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•Novel non-racemic pyrazolo1,5-apyrimidine derivatives.•Simple synthetic method and broad scope.•Facile manipulation of functional groups and scaffold.•Structural ...diversity.•Inhibition of cathepsin K.
A series of novel 7-aminoalkyl substituted pyrazolo1,5-apyrimidine derivatives were synthesized and tested for inhibition of cathepsin K. The synthetic methodology comprises cyclization of 5-aminopyrazoles with N-Boc-α-amino acid-derived ynones followed by transformation of the ester and the Boc-amino functions. It allows for easy diversification of the pyrazolo1,5-apyrimidine scaffold at various positions. Molecular docking studies with pyrazolo1,5-apyrimidine derivatives were also performed to elucidate the binding mode in the active site of cathepsin K. The synthesized compounds exhibited moderate inhibition activity (Ki ≥ 77 μM).
Cathepsin K is a highly potent collagenase and the predominant papain-like cysteine protease expressed in osteoclasts. Cathepsin K deficiencies in humans and mice have underlined the central role of ...this protease in bone resorption and, thus, have rendered the enzyme as an attractive target for anti-resorptive osteoporosis therapy. In the past decade, a lot of efforts have been made in developing highly potent, selective and orally applicable cathepsin K inhibitors. Some of these inhibitors have passed preclinical studies and are presently in clinical trials at different stages of advancement. The development of the inhibitors and preliminary results of the clinical trials revealed problems and lessons concerning the in situ specificity of the compounds and their tissue targeting. In this review, we briefly summarize the history of cathepsin K research and discuss the current development of cathepsin K inhibitors as novel anti-resorptives for the treatment of osteoporosis. We also discuss potential off-target effects of cathepsin K inhibition and alternative applications of cathepsin K inhibitors in arthritis, atherosclerosis, blood pressure regulation, obesity and cancer.
Pycnodysostosis is a rare autosomal recessive disorder with characteristic diagnostic manifestations. This study aims to phenotype and provide molecular characterization of Egyptian patients, with ...emphasis on identifying unusual phenotypes and raising awareness about pycnodysostosis with different presentations to avoid a mis- or under-diagnosis and consequent mismanagement. We report on 22 Egyptian pycnodysostosis patients, including 9 new participants, all descending from consanguineous families and their ages ranging from 6 to 15 years. In addition, prenatal diagnosis was performed in one family with affected siblings. They all presented with short stature, except for one patient who presented with pancytopenia as her primary complaint. Moreover, 41.2% of patients had sleep apnea, 14% presented with craniosynostosis, and 44.4% had failure of tooth development. Molecular analysis via direct exome sequencing of the cathepsin K gene revealed three novel mutations ((NM_000396.3) c.761_763delCCT, c.864_865delAA, and c.509G>T) as well as two previously reported mutations among nine new cases. The following is our conclusion: This study expands the molecular spectrum of pycnodysostosis by identifying three novel mutations and adds to the clinical and orodental aspects of the disease. The link between the
gene mutations and the failure of tooth development has not been established, and further studies could help to improve our understanding of the molecular pathology.
Cathepsin K (CatK) is a lysosomal cysteine protease whose highest expression is found in osteoclasts, which are the cells responsible for bone resorption. Investigations of the functions and ...physiological relevance of CatK have often relied on antibody-related techniques, which makes studying its activity patterns a challenging task. Hence, we developed a set of chemical tools for the investigation of CatK activity. We show that our probe is a valuable tool for monitoring the proteolytic activation of CatK during osteoclast formation. Moreover, we demonstrate that our inhibitor of CatK impedes osteoclastogenesis and bone resorption and that CatK is stored in its active form in osteoclasts within their lysosomal compartment and mainly in the ruffled borders of osteoclasts. Given that our probe recognizes active CatK within living cells without exhibiting any observed cytotoxicity in the several models tested, we expect that it would be well suited to theranostic applications in CatK-related diseases.
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•A toolbox for the investigation of osteoclast cathepsin K activity was developed•Active cathepsin K is localized in the ruffled borders of osteoclasts•Cathepsin K activity is associated with osteoclast formation
With a set of chemical reagents to monitor active CatK in osteoclasts, including substrate, inhibitor, and a quenched fluorescent activity-based probe, Janiszewski et al. demonstrate that active CatK is localized mainly in the ruffled borders of osteoclasts, plays a crucial role in bone loss, and is crucial in osteoclast formation.
•RA and periodontitis share many pathological features, such as deregulated cytokine production.•RA and periodontitis share common osteoimmune and innate immune responses.•Deficiency of Ctsk causes a ...radical reduction in TLRs, cytokine expression and bone erosion.•Deficiency of Ctsk results in decreased immune-cell infiltration and osteoclast numbers.•Ctsk may be targeted to treat RA and periodontitis simultaneously due to their shared pathogenesis.
Using rheumatoid arthritis (RA) and periodontitis mouse models, we demonstrate that RA and periodontitis share many pathological features, such as deregulated cytokine production, increased immune-cell infiltration, increased expression of Toll-like receptors (TLRs), and enhanced osteoclast activity and bone erosion. We reveal that genetic deletion of cathepsin K (Ctsk) caused a radical reduction in inflammation and bone erosion within RA joint capsules and periodontal lesions, a drastic decrease in immune-cell infiltration, and a significant reduction in osteoclasts, macrophages, dendritic and T-cells. Deficiency of Ctsk greatly decreased the expression of TLR-4, 5, and 9 and their downstream cytokines in periodontal gingival epithelial lesions and synovial RA lesions. Hence, Ctsk may be targeted to treat RA and periodontitis simultaneously due to its shared osteoimmune role.
