Real-time analysis of osteoclast resorption and fusion dynamics in response to bone resorption inhibitors.

Cathepsin K (CatK), an essential collagenase in osteoclasts (OCs), is a potential therapeutic target for the treatment of osteoporosis. Using live-cell imaging, we monitored the bone resorptive behaviour of OCs during dose-dependent inhibition of CatK by an ectosteric (Tanshinone IIA sulfonate) and an active site inhibitor (odanacatib). CatK inhibition caused drastic reductions in the overall resorption speed of OCs. At IC50 CatK-inhibitor concentration, OCs reduced about 40% of their trench-forming capacity and at fourfold IC50 concentrations, a > 95% reduction was observed. The majority of CatK-inhibited OCs (~ 75%) were involved in resorption-migration-resorption episodes forming adjacent pits, while ~ 25% were stagnating OCs which remained associated with the same excavation. We also observed fusions of OCs during the resorption process both in control and inhibitor-treated conditions, which increased their resorption speeds by 30-50%. Inhibitor IC50-concentrations increased OC-fusion by twofold. Nevertheless, more fusion could not counterweigh the overall loss of resorption activity by inhibitors. Using an activity-based probe, we demonstrated the presence of active CatK at the resorbing front in pits and trenches. In conclusion, our data document how OCs respond to CatK-inhibition with respect to movement, bone resorption activity, and their attempt to compensate for inhibition by activating fusion.

Extracellular Matrix Remodeling in Atopic Dermatitis Harnesses the Onset of an Asthmatic Phenotype and Is a Potential Contributor to the Atopic March.

The development of atopic dermatitis in infancy, and subsequent allergies, such as asthma in later childhood, is known as the atopic march. The mechanism is largely unknown, however the course of disease indicates an inter-epithelial crosstalk, through the onset of inflammation in the skin and progression to other mucosal epithelia. In this study, we investigated if and how skin-lung epithelial crosstalk contributes to the development of the atopic march. First, we emulated inter-epithelial crosstalk through indirect coculture of bioengineered atopic-like skin disease models and three-dimensional bronchial epithelial models triggering an asthma-like phenotype in the latter. A subsequent secretome analysis identified thrombospondin-1, CD44, complement factor C3, fibronectin, and syndecan-4 as potentially relevant skin-derived mediators. Because these mediators are extracellular matrix-related proteins, we then studied the involvement of the extracellular matrix, unveiling distinct proteomic, transcriptomic, and ultrastructural differences in atopic samples. The latter indicated extracellular matrix remodeling triggering the release of the above-mentioned mediators. In vivo mouse data showed that exposure to these mediators dysregulated activated circadian clock genes which are increasingly discussed in the context of atopic diseases and asthma development. Our data point toward the existence of a skin-lung axis that could contribute to the atopic march driven by skin extracellular matrix remodeling.

Lycopene Improves Bone Quality and Regulates AGE/RAGE/NF-кB Signaling Pathway in High-Fat Diet-Induced Obese Mice.

OBJECTIVE: This study was aimed at examining the effects of lycopene on bone metabolism in high-fat diet (HFD)- induced obese mice and to identify the potential underlying mechanisms. METHODS: Mice were fed a HFD for 12 weeks and then continue with or without lycopene intervention (15 mg/kg) for additional 10 weeks. The effects of lycopene on blood glucose and lipid metabolism, as well as serum levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and malondialdehyde (MDA) were determined by biochemical assays. Bone histomorphological features and osteoclast activity were assessed by hematoxylin/eosin and tartrate-resistant acid phosphatase staining. Bone microstructure at the proximal tibial metaphysis and diaphysis was determined by microcomputed tomography. Tibial biomechanical strength and material profiles were measured by a three-point bending assay and Fourier transform infrared spectroscopy. Protein expressions involved in the AGE/RAGE/NF-кB signaling pathway were determined by western blot and/or immunohistochemical staining. RESULTS: Lycopene consumption reduced body weight gain and improved blood glucose and lipid metabolism in HFD-induced obese mice. In addition, lycopene treatment preserved bone biomechanical strength, material profiles, and microarchitecture in obese mice. Moreover, these alterations were associated with an increase in serum levels of T-AOC and SOD, and a decline in serum levels of MDA, as well as a reduction of AGEs, RAGE, cathepsin K, and p-NF-кBp65 and NF-кBp65 expressions in the femurs and tibias of obese mice. CONCLUSION: Lycopene may improve bone quality through its antioxidant properties, which may be linked with the regulation of the AGE/RAGE/NF-кB signaling pathway in obese mice. These results suggest that lycopene consumption may be beneficial for the management of obesity-induced osteoporosis.

