed to Ni-coated plates for 24 h at 4 °C. After 3 washes to remove unbound material, bound IGD was exposed to 100 nM ADAMTS-4 for 2 h at 37 °C. After 3 more washes to remove N-terminal cleaved fragments, plate-bound IGD, intact or degraded, was desorbed from the wells, and a fraction separated by SDS-PAGE, in parallel with Mark 12 MW standards, and either stained by sypro ruby (A) or subjected to Western blot and probed by anti-ARGSV antibody (B).
0.25 μM without major loss of signal (data not shown). Linear neoepitope formation occurred during the first 30 min of reaction, using 0.25 μM aggrecan IGD for coating and ADAMTS-4 at 0.125 or 0.25 nM for substrate degradation (Fig. 4A). The signal obtained at 30 min was proportional to enzyme concentration in the range from 0.031 to 0.25 nM (Fig. 4B). Therefore, conditions selected for the screening test provided for coating with 0.25 μM aggrecan, and substrate degradation by 0.25 nM ADAMTS-4 for 30 min. Similar signals were obtained using as a primary antibody either antiARGSV (diluted 1 / 500) by HDM or anti-ARGSVIL (diluted 1 / 100) by Agro-bio. Using the conditions finally selected (see Section 4.2 for details), the aggrecanase inhibitor antagonized ADAMTS-4 with an IC50 value of 1.6 × 10− 7 (Fig. 5).
degraded, was desorbed from the wells and separated by SDSPAGE (Fig. 3A). The 20 kDa band of aggrecan IGD was converted to the 12 kDa form, which was confirmed to possess the ARGSV neoepitope by Western blot (Fig. 3B). It was then verified that degraded aggrecan IGD still attached to the plate by its poly-His tail could be detected in situ by the anti-ARGSV antibody. Bound primary antibody was detected using a Cy™5conjugated secondary antibody. Aggrecan IGD (0.75 μM) was adsorbed to the plate, then exposed to ADAMTS-4. It was found that enzyme concentration of 100 nM, well adapted to the degradation of solubilised aggrecan IGD, was in large excess when used against the immobilized substrate. The signal detected after 1 h of reaction was strongly decreased after 2 h and had almost disappeared by 4 h (data not shown). This could be due to further degradation of the substrate and shedding of the neoepitope, as suggested by the fact that further dilutions of ADAMTS-4 in the range from 0.25 to 0.031 nM allowed a time-dependent increase of neoepitope formation. It was also found that within this range of enzyme concentrations, aggrecan-IGD coating solution could be diluted from 0.75 to
3. Discussion We describe in this paper the development of a rapid and simple immunoassay for screening of ADAMTS-4 inhibitors. When examining the conditions of aggrecan IGD cleavage by
A
B
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Fig. 4. A: Time course of neoepitope signal generation. Aggrecan IGD (0.250 μM) was adsorbed to 96-well Ni-coated plates, then exposed to ADAMTS-4 at concentrations of 0.031 (squares), 0.063 (triangles), 0.125 (circles) and 0.250 (diamonds) nM, for 15, 30, 45 and 60 min at 37 °C. B: Data in A were replotted to show fluorescence vs. ADAMTS-4 concentration at 15 (squares), 30 (circles), 45 (triangles), and 60 min (triangles).
M. Thomas et al. / Matrix Biology 25 (2006) 261–267
30000 IC50=1.6x10-7 M
Fluorescence unit
25000 20000 15000 10000 5000 0 0
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-8
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Inhibitor concentration (Log M) Fig. 5. Inhibition of ADAMTS-4 activity by the aggrecanase inhibitor. Aggrecan (0.25 μM) was preadsorbed to Ni-Coated plates. The inhibitor was added at final concentrations between 10− 9 and 10− 3 M, followed by ADAMTS-4 at the final concentration of 0.25 nM for 30 min at 37 °C (see Section 4.2 for details). Data are means ± ESM; n = 3 points at each concentration.
