Pharmacological characterization of GLPG1972/S201086, a potent and selective small-molecule inhibitor of ADAMTS5.

OBJECTIVE: A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) is a key enzyme in degradation of cartilage in osteoarthritis (OA). We report the pharmacological characterization of GLPG1972/S201086, a new, potent and selective small-molecule ADAMTS5 inhibitor. METHODS: Potency and selectivity of GLPG1972/S201086 for ADAMTS5 were determined using fluorescently labeled peptide substrates. Inhibitory effects of GLPG1972/S201086 on interleukin-1α-stimulated glycosaminoglycan release in mouse femoral head cartilage explants and on interleukin-1ß-stimulated release of an ADAMTS5-derived aggrecan neoepitope (quantified with ELISA) in human articular cartilage explants were determined. In the destabilization of the medial meniscus (DMM) mouse and menisectomized (MNX) rat models, effects of oral GLPG1972/S201086 on relevant OA histological and histomorphometric parameters were evaluated. RESULTS: GLPG1972/S201086 inhibited human and rat ADAMTS5 (IC50 ± SD: 19 ± 2 nM and <23 ± 1 nM, respectively), with 8-fold selectivity over ADAMTS4, and 60->5,000-fold selectivity over other related proteases in humans. GLPG1972/S201086 dose-dependently inhibited cytokine-stimulated aggrenolysis in mouse and human cartilage explants (100% at 20 µM and 10 µM, respectively). In DMM mice, GLPG1972/S201086 (30-120 mg/kg b.i.d) vs vehicle reduced femorotibial cartilage proteoglycan loss (23-37%), cartilage structural damage (23-39%) and subchondral bone sclerosis (21-36%). In MNX rats, GLPG1972/S201086 (10-50 mg/kg b.i.d) vs vehicle reduced cartilage damage (OARSI score reduction, 6-23%), and decreased proteoglycan loss (∼27%) and subchondral bone sclerosis (77-110%). CONCLUSIONS: GLPG1972/S201086 is a potent, selective and orally available ADAMTS5 inhibitor, demonstrating significant protective efficacy on both cartilage and subchondral bone in two relevant in vivo preclinical OA models.

Hydrogen sulphide-releasing diclofenac derivatives inhibit breast cancer-induced osteoclastogenesis in vitro and prevent osteolysis ex vivo.

BACKGROUND AND PURPOSE: Hydrogen sulphide (H(2)S) and prostaglandins are both involved in inflammation, cancer and bone turnover, and non-steroidal anti-inflammatory drugs (NSAIDs) and H(2)S donors exhibit anti-inflammatory and anti-tumour properties. H(2)S-releasing diclofenac (S-DCF) derivatives are a novel class of NSAIDs combining the properties of a H(2)S donor with those of a conventional NSAID. EXPERIMENTAL APPROACH: We studied the effects of the S-DCF derivatives ACS15 and ACS32 on osteoclast and osteoblast differentiation and activity in vitro, human and mouse breast cancer cells support for osteoclast formation and signalling in vitro, and osteolysis ex vivo. KEY RESULTS: The S-diclofenac derivatives ACS15 and ACS32 inhibited the increase in osteoclast formation induced by human MDA-MB-231 and MCF-7 and mouse 4T1 breast cancer cells without affecting breast cancer cell viability. Conditioned media from human MDA-MB-231 cells enhanced IκB phosphorylation and osteoclast formation and these effects were significantly inhibited following treatment by ACS15 and ACS32, whereas the parent compound diclofenac had no effects. ACS15 and ACS32 inhibited receptor activator of NFκB ligand-induced osteoclast formation and resorption, and caused caspase-3 activation and apoptosis in mature osteoclasts via a mechanism dependent on IKK/NFκB inhibition. In calvaria organ culture, human MDA-MB-231 cells caused osteolysis, and this effect was completely prevented following treatment with ACS15 and ACS32. CONCLUSIONS AND IMPLICATIONS: S-diclofenac derivatives inhibit osteoclast formation and activity, suppress breast cancer cell support for osteoclastogenesis and prevent osteolysis. This suggests that H(2)S-releasing diclofenac derivatives exhibit anti-resorptive properties, which might be of clinical value in the treatment of osteolytic bone disease.

Sonic hedgehog increases the commitment of pluripotent mesenchymal cells into the osteoblastic lineage and abolishes adipocytic differentiation.