Antibody-induced pain-like behavior and bone erosion: links to subclinical inflammation, osteoclast activity, and acid-sensing ion channel 3-dependent sensitization.

ABSTRACT: Several bone conditions, eg, bone cancer, osteoporosis, and rheumatoid arthritis (RA), are associated with a risk of developing persistent pain. Increased osteoclast activity is often the hallmark of these bony pathologies and not only leads to bone remodeling but is also a source of pronociceptive factors that sensitize the bone-innervating nociceptors. Although historically bone loss in RA has been believed to be a consequence of inflammation, both bone erosion and pain can occur years before the symptom onset. Here, we have addressed the disconnection between inflammation, pain, and bone erosion by using a combination of 2 monoclonal antibodies isolated from B cells of patients with RA. We have found that mice injected with B02/B09 monoclonal antibodies (mAbs) developed a long-lasting mechanical hypersensitivity that was accompanied by bone erosion in the absence of joint edema or synovitis. Intriguingly, we have noted a lack of analgesic effect of naproxen and a moderate elevation of few inflammatory factors in the ankle joints suggesting that B02/B09-induced pain-like behavior does not depend on inflammatory processes. By contrast, we found that inhibiting osteoclast activity and acid-sensing ion channel 3 signaling prevented the development of B02/B09-mediated mechanical hypersensitivity. Moreover, we have identified secretory phospholipase A2 and lysophosphatidylcholine 16:0 as critical components of B02/B09-induced pain-like behavior and shown that treatment with a secretory phospholipase A2 inhibitor reversed B02/B09-induced mechanical hypersensitivity and bone erosion. Taken together, our study suggests a potential link between bone erosion and pain in a state of subclinical inflammation and offers a step forward in understanding the mechanisms of bone pain in diseases such as RA.

The abnormal accumulation of heparan sulfate in patients with mucopolysaccharidosis prevents the elastolytic activity of cathepsin V.

Mucopolysaccharidosis (MPS) are rare inherited diseases characterized by accumulation of lysosomal glycosaminoglycans, including heparan sulfate (HS). Patients exhibit progressive multi-visceral dysfunction and shortened lifespan mainly due to a severe cardiac/respiratory decline. Cathepsin V (CatV) is a potent elastolytic protease implicated in extracellular matrix (ECM) remodeling. Whether CatV is inactivated by HS in lungs from MPS patients remained unknown. Herein, CatV colocalized with HS in MPS bronchial epithelial cells. HS level correlated positively with the severity of respiratory symptoms and negatively to the overall endopeptidase activity of cysteine cathepsins. HS bound tightly to CatV and impaired its activity. Withdrawal of HS by glycosidases preserved exogenous CatV activity, while addition of Surfen, a HS antagonist, restored elastolytic CatV-like activity in MPS samples. Our data suggest that the pathophysiological accumulation of HS may be deleterious for CatV-mediated ECM remodeling and for lung tissue homeostasis, thus contributing to respiratory disorders associated to MPS diseases.

Green asymmetric synthesis of epoxypeptidomimetics and evaluation as human cathepsin K inhibitors.

Cathepsin K (CatK) is a cysteine protease known for its potent collagenolytic activity, being recognized as an important target to the development of therapies for the treatment of bone disorders. Epoxypeptidomimetics have been reported as potent inhibitors of cathepsins, thus in this work we present a green synthesis of new peptidomimetics by using a one-pot asymmetric epoxidation/Ugi multicomponent reaction. The compounds were evaluated against CatK showing selectivity when compared with cathepsin L, with an inhibition profile in the low micromolar IC50 range. Investigation of the mechanism of action carried out for compounds LSPN428 and LSPN694 suggested a mixed inhibition mode and docking studies allowed a better understanding about interactions of inhibitors with the enzyme.

Expression of elastolytic cathepsins in human skin and their involvement in age-dependent elastin degradation.

