CatK Inhibitor Research Articles

Immune regulatory and anti-resorptive activities of tanshinone IIA sulfonate attenuates rheumatoid arthritis in mice.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and painful joint destruction. Current treatments are helpful in RA remission, but strong immunosuppressive activity and patient resistance are clinical issues. This study explores a dual-action inhibitor, possessing both anti-inflammatory and anti-resorptive properties, as a novel treatment for RA. Therapeutic efficacy and mechanisms of ectosteric (tanshinone IIA sulfonate [T06]) and active site-directed (odanacatib [ODN]) inhibitors of cathepsin K (CatK) were evaluated in RA mouse models. Pathology was assessed through biochemical analyses and histopathological examination. Flow cytometry analysis was performed to characterize immune cells. Anti-inflammatory effects of T06 on nuclear factor kappa beta (NF-κB) pathway were studied in macrophages. T06 effectively lowered the number of joint-resident immune cells, accompanied by significantly reduced production of inflammatory cytokines and collagenolytic proteases. This also included the suppression of Th17 cells and IL-17, resulting in the reduction of osteoclasts in arthritic joints and amplification of the overall anti-resorptive effect of T06, which has been attributed to its selective inhibition of the collagenolytic activity of CatK by preventing its oligomerization. The anti-inflammatory mechanism of T06 was based on blocking the phosphorylation of IκBα in the NF-κB pathway, resulting in reduced activation and expression of inflammatory cytokines. In contrast, ODN had no effect on inflammation and disease progression and was limited to the inhibition of CatK. The combined anti-resorptive and anti-inflammatory activities characterize T06 as a novel therapeutic agent for RA.

Deciphering Cathepsin K inhibitors: a combined QSAR, docking and MD simulation based machine learning approaches for drug design

ABSTRACT Cathepsin K (CatK), a lysosomal cysteine protease, contributes to skeletal abnormalities, heart diseases, lung inflammation, and central nervous system and immune disorders. Currently, CatK inhibitors are associated with severe adverse effects, therefore limiting their clinical utility. This study focuses on exploring quantitative structure–activity relationships (QSAR) on a dataset of CatK inhibitors (1804) compiled from the ChEMBL database to predict the inhibitory activities. After data cleaning and pre-processing, a total of 1568 structures were selected for exploratory data analysis which revealed physicochemical properties, distributions and statistical significance between the two groups of inhibitors. PubChem fingerprinting with 11 different machine-learning classification models was computed. The comparative analysis showed the ET model performed well with accuracy values for the training set (0.999), cross-validation (0.970) and test set (0.977) in line with OECD guidelines. Moreover, to gain structural insights on the origin of CatK inhibition, 15 diverse molecules were selected for molecular docking. The CatK inhibitors (1 and 2) exhibited strong binding energies of −8.3 and −7.2 kcal/mol, respectively. MD simulation (300 ns) showed strong structural stability, flexibility and interactions in selected complexes. This synergy between QSAR, docking, MD simulation and machine learning models strengthen our evidence for developing novel and resilient CatK inhibitors.

Novel selective proline-based peptidomimetics for human cathepsin K inhibition

Human cathepsin K (CatK) stands out as a promising target for the treatment of osteoporosis, considering its role in degrading the bone matrix. Given the small and shallow S2 subsite of CatK and considering its preference for proline or hydroxyproline, we now propose the rigidification of the leucine fragment found at the P2 position in a dipeptidyl-based inhibitor, generating rigid proline-based analogs. Accordingly, with these new proline-based peptidomimetics inhibitors, we selectively inhibited CatK against other human cathepsins (B, L and S). Among these new ligands, the most active one exhibited a high affinity (pKi = 7.3 – 50.1 nM) for CatK and no inhibition over the other cathepsins. This specific inhibitor harbors two novel substituents never employed in other CatK inhibitors: the trifluoromethylpyrazole and the 4-methylproline at P3 and P2 positions. These results broaden and advance the path toward new potent and selective inhibitors for CatK.