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)aminoethyl-7,8-diethylbenzogpteridine-2,4-dione, (NSC-374902), displayed an inhibition of bone resorption with an IC50-value of approximately 300 nM and no cell toxicity effects.
Matrix-metalloproteinases 9 (MMP-9) belongs to the class of matrix metalloproteinases whose main function is to degrade and remodel the extracellular matrix (ECM). MMP-9 has been shown to be an ...integral part of many diseases where modulation of the ECM is a key step such as cancer, osteoporosis and fibrosis. MMP-9 is secreted as a latent pro-enzyme that requires activation in the extracellular space. Therefore, identifying physiological and molecular contexts, which can activate MMP-9 is important.
Acidification of osteoclast-conditioned media to pH 5 resulted in a fragment with a size corresponding to active MMP-9. Also, treatment of recombinant proMMP-9 with recombinant cathepsin K (CTSK) at pH 5 yielded a fragment that corresponded to the molecular weight of active MMP-9, and showed MMP-9 activity. This activation was abrogated in the presence of CTSK inhibitor indicating that CTSK was responsible for the activation of pro-MMP-9. Knocking down CTSK in MDA-MB-231 cells also diminished MMP-9 activity compared to wild type control.
Here we provide the first evidence that CTSK can cleave and activate MMP-9 in acidic environments such as seen in tumors and during bone resorption. This finding provides a key link between CTSK expression in tumors and bone and ECM remodeling, through MMP-9 activation. This novel mechanism to activate MMP-9 through extracellular physiological changes elucidated in this study reveals a protease-signaling network involving CTSK and MMP-9 and provides the impetus to explore ECM proteases as physiological markers and pharmacological targets.
Periodontitis is a highly prevalent disease leading to uncontrolled osteoclastic jawbone resorption and ultimately edentulism; however, the disease onset mechanism has not been fully elucidated. Here ...we propose a mechanism for initial pathology based on results obtained using a recently developed Osteoadsorptive Fluogenic Sentinel (OFS) probe that emits a fluorescent signal triggered by cathepsin K (Ctsk) activity. In a ligature-induced mouse model of periodontitis, a strong OFS signal is observed before the establishment of chronic inflammation and bone resorption. Single cell RNA sequencing shows gingival fibroblasts to be the primary cellular source of early Ctsk. The in vivo OFS signal is activated when Toll-Like Receptor 9 (TLR9) ligand or oral biofilm extracellular DNA (eDNA) is topically applied to the mouse palatal gingiva. This previously unrecognized interaction between oral microbial eDNA and Ctsk of gingival fibroblasts provides a pathological mechanism for disease initiation and a strategic basis for early diagnosis and treatment of periodontitis.
Extracellular matrix metabolism and cardiac cell death participate centrally in myocardial infarction (MI). This study tested the roles of collagenolytic cathepsin K (CatK) in post-MI left ...ventricular remodeling.
Patients with acute MI had higher plasma CatK levels (20.49 ± 7.07 pmol/L, n = 26) than those in subjects with stable angina pectoris (8.34 ± 1.66 pmol/L, n = 28, P = .01) or those without coronary heart disease (6.63 ± 0.84 pmol/L, n = 93, P = .01). CatK protein expression increases in mouse hearts at 7 and 28 days post-MI. Immunofluorescent staining localized CatK expression in cardiomyocytes, endothelial cells, fibroblasts, macrophages, and CD4+ T cells in infarcted mouse hearts at 7 days post-MI. To probe the direct participation of CatK in MI, we produced experimental MI in CatK-deficient mice (Ctsk−/−) and their wild-type (Ctsk+/+) littermates. CatK-deficiency yielded worsened cardiac function at 7 and 28 days post-MI, compared to Ctsk+/+ littermates (fractional shortening percentage: 5.01 ± 0.68 vs. 8.62 ± 1.04, P < .01, 7 days post-MI; 4.32 ± 0.52 vs. 7.60 ± 0.82, P < .01, 28 days post-MI). At 7 days post-MI, hearts from Ctsk−/− mice contained less CatK-specific type-I collagen fragments (10.37 ± 1.91 vs. 4.60 ± 0.49 ng/mg tissue extract, P = .003) and more fibrosis (1.67 ± 0.93 vs. 0.69 ± 0.20 type-III collagen positive area percentage, P = .01; 14.25 ± 4.12 vs. 6.59 ± 0.79 α-smooth muscle actin-positive area percentage, P = .016; and 0.82 ± 0.06 vs. 0.31 ± 0.08 CD90-positive area percentage, P = .008) than those of Ctsk+/+ mice. Immunostaining demonstrated that CatK-deficiency yielded elevated cardiac cell death but reduced cardiac cell proliferation. In vitro studies supported a role of CatK in cardiomyocyte survival.
Plasma CatK levels are increased in MI patients. Heart CatK expression is also elevated post-MI, but CatK-deficiency impairs post-MI cardiac function in mice by increasing myocardial fibrosis and cardiomyocyte death.
•Plasma levels of CatK increase in patients with CHD particularly during AMI, compared to controls.•CatK is expressed in cardiomyocytes, endothelial cell, fibroblast, macrophage, and CD4+ T-cell from post-MI mouse heart.•In post-MI heart, CatK-deficiency increases fibrosis and cell death, and impairs cell proliferation and cardiac function.•CatK inhibition or deficiency increases cardiomyocyte death, but suppresses CD4+ T-cell and macrophage death.
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