BACKGROUND: Skin ageing is associated with structure-functional changes in the extracellular matrix, which is in part caused by proteolytic degradation. Since cysteine cathepsins are major matrix protein-degrading proteases, we investigated the age-dependent expression of elastolytic cathepsins K, S, and V in human skin, their in vitro impact on the integrity of the elastic fibre network, their cleavage specificities, and the release of bioactive peptides. METHODS: Cathepsin-mediated degradation of human skin elastin samples was assessed from young to very old human donors using immunohistochemical and biochemical assays, scanning electron microscopy, and mass spectrometry. RESULTS: Elastin samples derived from patients between 10 and 86 years of age were analysed and showed an age-dependent deterioration of the fibre structure from a dense network of thinner fibrils into a beaded and porous mesh. Reduced levels of cathepsins K, S, and V were observed in aged skin with a predominant epidermal expression. Cathepsin V was the most potent elastase followed by cathepsin K and S. Biomechanical analysis of degraded elastin fibres corroborated the destructive activity of cathepsins. Mass spectrometric determination of the cleavage sites in elastin revealed that all three cathepsins predominantly cleaved in hydrophobic domains. The degradation of elastin was efficiently inhibited by an ectosteric inhibitor. Furthermore, the degradation of elastin fibres resulted in the release of bioactive peptides, which have previously been associated with various pathologies. CONCLUSION: Cathepsins are powerful elastin-degrading enzymes and capable of generating a multitude of elastokines. They may represent a viable target for intervention strategies to reduce skin ageing.

Characterization of cathepsin S exosites that govern its elastolytic activity.

We have previously determined that the elastolytic activities of cathepsins (Cat) K and V require two exosites sharing the same structural localization on both enzymes. The structural features involved in the elastolytic activity of CatS have not yet been identified. We first mutated the analogous CatK and V putative exosites of CatS into the elastolytically inactive CatL counterparts. The modification of the exosite 1 did not affect the elastase activity of CatS whilst mutation of the Y118 of exosite 2 decreased the cleavage of elastin by ∼70% without affecting the degradation of other macromolecular substrates (gelatin, thyroglobulin). T06, an ectosteric inhibitor that disrupt the elastolytic activity of CatK, blocked ∼80% of the elastolytic activity of CatS without blocking the cleavage of gelatin and thyroglobulin. Docking studies showed that T06 preferentially interacts with a binding site located on the Right domain of the enzyme, outside of the active site. The structural examination of this binding site showed that the loop spanning the L174N175G176K177 residues of CatS is considerably different from that of CatL. Mutation of this loop into the CatL-like equivalent decreased elastin degradation by ∼70% and adding the Y118 mutation brought down the loss of elastolysis to ∼80%. In addition, the Y118 mutation selectively reduced the cleavage of the basement membrane component laminin by ∼50%. In summary, our data show that the degradation of elastin by CatS requires two exosites where one of them is distinct from those of CatK and V whilst the cleavage of laminin requires only one exosite.

Fructus Ligustri Lucidi preserves bone quality through the regulation of gut microbiota diversity, oxidative stress, TMAO and Sirt6 levels in aging mice.

Gut dysbiosis and oxidative stress may trigger senile osteoporosis. Fructus Ligustri Lucidi (FLL) has bone-preserving properties and affects the intestinal microecology. However, the mechanism of the anti-osteoporotic effect of FLL and its link to the gut microbiota remains to be elucidated. Here, we demonstrated that sustained exposure of ICR mice to D-galactose / sodium nitrite for 90 days causes aging-related osteoporosis and reduced cognitive performance. The aging phenotype is also characterized by increased oxidative stress in serum. This is likely triggered by abnormal changes in the gut microbiota population of Bifidobacterium and the ratio of Firmicutes/ Bacteroidetes that resulted in increased levels of flavin-containing monooxygenase-3 and trimethylamine-N-oxide (TMAO). Moreover, the increased oxidative stress further accelerated aging by increasing tumor necrosis factor-α levels in serum and reducing Sirtuin 6 (Sirt6) expression in long bones, which prompted nuclear factor kappa-B acetylation as well as over-expression and activation of cathepsin K. FLL-treated aging mice revealed a non-osteoporotic bone phenotype and an improvement on the cognitive function. The mechanism underlying these effects may be linked to the regulation of gut microbiota diversity, antioxidant activity, and the levels of TMAO and Sirt6. FLL may represent a potential source for identifying anti-senile osteoporotic drug candidates.

Elastolytic activity of cysteine cathepsins K, S, and V promotes vascular calcification.