Potential Role of Odanacatib in the Treatment of Postmenopausal Intervertebral Disc Degeneration

Odanacatib (ODN), a cathepsin K (CatK) inhibitor, is widely used for the treatment of postmenopausal osteoporosis. However, whether ODN is efficient in delaying intervertebral disc degeneration (IVDD) remaining unknown. We aimed to explore the effect of ODN in the postmenopausal IVDD using an ovariectomized (OVX) mouse model. We collected the disc tissues from female spinal fracture patients and analyzed the CatK expression in different estrogen levels. Besides, we injected ranged concentration of OND subcutaneously in the OVX mice and tested the disc height index (DHI), aggrecan positive area, collagen I, collagen II, inflammatory factors, and apoptosis-related gene expression comparing to control. The intervertebral disc degraded and the CatK gene expression decreased in the disc lacking of estrogen. OVX method increased the collagen I, MMP-3, MMP-13, caspase-3, and caspase-8 expression, but reduced DHI, and the content of aggrecan and collagen II, indicating an IVDD tendency. However, the ODN treatment could suppress MMP-3, MMP-13, caspase-3, and caspase-8 and protect the stability of extracellular matrix (ECM). ODN is a potential drug to delay the IVDD by suppressing apoptosis and ECM degradation.

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.

Effects of Cathepsin K Inhibitors on Dentin Erosion: An in vitro Study

Cathepsin K (catK) modulates the degradation of dentin collagen. This study aimed to evaluate the effects of catK inhibitors on dentin erosion. Dentin beams were eroded (4 times/d for 5 days) and immersed in deionized water (negative control), 0.1 <sc>M</sc> NaCl, 0.3 <sc>M</sc> NaCl, 0.5 <sc>M</sc> NaCl, or 1 μ<sc>m</sc> odanacatib (each n = 16) for 30 min after each erosive challenge. Erosive dentin loss (EDL) and demineralized organic matrix (DOM) thickness were evaluated profilometrically. Additionally, dentin beams were demineralized, immersed in the respective solutions for 30 min each (n = 5), and then incubated in artificial saliva for 5 days. Dentin collage degradation was evaluated by quantifying the levels of the C-terminal peptide of type I collagen (CTX), C-terminal cross-linked telopeptide of type I collagen (ICTP), and hydroxyproline (HYP) in the incubation media. Significantly lower EDL and dentin collagen degradation (CTX, ICTP, and HYP) and thicker DOM layers were observed in the samples treated with 0.3 <sc>m</sc> NaCl and 1 μ<sc>m</sc> odanacatib than in those treated with deionized water (all p < 0.05). The samples treated with 1 μ<sc>m</sc> odanacatib showed significantly lower levels of CTX and HYP than those treated with 0.3 <sc>M</sc> NaCl (all p < 0.05). The present findings support the potential use of catK inhibitors in controlling dentin erosion.

Do matrix metalloproteinase and cathepsin K inhibitors work synergistically to reduce dentin erosion?

To evaluate the effects of matrix metalloproteinase (MMP) and cathepsin K (catK) inhibitors on resistance to dentin erosion. A total of 96 dentin specimens (3×3×2 mm) were prepared and randomly assigned into four groups (n=24): deionized water (DW); 1 µM odanacatib (ODN, catK inhibitor); 1 mM 1,10-phenanthroline (PHEN, MMP inhibitor); and 1 µM odanacatib + 1 mM 1,10-phenanthroline (COM). Each group was further divided into two subgroups for the application of treatment solutions before (PRE) and after erosive challenges (POST). All specimens were subjected to four daily erosive challenges for 5 d. For each erosive challenge, the specimens in subgroup PRE were immersed in the respective solutions before cola drinks, while the specimens in subgroup POST were immersed in the respective solutions after cola drinks (the immersion duration was 5 min in both cases). All specimens were stored in artificial saliva at 37°C between erosive challenges. The erosive dentin loss (EDL) was measured by profilometry. The residual demineralized organic matrix (DOM) of specimens was removed using type VII collagenase and evaluated by profilometry. Both the EDL and thickness of the residual DOM were statistically analyzed by two-way analysis of variance (ANOVA) and Bonferroni's test (α=0.05). The surface topography and transverse sections of the specimens were observed using SEM. MMPs and catK were immunolabeled in the eroded dentin and in situ zymography was performed to evaluate the enzyme activity. Significantly lower EDL was found in the groups ODN, PHEN, and COM than in the control group (all p<0.05), while no significant difference in EDL was found among the groups ODN, PHEN, and COM (all p>0.05). The application sequence showed no significant effect on the EDL of the tested groups (p=0.310). A significantly thicker DOM was observed in the group ODN than in the control group regardless of the application sequence (both p<0.05). The treatment with ODN, PHEN, and COM inhibited the gelatinolytic activity by approximately 46.32%, 58.6%, and 74.56%, respectively. The inhibition of endogenous dentinal MMPs and catK increases the acid resistance of human dentin but without an apparent synergistic effect. The inhibition of MMPs and catK is equally effective either before or after the acid challenge.