The proteins of the hedgehog (Hh) family regulate various aspects of development. Recently, members of this family have been shown to regulate skeletal formation in vertebrates and to control both chondrocyte and osteoblast differentiation. In the present study, we analyzed the effect of Sonic hedgehog (Shh) on the osteoblastic and adipocytic commitment/differentiation. Recombinant N-terminal Shh (N-Shh) significantly increased the percentage of both the pluripotent mesenchymal cell lines C3H10T1/2 and ST2 and calvaria cells responding to bone morphogenetic protein 2 (BMP-2), in terms of osteoblast commitment as assessed by measuring alkaline phosphatase (ALP) activity. This synergistic effect was mediated, at least partly, through the positive modulation of the transcriptional output of BMPs via Smad signaling. Furthermore, N-Shh was found to abolish adipocytic differentiation of C3H10T1/2 cells both in the presence or absence of BMP-2. A short treatment with N-Shh was sufficient to dramatically reduce the levels of the adipocytic-related transcription factors C/EBPalpha and PPARgamma in both C3H10T1/2 and calvaria cell cultures. Given the inverse relationship between marrow adipocytes and osteoblasts with aging, agonists of the Hh signaling pathway might constitute potential drugs for preventing and/or treating osteopenic disorders.

Endogenous glutathione-binding proteins of insect cell lines: characterization and removal from glutathione S-transferase (GST) fusion proteins.

After affinity purification on immobilized glutathione, insect-cell-derived glutathione S-transferase (GST) fusion proteins contain variable amounts of protein contaminants of about 23-24 kDa. We have isolated these glutathione-binding proteins from the widely used Sf9 and Hi5 insect cell lines and characterized them by LC-MS and N-terminal sequencing. Based on the observation that these proteins have higher affinity for glutathione than GST fusions, we have found that by using differential elution conditions the amount of such contaminants in GST fusion preparations can be strongly reduced directly during the affinity purification step. The main interest of these results is that they are not restricted to a specific construct, but rather they seem to apply to various insect-cell-derived GST fusions.

Differential roles of JNK and Smad2 signaling pathways in the inhibition of c-Myc-induced cell death by TGF-beta.

The transforming growth factor beta (TGF-beta) plays an important role in constraining cellular proliferation, but it is also a potent inducer of programmed cell death or apoptosis. Here, we demonstrate that TGF-beta can have an opposite effect, acting as a survival factor to prevent c-Myc-induced cell death in Rat-1 fibroblasts. However, in marked contrast to TGF-beta, Smad2, which is a critical intracellular mediator of the TGF-beta signaling pathway, functions as an antagonist to induce increased cell death. The protective activity of TGF-beta was associated with the activation of c-Jun N-terminal Kinase (JNK) and was not linked to the ability of TGF-beta to promote cell cycle progression. Expression of dominant-interfering forms of various components of the JNK signaling pathway, including Rac1, Cdc42, mitogen-activated protein kinase kinase 4 (MKK4), and c-Jun, abolished TGF-beta-mediated cell survival. Furthermore, overexpression of the constitutively activated mutant RacL61F37A, which selectively stimulates JNK cascade but not G1 cell cycle progression or actin polymerization, was sufficient to prevent apoptosis induced by c-Myc. These findings describe a differential effect of two separated signaling pathways of TGF-beta and indicate for the first time that Smad2 can act as antagonist to suppress TGF-beta-dependent cell survival. Oncogene (2000) 19, 1277 - 1287.

IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line.

Activation of the Jak/STAT pathway by cytokines has been shown to regulate differentiation, proliferation or apoptosis in hematopoeitic cells. Among the Stat proteins, STAT5 is activated by a broad range of cytokines. In order to study the role of STAT5 in hematopoietic cells, we stably expressed a dominant negative form of STAT5 (STAT5A delta749) in the IL-3 dependent bone marrow derived Ba/F3 cell line. Ba/F3 cells expressing STAT5A delta749 were found to be more sensitive to apoptosis than parental or control Ba/F3 cells after IL-3 withdrawal. Analysis of the expression of the cell death regulators, Bcl-2 and Bcl-x, revealed that the level of Bcl-x was lower in Ba/F3 cells expressing STAT5A delta749 than in control cells. IL-3 regulation of Bcl-x expression at protein and mRNA levels was impaired in these cells while that of Bcl-2 expression was unaffected. We further demonstrated that the Bcl-x gene promoter contained a proximal STAT consensus sequence that bound STAT5. Transactivation of a Bcl-x gene promoter reporter construct by STAT5 was observed in Ba/F3 cells. Introduction of a mutation in the STAT binding site abolished this transactivation. These data indicate that Bcl-x is probably a STAT5 target gene. They also support the involvement of STAT5 in hematopoietic cell survival.

Species specificity and organ, cellular and subcellular localization of the 100 kDa Ras GTPase activating protein.