Elastin plays an important role in maintaining blood vessel integrity. Proteolytic degradation of elastin in the vascular system promotes the development of atherosclerosis, including blood vessel calcification. Cysteine cathepsins have been implicated in this process, however, their role in disease progression and associated complications remains unclear. Here, we showed that the degradation of vascular elastin by cathepsins (Cat) K, S, and V directly stimulates the mineralization of elastin and that mineralized insoluble elastin fibers were ~25-30% more resistant to CatK, S, and V degradation when compared to native elastin. Energy dispersive X-ray spectroscopy investigations showed that insoluble elastin predigested by CatK, S, or V displayed an elemental percentage in calcium and phosphate up to 8-fold higher when compared to non-digested elastin. Cathepsin-generated elastin peptides increased the calcification of MOVAS-1 cells acting through the ERK1/2 pathway by 34-36%. We made similar observations when cathepsin-generated elastin peptides were added to ex vivo mouse aorta rings. Altogether, our data suggest that CatK-, S-, and V-mediated elastolysis directly accelerates the mineralization of the vascular matrix by the generation of nucleation points in the elastin matrix and indirectly by elastin-derived peptides stimulating the calcification by vascular smooth muscle cells. Both processes inversely protect against further extracellular matrix degradation.

Identification of substrate-specific inhibitors of cathepsin K through high-throughput screening.

Cathepsin K (CatK) is a cysteine protease and drug target for skeletal disorders that is known for its potent collagenase and elastase activity. The formation of oligomeric complexes of CatK in the presence of glycosaminoglycans has been associated with its collagenase activity. Inhibitors that disrupt these complexes can selectively block the collagenase activity without interfering with the other regulatory proteolytic activities of the enzyme. Here, we have developed a fluorescence polarization (FP) assay to screen 4761 compounds for substrate-specific ectosteric collagenase inhibitors of CatK. A total of 38 compounds were identified that block the collagenase activity without interfering with the hydrolysis of active site substrates such as the synthetic peptide substrate, benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, and gelatin. The identified inhibitors can be divided into two main classes, negatively charged and polyaromatic compounds which suggest the binding to different ectosteric sites. Two of the inhibitors were highly effective in preventing the bone-resorption activity of CatK in osteoclasts. Interestingly, some of the ectosteric inhibitors were capable of differentiating between the collagenase and elastase activity of CatK depending on the ectosteric site utilized by the compound. Owing to their substrate-specific selectivity, ectosteric inhibitors represent a viable alternative to side effect-prone active site-directed inhibitors.

A Mild Inhibition of Cathepsin K Paradoxically Stimulates the Resorptive Activity of Osteoclasts in Culture.

Cathepsin K (CatK) inhibition allows reducing bone resorption with specific advantages compared to the existing anti-osteoporosis drugs. Its clinical use appears even more promising with the recent development of ectosteric inhibitors. A confusing observation, however, is that a low dose of the active site CatK inhibitor odanacatib (ODN) was reported to decrease bone mineral density and increase serum levels of the bone resorption marker carboxy-terminal collagen crosslinks (CTX). The present study provides a possible explanation for this paradox. The resorptive activity of human osteoclasts seeded on bone slices was inhibited when subjected to ODN at doses of 20 nM, but about 100-fold lower doses induced a significant increase in CTX levels and in eroded surface (12 repeats). This low-dose-induced stimulation was prevented by inhibition of non-CatK cysteine proteinases, thereby indicating that the stimulation results from an interplay between CatK and other cysteine proteinases. Effective interplay between these proteinases was also shown in enzymatic assays where the CatK-mediated degradation of collagen was enhanced upon addition of cathepsins B or L. Furthermore, extracts of osteoclasts subjected to a low dose of ODN showed higher levels of cathepsin B compared with extracts of control osteoclasts. In conclusion, the low-dose-induced stimulation of resorption observed in the clinical study can be reproduced in osteoclasts cultured in the absence of any other cell. Our data support an osteoclast-intrinsic mechanism where a mild inhibition of CatK results in increased levels of other proteinases contributing to the collagen degradation process.

Collagen type I degradation fragments act through the collagen receptor LAIR-1 to provide a negative feedback for osteoclast formation.

The major organic component of bone is collagen type I. Osteoclasts are terminally differentiated multinucleated cells of hematopoietic origin that are essential for physiological development of bone and teeth. We examined if osteoclast differentiation from murine bone marrow precursors is affected by collagen type I, or by its degradation products produced by human recombinant cathepsin K. Osteoclasts formation was dose-dependently inhibited in the presence of full length collagen type I or its 30-75 kDa degradation products added to the osteoclast differentiation media for the duration of an experiment. Collagen degradation fragments signaled through SH-2 phosphatases, inhibiting calcium signaling and NFATc1 translocation in osteoclast precursors. Osteoclasts and their precursors expressed a collagen receptor of leukocyte receptor complex family, LAIR-1. Importantly, collagen fragments failed to inhibit osteoclast formation from LAIR-1 deficient murine osteoclast precursors. This study demonstrates that collagen degradation fragments inhibit osteoclast formation acting through LAIR-1, providing a novel mechanism for the physiologically-relevant negative control of osteoclastogenesis.