Effects of Cathepsin K on spatial learning and memory in rats

To investigate the effects of Cathepsin K(CatK) on spatial learning and memory in rat hippocampus and its mechanisms. Twenty male SD rats were randomly divided into Control group and CatK inhibitor group(CatKⅡ group), which were microinjected with Cathepsin K specific inhibitor(0.5 μg/μl) and artificial cerebrospinal fluid in hippocampal DG area respectively with 5 days. The cultured hippocampal neuron cells were divided into control group (CON group), negative control group(NC group), siRNA interference group(siCatK group). Three re-wells were set for each group, and samples were collected 18～20 h after siRNA transfection. Morris water maze was used to evaluate spatial learning and memory function of rats. Meanwhile, dynamic changes of glutamate(Glu) content in extracellular fluid of DG region during learning and memory were observed by microdialysis and high performance liquid chromatography in conscious rats. Western blot was used to detect CatK-mediated Notch1 activation and other signal molecules. Animal experiments showed that compared with the control group, the spatial learning and memory ability were decreased significantly in CatKII group, and the hippocampus protein expressions of c-Notch1, p-Akt, p-CREB and BDNF were also decreased significantly(P＜0.05); the levels of Glu in DG area of control group and CatK II group were increased significantly with Morris water maze training days, but the increase of CatK II group was significantly weaker than that of control group(P＜ 0.05). The results of cell experiment showed that the expressions of CatK, c-Notch1, p-CREB and BDNF in siCatK group were significantly lower than other groups (P＜0.05). CatK can affect the spatial learning and memory function of rats by activating Notch1 and its memory related signal protein in hippocampus.

Cathepsin K Deficiency Prevented Kidney Damage and Dysfunction in Response to 5/6 Nephrectomy Injury in Mice With or Without Chronic Stress.

Background:Chronic psychological stress is a risk factor for kidney disease, including kidney dysfunction and hypertension. Lysosomal CatK (cathepsin K) participates in various human pathobiologies. We investigated the role of CatK in kidney remodeling and hypertension in response to 5/6 nephrectomy injury in mice with or without chronic stress.Methods:Male 7-week-old WT (wild type; CatK+/+) and CatK-deficient (CatK−/−) mice that were or were not subjected to chronic stress underwent 5/6 nephrectomy. At 8 weeks post-stress/surgery, the stress was observed to have accelerated injury-induced glomerulosclerosis, proteinuria, and blood pressure elevation.Results:Compared with the nonstressed mice, the stressed mice showed increased levels of TLR (Toll-like receptor)-2/4, p22phox, gp91phox, CatK, MMP (matrix metalloproteinase)-2/9, collagen type I and III genes, PPAR-γ (peroxisome proliferator-activated receptor-gamma), NLRP-3 (NOD-like receptor thermal protein domain associated protein 3), p21, p16, and cleaved caspase-8 proteins, podocyte foot process effacement, macrophage accumulation, apoptosis, and decreased levels of Bcl-2 (B cell lymphoma 2) and Sirt1, as well as decreased glomerular desmin expression in the kidneys. These harmful changes were retarded by the genetic or pharmacological inhibition of CatK. Consistently, CatK inhibition ameliorated 5/6 nephrectomy–related kidney injury and dysfunction. In mesangial cells, CatK silencing or overexpression, respectively, reduced or increased the PPAR-γ and cleaved caspase-8 protein levels, providing evidence and a mechanistic explanation of CatK’s involvement in PPAR-γ/caspase-8–mediated cell apoptosis in response to superoxide and stressed serum.Conclusions:These results demonstrate that CatK plays an essential role in kidney remodeling and hypertension in response to 5/6 nephrectomy or stress, possibly via a reduction of glomerular inflammation, apoptosis, and fibrosis, suggesting a novel therapeutic strategy for controlling kidney injury in mice under chronic psychological stress conditions.