ADAMTS-4 (p40 form) in solution, reaction kinetics could not be determined, for lack of an analytical system of the necessary sensitivity for the measurement of the substrate and the reaction products. It was however verified that IGD, which showed an apparent MW of 20 kDa in SDS-PAGE, could be cleaved by truncated ADAMTS-4 and form a fragment of 12 kDa, exhibiting the ARGSVIL neoepitope, which is a specific marker of aggrecanase activity. Formation of the smaller, N-terminal fragment, with a calculated MW of 4.8 kDa, could not be monitored by SDS-PAGE on a 6–12% gel, possibly also because of secondary cleavage at the MMP site N341–F342 (corresponding to position N360–F361 of the full-length human sequence) (Westling et al., 2002), further decreasing MW to 3.7 kDa. Cleavage of IGD and production of the 12 kDa fragment could be inhibited by a specific aggrecanase inhibitor. Results obtained by SDS-PAGE and Western blot were supported by studies with high performance capillary electrophoresis, which confirmed both IGD degradation by ADAMTS-4 and cleavage inhibition by the anti-aggrecanase compound. That aggrecan IGD, consisting of G1–G2 region only, could be cleaved by truncated ADAMTS-4 was not obvious, in light of the paper by Tortorella et al. (2000) showing that the G2–G3 region of aggrecan, rich in glycosaminoglycan, is important for substrate recognition by ADAMTS-4, via the C-terminal thrombospondin-1 region (TSP-1) of the enzyme. However, the lack of the G2–G3 region in the substrate was probably offset by less stringent substrate recognition by the enzyme, due to the absence of the C-terminal cys-rich and spacer domains in the p 40 form of ADAMTS-4 (Gao et al., 2002, 2004; Kashiwagi et al., 2004; Flannery et al., 2002). After verification of substrate cleavage in solution, the conditions were defined under which aggrecan IGD could be adsorbed to the Ni-coated plates via its poly-His tail, and degraded by ADAMTS-4 with generation of ARGSVIL neoepitope. Compared with the conditions used for substrate degradation in solution, it was necessary to decrease
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ADAMTS-4 concentration by at least 400 times in order to assure linear generation of neoepitope signal. Moreover, aggrecan IGD concentration for substrate adsorption could be limited to 250 nM. Exposure of plate-adsorbed aggrecan IGD to 100 nM ADAMTS-4 produced a transient signal, probably because of further degradation of the plate-bound substrate. It is known that the G1-carrying fragment deriving from cleavage of aggrecan IGD at E373–A374 can be secondarily attacked by ADAMTS-4 at the N341–F342 region, which is normally recognized by MMPs (Westling et al., 2002). However, no secondary cleavage has been so far reported for the G2 carrying fragment. Further degradation at the A374 N-extremity could be due to altered substrate conformation due to the presence of the poly-His tail linked to the well surface. Another possibility could be that the enzyme competed the substrate out of the Ni-coated suface, since they both possess a C-terminal His-tag. However, addition of the aggrecanase inhibitor blocked the disappearance of the signal (not shown), suggesting that the enzyme's poly-His tail does not interfere with substrate binding to the plate. The conditions selected for the test allowed signal generation that was linear over 30 min, and proportional to enzyme concentration in the 0.031 to 0.25 nM range. When a specific aggrecanase inhibitor (Yao et al., 2001) was tried on the plate test, the IC50 found (1.6 × 10− 7 M) was of the same order as that determined when the reaction was carried out in a test tube with the substrate in solution, and the analysis carried out by capillary electrophoresis (3.5 × 10− 7 M). This value was however higher than that (6.4 × 10− 8 M) (Yao et al., 2001) found when the inhibitor was tested according to a protocol using full-length human ADAMTS-4 and purified bovine aggrecan (Tortorella et al., 2000). Most of this difference could be due to the chiral nature of the compound we used, if only one of the two forms present were able, as it is often the case, to inhibit enzyme activity. This difference could be also explained by changes of affinity between ADAMTS-4 and aggrecan, when truncated