Substrate-derived triazolo- and azapeptides as inhibitors of cathepsins K and S.

Cathepsin (Cat) K is a critical bone-resorbing protease and is a relevant target for the treatment of osteoporosis and bone metastasis, while CatS is an attractive target for drugs in autoimmune diseases (e.g. rheumatoid arthritis), emphysema or neuropathic pain. Despite major achievements, current pharmacological inhibitors are still lacking in safety and may have damaging side effects. A promising strategy for developing safer reversible and competitive inhibitors as new lead compounds could be to insert non-cleavable bonds at the scissile P1-P1' position of selective substrates of CatS and CatK. Accordingly, we introduced a 1,4-disubstituted 1,2,3-triazole heterocycle that mimics most of the features of a trans-amide bond, or we incorporated a semicarbazide bond (azaGly residue) by replacing the α-carbon of the glycyl residue at P1 by a nitrogen atom. AzaGly-containing peptidomimetics inhibited powerfully their respective target proteases in the nM range, while triazolopeptides were weaker inhibitors (Ki in the µM range). The selectivity of the azaGly CatS inhibitor (1b) was confirmed by using spleen lysates from wild-type vs CatS-deficient mice. Alternatively, the azaGly bradykinin-derived CatK inhibitor (2b) potently inhibited CatK (Ki = 9 nM) and impaired its kininase activity in vitro. Molecular modeling studies support that the semicarbazide bond of 2b is more favorable than the 1,2,3-triazole linkage of the bradykinin-derived pseudopeptide 2a to preserve an effective affinity towards CatK, its protease target.

Tanshinones that selectively block the collagenase activity of cathepsin K provide a novel class of ectosteric antiresorptive agents for bone.

BACKGROUND AND PURPOSE: Attempts to generate active site-directed cathepsin K (CatK) inhibitors for the treatment of osteoporosis have failed because of side effects. We have previously shown that an ectosteric tanshinone CatK inhibitor isolated from Salvia miltiorrhiza blocked, selectively, the collagenase activity of CatK, without affecting the active site and demonstrated its bone-preserving activity in vivo. Here, we have characterize the antiresorptive potential of other tanshinones, which may provide a scaffold for side effect-free CatK inhibitors. EXPERIMENTAL APPROACH: Thirty-one tanshinones were tested for their activity against CatK in enzymic and cell-based assays. The inhibitory potency against triple helical and fibrillar collagen degradation was determined in enzymic assays, by scanning electron microscopy and mechanical strength measurements. Human osteoclast assays were used to determine the effects of the inhibitors on bone resorption, its reversibility and osteoclastogenesis. Binding sites were characterized by molecular docking. KEY RESULTS: Twelve compounds showed highly effective anti-collagenase activity and protected collagen against destruction and mechanical instability without inhibiting the hydrolysis of non-collagenous substrates. Six compounds were highly effective in osteoclast bone resorption assays with IC50 values of <500 nM. None of these tanshinones had effects on cell viability, reversibility of bone resorption inhibition and osteoclastogenesis. The core pharmacophore of the tanshinones appears to be the three-ring system with either a para- or ortho-quinone entity. CONCLUSIONS AND IMPLICATIONS: Our study identified several potent ectosteric antiresorptive CatK inhibitors from the medicinal plant, S. miltiorrhiza, which may avoid side effects seen with active site-directed inhibitors in clinical trials.

Aging-associated modifications of collagen affect its degradation by matrix metalloproteinases.

The natural aging process and various pathologies correlate with alterations in the composition and the structural and mechanical integrity of the connective tissue. Collagens represent the most abundant matrix proteins and provide for the overall stiffness and resilience of tissues. The structural changes of collagens and their susceptibility to degradation are associated with skin wrinkling, bone and cartilage deterioration, as well as cardiovascular and respiratory malfunctions. Here, matrix metalloproteinases (MMPs) are major contributors to tissue remodeling and collagen degradation. During aging, collagens are modified by mineralization, accumulation of advanced glycation end-products (AGEs), and the depletion of glycosaminoglycans (GAGs), which affect fiber stability and their susceptibility to MMP-mediated degradation. We found a reduced collagenolysis in mineralized and AGE-modified collagen fibers when compared to native fibrillar collagen. GAGs had no effect on MMP-mediated degradation of collagen. In general, MMP digestion led to a reduction in the mechanical strength of native and modified collagen fibers. Successive fiber degradation with MMPs and the cysteine-dependent collagenase, cathepsin K (CatK), resulted in their complete degradation. In contrast, MMP-generated fragments were not or only poorly cleaved by non-collagenolytic cathepsins such as cathepsin V (CatV). In conclusion, our data indicate that aging and disease-associated collagen modifications reduce tissue remodeling by MMPs and decrease the structural and mechanic integrity of collagen fibers, which both may exacerbate extracellular matrix pathology.