A patent review on cathepsin K inhibitors to treat osteoporosis (2011 – 2021)

Introduction Cathepsin K (CatK) is a lysosomal cysteine protease and the predominant cathepsin expressed in osteoclasts, where it degrades the bone matrix. Hence, CatK is an attractive therapeutic target related to diseases characterized by bone resorption, like osteoporosis. Areas covered This review summarizes the patent literature from 2011 to 2021 on CatK inhibitors and their potential use as new treatments for osteoporosis. The inhibitors were classified by their warheads, with the most explored nitrile-based inhibitors. Promising in vivo results have also been disclosed. Expert opinion As one of the most potent lysosomal proteins whose primary function is to mediate bone resorption, cathepsin K remains an excellent target for therapeutic intervention. Nevertheless, there is no record of any approved drug that targets CatK. The most notable cases of drug candidates targeting CatK were balicatib and odanacatib, which reached Phase II and III clinical trials, respectively, but did not enter the market. Further developments include exploring new chemical entities beyond the nitrile-based chemical space, with improved ADME and safety profiles. In addition, CatK’s role in cancer immunoexpression and its involvement in the pathophysiology of osteo- and rheumatoid arthritis have raised the race to develop activity-based probes with excellent potency and selectivity.

Highly potent inhibitors of cathepsin K with a differently positioned cyanohydrazide warhead: structural analysis of binding mode to mature and zymogen-like enzymes

Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.

Abstract 9561: Loss of Full Length Pumilio 1 Abrogates Gene P27 Silencing-Mediated Cell Proliferation and Secures Mirna-221 Mimics Therapeutics in the Heart

Introduction: MiR-221 mimics have been demonstrated to be cardioprotective and anti-fibrotic in the treatment of myocardium infarction and heart failure. However, miR-221 is a known oncomiR as the upregulation of miR-221 and the downregulation of its target p27 are associated with tumor progression and poor prognosis. Therefore the treatment of miR-221 mimics raise safety concerns. Hypothesis: miR-221 fails to trigger P27 mediated neoplastic cellular proliferation due to loss of full length pumilio-1 (FL PUM-1) expression in vivo . Methods: H9c2, Hela and primary cardiac fibroblasts were used to study the regulations of miR-221 mimics on P27 by RT-qPCR and Western blot, cell proliferation by cell number, CCk-8 and Ki67 staining. The interaction with PUM1 was assessed by siRNA transfection, p27 3’UTR cloning and luciferase reporter assay. In vivo 2 days after miR-221 mimic (1mg/kg and 4mg/kg) i.v. injection in the rats, the liver, heart, kidney and muscle were collected to measure p27 and PUM1 by RT-qPCR and WB. Pum1 cDNA was cloned using primers flanking its start and stop codons, and inserted into pcDNA3.1 vector. 10 colonies from each tissue were sequenced to verify mRNA splicing variants. PUM1 cleavage were verified by the incubations of cathepsin K (CatK) inhibitor odanacatib (ODN). Results: in vitro miR-221 mimics significantly downregulated the expression of P27 and increased cell proliferation. FL PUM1 (140kDa) and isoforms of 105 and 90 kDa were detected. The transfections of siRNAs to knockdown PUM1 but not PMU2 abolished miR-221-mediated effects and direct binding of miR-221 on p27 3’UTR. In vivo following two dosages miR-221 mimic treatments, the expressions of miR-221 increased in the heart, liver, kidney and muscle for 6.8 and 17.3 fold, 33.4 and 506.5 fold, 2.2 and 3.5 fold, and no significant increases respectively. P27 was not affected. FL-PUM1 was not detected in all tissues, but only isoforms. 105 kDa was generated through alternative RNA slicing and 90 kDa by CatK cleavage. Conclusions: This is the first study to address the safety concerns with respect to the possible pro-proliferative effects of miR-221 mimic therapeutics in cardiovascular applications. It provides essential insights of the context-dependent nature of miRNA functionality.