instead of full-length forms are used (Gao et al., 2002; 2004; Kashiwagi et al., 2004). Moreover, different concentrations of substrate and enzyme were used in the two assays, which would also contribute to IC50 changes. It can also be added that the absence of the cysrich and spacer domain in ADAMTS-4 p40 does not allow interactions between the inhibitor and regions that are otherwise important for substrate recognition in p53 and p68 forms of the enzyme. Compared with already published protocols (Miller et al., 2003; Tortorella et al., 2000) this assay has the advantage of being based on commercially available reagents and therefore offers the possibility of a rapid set-up. A possibly limiting factor in the routine use of this protocol is the primary antibody, due to the limited dilution (1 / 100 for Agro-bio; 1 / 500 for HDM antibodies) that has to be used for the detection of the aggrecanase-generated neoepitope. However, we easily produced different batches of rabbit affinity-purified anti-ARGSVIL of consistent affinity for the epitope. Furthermore, it also has the advantage of not being based on a proprietary technology, as is the already described HTS assay (Peppard et al., 2003).
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In conclusion, we present in this paper an immunoassay well suited for rapid screening of aggrecanase inhibitors in chemical libraries. Initially set up for manual procedure, it has been easily automatized, thanks to its limited number of steps, opening the route towards a large scale screen for the discovery of aggrecanase inhibitors. 4. Experimental procedures 4.1. Reagents Human recombinant aggrecan IGD and ADAMTS-4 were from Invitek (Berlin, Germany). Aggrecan IGD consisted of aminoacids T331–G458, which correspond to residues T350–G477 of the full-length human sequence including the leader sequence, and a C-terminal poly-His tail, with a calculated molecular weight (MW) of 15.5 kDa. According to the manufacturer, this peptide has an apparent MW of 20 kDa in sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE). ADAMTS-4 consisted of aminoacids F213–A579 and a C-terminal poly-His tail, with a calculated MW of 40.4 kDa. According to the manufacturer, this peptide has an apparent MW in SDS-PAGE of 44 kDa. This fragment is the equivalent of the p40 form described by others, which consists of the catalytic, disintegrin and thrombospondin domains, and cleaves aggrecan in both the CS-rich domain and the IGD (Gao et al., 2004; Kashiwagi et al., 2004; Flannery et al., 2002). This form has wider substrate competence than the p68 form, due to the absence of the cysteine-rich and spacer domains, which play an important role in substrate recognition. The aggrecanase inhibitor is the molecule identified as compound 8 in the paper by Yao et al. (2001), and was synthesized, as a mixture of two chiral isomers, at Institut de Recherches Servier. Its formula is: N4-hydroxy-2-(3-hydroxybenzyl) N1-[(1S,2R) 2-hydroxy-2,3dihydro-1H-inden-1-yl] succinamide. This compound inhibited aggrecanase activity with an IC50 of 6.4 × 10− 8 M, when tested according to the protocol by Tortorella et al. (2000). Bovine serum albumin (BSA) of RIA grade, Brij, Tris-buffered saline (TBS), phosphate-buffered saline (PBS), Tween 20, and HISselect™ nickel-coated plates of high binding capacity were from Sigma (St. Louis, MO, USA). Two different antibodies were used to detect the aggrecanase-generated neoepitope in aggrecan IGD. Rabbit anti-ARGSV serum (antibody 2171) was from HDM (Montreal, Canada). Rabbit anti-ARGSVIL was produced by Agro-bio (La Ferté St Aubin, France), using as antigen the peptide consisting of ARGSVIL plus a C-terminal TC dimer conjugated to keyhole limpet hemocyanin (KLH). The anti-ARGSVIL antiserum was affinity-purified by antigenconjugated beads. Cy™5-conjugated donkey anti-rabbit IgG was from Jackson Immunoresearch (Baltimore, MD, USA). The solution used to detach aggrecan IGD from Ni-coated plates was made of NuPage LDS sample buffer and NuPage sample reducing agent (both from Invitrogen) diluted 4 and 10 times, respectively, into TBS (Sigma). 50 μl/well were added to the plates, incubated for 30 min on an orbital shaker at room temperature, then reduced and separated by SDS-PAGE as described below.