A composite docking approach for the identification and characterization of ectosteric inhibitors of cathepsin K.

Cathepsin K (CatK) is a cysteine protease that plays an important role in mammalian intra- and extracellular protein turnover and is known for its unique and potent collagenase activity. Through studies on the mechanism of its collagenase activity, selective ectosteric sites were identified that are remote from the active site. Inhibitors targeting these ectosteric sites are collagenase selective and do not interfere with other proteolytic activities of the enzyme. Potential ectosteric inhibitors were identified using a computational approach to screen the druggable subset of and the entire 281,987 compounds comprising Chemical Repository library of the National Cancer Institute-Developmental Therapeutics Program (NCI-DTP). Compounds were scored based on their affinity for the ectosteric site. Here we compared the scores of three individual molecular docking methods with that of a composite score of all three methods together. The composite docking method was up to five-fold more effective at identifying potent collagenase inhibitors (IC50 < 20 µM) than the individual methods. Of 160 top compounds tested in enzymatic assays, 28 compounds revealed blocking of the collagenase activity of CatK at 100 µM. Two compounds exhibited IC50 values below 5 µM corresponding to a molar protease:inhibitor concentration of <1:12. Both compounds were subsequently tested in osteoclast bone resorption assays where the most potent inhibitor, 10-[2-[bis(2-hydroxyethyl)amino]ethyl]-7,8-diethylbenzo[g]pteridine-2,4-dione, (NSC-374902), displayed an inhibition of bone resorption with an IC50-value of approximately 300 nM and no cell toxicity effects.

Corrigendum: Antioxidant Effect of Fructus Ligustri Lucidi Aqueous Extract in Ovariectomized Rats Is Mediated through Nox4-ROS-NF-κB Pathway.

[This corrects the article on p. 266 in vol. 8, PMID: 28588482.].

Effect of conditioning solutions containing ferric chloride on dentin bond strength and collagen degradation.

OBJECTIVE: To investigate the effects of conditioning solutions containing ferric chloride (FeCl3) on resin-dentin bond strength; on protection of dentin collagen against enzymatic degradation and on cathepsin-K (CT-K) activity. METHODS: Conditioning solutions were prepared combining citric acid (CA) and anhydrous ferric chloride (FeCl3) in different concentrations. The solutions were applied to etch flat dentin surfaces followed by bonding with adhesive resin. Phosphoric acid (PA) gel etchant was used as control. The microtensile bond strength (µTBS) was tested after 24h of storage in water and after 9 months of storage in phosphate buffer saline. Dentin slabs were demineralized in 0.5M EDTA, pre-treated or not with FeCl3 and incubated with CT-K. The collagenase activity on dentin collagen matrix was examined and characterized by SEM. Additional demineralized dentin slabs were treated with the conditioning solutions, and the amount of Fe bound to collagen was determined by EDX. The activity of CT-K in the presence of FeCl3 was monitored fluorimetrically. Data were analyzed by ANOVA followed by post-hoc tests as required (α=5%). RESULTS: Slightly higher bond strengths were obtained when dentin was conditioned with 5% CA/0.6% FeCl3 and 5% CA-1.8%FeCl3 regardless of storage time. Bond strengths reduced significantly for all tested conditioners after 9 months of storage. Treating dentin with 1.8% FeCl3 was effective to preserve the structure of collagen against CT-K. EDX analysis revealed binding of Fe-ions to dentin collagen after 15s immersion of demineralized dentin slabs into FeCl3 solutions. FeCl3 at concentration of 0.08% was able to suppress CT-K activity. SIGNIFICANCE: This study shows that FeCl3 binds to collagen and offers protection against Cat-K degradation. Mixed solutions of CA and FeCl3 may be used as alternative to PA to etch dentin in resin-dentin bonding with the benefits of preventing collagen degradation.