A Combined approach of QSAR study, molecular docking and pharmacokinetics prediction of promising Amide-Ac6-aminoacetonitriles Cathepsin K inhibitors

• Combined use of QSAR, molecular docking and pharmacokinetics prediction approaches. • One promising Cathepsin K inhibitor was identified. Cathepsin K (Cat-K) highly expressed in osteoclasts, is considered to be a promising target for osteoclast-related maladies. In the present study, quantitative structure–activity relationships (QSAR) and molecular docking approaches were used to identify promising Cat-K inhibitors from a series of Amide-Ac6-aminoacetonitriles analogs. During the two-dimensional QSAR study, the statistical model PLS_MDL was chosen (r 2 = 0.823, q 2 (cross-validation) = 0.732). Descriptors with positive or negative contributions were derived from model PLS_MDL. In order to build robust three-dimensional QSAR models, three different molecular fingerprints were used as analysis precepts for clustering and three different minimization algorithms were employed for generation of molecular poses. The best database which used molecular fingerprint of FCFP and minimization algorithm of Smart Minimizer was selected for further QSAR model construction. The ideal CoMFA and CoMSIA models were acquired (CoMFA: r 2 = 0.963, q 2 = 0.674, r 2 pred = 0.702 and Q 2 (F2) = 0.616; CoMSIA_SEHA: r 2 = 0.989, q 2 = 0.622, r 2 pred = 0.719 and Q 2 (F2) = 0.638). Physicochemical, structural, electrostatic, and steric properties were derived from the created QSAR models and then applied to portray a design scheme. Furthermore, after a selecting of docking scheme, docking studies were carried out to measure the binding affinity of eligible query compounds and to reveal the detailed interactions between ligand and the target protein. Amongst these designed compounds, compound19OX32 was focused for its preferable predicted activity and docking score. Strong interactions and extra hydrogen bonds which echoed the previous summarized design scheme were also observed via a deep investigation of docking result. Consequently, compound 19OX32 was identified as promising Cat-K inhibitor.

Regulatory properties of vitronectin and its glycosylation in collagen fibril formation and collagen-degrading enzyme cathepsin K activity

Vitronectin (VN) is a glycoprotein found in extracellular matrix and blood. Collagen, a major extracellular matrix component in mammals, is degraded by cathepsin K (CatK), which is essential for bone resorption under acidic conditions. The relationship between VN and cathepsins has been unclear. We discovered that VN promoted collagen fibril formation and inhibited CatK activity, and observed its activation in vitro. VN accelerated collagen fibril formation at neutral pH. Collagen fibers formed with VN were in close contact with each other and appeared as scattered flat masses in scanning electron microscopy images. VN formed collagen fibers with high acid solubility and significantly inhibited CatK; the IC50 was 8.1–16.6 nM and competitive, almost the same as those of human and porcine VNs. VN inhibited the autoprocessing of inactive pro-CatK from active CatK. DeN-glycosylation of VN attenuated the inhibitory effects of CatK and its autoprocessing by VN, but had little effect on acid solubilization of collagen and VN degradation via CatK. CatK inhibition is an attractive treatment approach for osteoporosis and osteoarthritis. These findings suggest that glycosylated VN is a potential biological candidate for CatK inhibition and may help to understand the molecular mechanisms of tissue re-modeling.