4.2. Test of aggrecanase inhibition Nickel-coated 96-well plates (His Select™, Sigma) were coated with 200 μl/well of 0.250 μM recombinant His-tagged aggrecan IGD in TBS for 24 h at 4 °C or 2 h at 37 °C. The wells were washed 3 times with 300 μl/well of 0.05% Tween TBS, then filled with 10 μl of inhibitor or vehicle plus 190 μl ADAMTS-4 (0.25 nM final concentration) in 10 mM CaCl2, 0.05% Brij TBS, and incubated at 37 °C for 30 min, at the end of which the supernatants were discarded. After saturation with 200 μl of 1% BSA, 0.1% Tween PBS for 1 h at room temperature, and 3 washes with 300 μl of 0.05% Tween TBS, the plates were incubated overnight at 4 °C with 200 μl/well of 1 / 100 rabbit anti-ARGSVIL antibody (Agro-bio) or 1 / 500 anti-ARGSV (HDM) in 1% BSA PBS. After 3 more washes, 100 μl of 1/125 Cy™5-conjugated donkey anti rabbit IgG was added. After 2-h incubation at room temperature, and 3 washes with 300 μl of 0.05% Tween TBS, wells were emptied and fluorescence was read at excitation and emission wavelengths of 650 and 680 nm, respectively using a Synergy HT microplate reader (Bio-Tek, Winooski, VT, USA). 4.3. High performance capillary electrophoresis (HPCE) Experiments were carried out on a ProteomeLab TM PA-800 system with photodiode array detection (Beckman Coulter Inc. Fullerton, CA, USA), using a Fused Silica Capillary 50 μm ID × 30.2 cm (20 cm to detector). Samples were electrokinetically introduced by applying voltage at − 5 kV for 60 s. Electrophoresis was performed at constant voltage, with an applied field strength of (−) 497 V/cm with a capillary thermostated to 25 °C using recirculating liquid coolant. UV detection was performed at 220 nm. 32 Karat software version 7.0 was used for instrument control and for data collecting and processing. Between two consecutive injections, the capillary was rinsed with 0.1 N NaOH (70 psi × 3 min), 0.1 N HCl (70 psi × 1 min), deionized water (70 psi × 1 min) and SDS gel buffer (70 psi × 6 min). Reagents : SDS-MW Gel buffer, SDS sample buffer (100 mM Tris HCl pH = 9–1% SDS) were all manufactured at Beckman Coulter Inc., Fullerton, CA, USA. 4.4. SDS-PAGE and Western blots Samples were reduced at 90 °C for 5 min by NuPAGE reducing agent (Invitrogen), and separated by SDS-PAGE on 4– 12% gels (Invitrogen) in parallel with either Magic Mark or Mark 12 MW markers from Invitrogen. Separated samples were then either stained (by sypro ruby protein gel stain, or by colloidal blue staining kit, both from Invitrogen) or transferred onto Hybond ECL membranes (Amersham Pharmacia), using an Xcell system from Invitrogen. Membranes were saturated with 1% BSA, 0.1% Tween PBS for 1 h, then probed with anti ARGSV antiserum from HDM (1 / 1000) in 1% BSA PBS for 1 h at room temperature. After 3 washes, membranes were probed with 1 / 2000 peroxidase-labeled anti rabbit IgG in 1% BSA PBS for 1 h at room temperature. After 4 washes, signals were visualized using an ECL detection kit from Amersham
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