A p100-GAP isoform, generated by an alternative splicing mechanism that eliminates the 180 hydrophobic amino acids at the amino terminus of p120-GAP, has been described in human placenta, in addition to the known p120GAP and neurofibromin. This p100-GAP possesses full Ras-GTPase stimulating activity. p120-GAP is ubiquitously localized in the cytosol while the localization of p100-GAP is unknown. Here we have explored the precise localization of p100-GAP and show that p100-GAP is present only in extracts of primate placenta. It is abundant in both human and Maccaca Rhesus placentae, where it is present in far larger amounts than p120-GAP. The p100-GAP is species-specific since it was not detected in the placenta of pig, sheep, mouse or rat. p100-GAP was also found to be organ-specific, since it was not detectable in organs other than the placenta. In this connection, we substantiated our previous finding that p100-GAP is mainly localized in the trophoblasts. Both subcellular trophoblast fractionation and immunofluorescence analyses showed that this protein was distributed between the cytosol, plasma membrane and a fraction bound to the nucleus, but not inside it. This highly restrictive specificity of p100-GAP localization in relation to species, organ and cell type, confirms the extreme singularity of this protein, and strongly suggests a particular specific function in the trophoblast.

Comparison of the biochemical properties of unprocessed and processed forms of the small GTP-binding protein, rab6p.

The rab6 protein (rab6p) belongs to a large family of ras-like low-molecular-mass GTP-binding proteins thought to be involved in the regulation of intracellular transport in mammalian cells. When expressed in the baculovirus/insect cell system, two major forms of rab6p are obtained; a 24-kDa cytosolic unprocessed form and a 23-kDa membrane-bound form which represents the processed lipid-modified protein. Here, we have purified both forms to homogeneity and we have studied and compared their biochemical properties. Unprocessed and processed rab6p display similar binding-rate constants (kon) for GDP and GTP (1-1.9 microM-1 min-1). However, significant differences exist in the dissociation constants of bound guanine nucleotides. Processed rab6p in low and high magnesium solutions displays similar koff values for GTP and GDP. However, unprocessed rab6p has a koff value higher for GDP than for GTP in both low and high magnesium solutions. Their intrinsic GTPase activities also differ; unprocessed rab6p has an almost undetectable GTPase activity, whereas that of processed rab6p is in the same range as that reported for other ras and ras-like GTP-binding proteins (0.012 +/- 0.002 min-1). These results suggest that post-translational modifications of rab6p might induce subtle changes in the three-dimensional structure of the protein which affect the guanine-nucleotide-binding/hydrolysis activity.

Purification, characterization, and cellular localization of the 100-kDa human placental GTPase-activating protein.

Human placenta contains, in addition to the ubiquitous p120-GTPase-activating protein (GAP), another isoform of 100 kDa, which is specific to this organ. We have established a method for purifying this placental p100-GAP to near homogeneity. The purified p100-GAP allowed the preparation of polyclonal and monoclonal anti Ras-GAP antibodies. Two monoclonal antibodies were selected for a two-site enzyme immunoassay. This simple and accurate assay in turn facilitated the detection of the GAPs during purification. The purified p100-GAP has a specific activity identical to and catalytic properties similar to those of native p120-GAP. Sequence analysis of p100-GAP revealed almost total identity to the known corresponding sequences predicted by the cDNA. The purified p100-GAP kept its activity for 1 year when stored at -80 degrees C. Our immunometric assay showed GAP to be present in human placental extracts at the exceptional abundance of about 0.1% of the total protein content. Quantitative assays showed p100-GAP to be up to 10 times more abundant than p120-GAP. Use of our antibodies allowed the specific localization of placental GAPs to cytotrophoblasts and in the syncytiotrophoblast barrier. Hence p100-GAP is shown to be found only in trophoblasts. The large quantity of p100-GAP in trophoblasts suggests that it may play a regulatory role in the proliferation or the differentiation of this cell type.

Non-neutralizing monoclonal antibodies against Ras GTPase-activating protein: production, characterization and use in an enzyme immunometric assay.

We studied several monoclonal antibodies (mAbs) raised against the 100 kD Ras GTPase activating protein (p100-GAP), which was purified from human placenta. These antibodies recognized p120-GAP and p100-GAP in native and in denatured forms. The most reactive, GP15 and GP200, both recognized distinct epitopes and did not neutralize GTPase stimulatory activity. These two mAbs were selected for a two-site enzyme immunoassay, using covalent conjugates of the antibodies coupled to the tetrameric form of acetylcholinesterase as tracer. This assay was used to quantify Ras-GAP in both normal and tumor tissues and cell extracts.