Preclinical evaluation of [11 C]L-235 as a radioligand for Positron Emission Tomography cathepsin K imaging in bone.

The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.

Cathepsin K: The Action in and Beyond Bone.

Cathepsin K (CatK) is one of the most potent proteases in lysosomal cysteine proteases family, of which main function is to mediate bone resorption. Currently, CatK is among the most attractive targets for anti-osteoporosis drug development. Although many pharmaceutical companies are working on the development of selective inhibitors for CatK, there is no FDA approved drug till now. Odanacatib (ODN) developed by Merck & Co. is the only CatK inhibitor candidate which demonstrated high therapeutic efficacy in patients with postmenopausal osteoporosis in Phase III clinical trials. Unfortunately, the development of ODN was finally terminated due to the cardio-cerebrovascular adverse effects. Therefore, it arouses concerns on the undesirable CatK inhibition in non-bone sites. It is known that CatK has far-reaching actions throughout various organs besides bone. Many studies have also demonstrated the involvement of CatK in various diseases beyond the musculoskeletal system. This review not only summarized the functional roles of CatK in bone and beyond bone, but also discussed the potential relevance of the CatK action beyond bone to the adverse effects of inhibiting CatK in non-bone sites.

Deficiency of cysteinyl cathepsin K suppresses the development of experimental intimal hyperplasia in response to chronic stress.

Chronic psychological stress (CPS) is linked to cardiovascular disease initiation and progression. Given that cysteinyl cathepsin K (CatK) participates in vascular remodeling and atherosclerotic plaque growth in several animal models, we investigated the role of CatK in the development of experimental neointimal hyperplasia in response to chronic stress. At first, male wild-type (CatK) mice that underwent carotid ligation injury were subjected to chronic immobilization stress. On postoperative and stressed day 14, the results demonstrated that stress accelerated injury-induced neointima hyperplasia. On day 4, stressed mice showed following: increased levels of monocyte chemoattractant protein-1, gp91phox, toll-like receptor-2 (TLR2), TLR4, and CatK mRNAs or/and proteins, oxidative stress production, aorta-derived smooth muscle cell (SMC) migration, and macrophage infiltration as well as targeted intracellular proliferating-related molecules. Stressed mice showed increased matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA expressions and activities and elastin disruption in the injured carotid arteries. Second, CatK and CatK deficiency (CatK) mice received ligation injury and stress to explore the role of CatK. The stress-induced harmful changes were prevented by CatK. Finally, CatK mice that had undergone ligation surgery were randomly assigned to one of two groups and administered vehicle or CatK inhibitor for 14 days. Pharmacological CatK intervention produced a vascular benefit. These data indicate that CatK deletion protects against the development of experimental neointimal hyperplasia via the attenuation of inflammatory overaction, oxidative stress production, and VSMC proliferation, suggesting that CatK is a novel therapeutic target for the management of CPS-related restenosis after intravascular intervention therapies.

New Synthetic Quinolines as Cathepsin K Inhibitors

Cathepsin K is a papain-like cysteine protease and is responsible for collagen degradation in bone tissue and thus represents an important target for the development of new therapies for treating diseases such as osteoporosis. Quinolines are an important class of heterocyclic molecular leads with a great pharmacological potential and represent a relevant scaffold to explore the chemical space of cathepsin K (CatK) inhibitors. This study presents the synthesis of nine 2,4-diphenylquinolines, including five phthalonitrile quinolines dyads, and the evaluation of their CatK inhibitory activity. Among the evaluated compounds, 4b was the most potent inhibitor with an IC50 (half-maximal inhibitory concentration) value of 1.55 µM (against Z-Phe-Arg-MCA substrate) acting in an uncompetitive inhibition mode. Molecular docking studies provided important information on the interaction of the inhibitor with the enzyme showing that these quinoline derivatives can play an important role as CatK inhibitors.

Targeting cathepsin K diminishes prostate cancer establishment and growth in murine bone

BackgroundThe processes of prostate cancer (PCa) invasion and metastasis are facilitated by proteolytic cascade involving multiple proteases, such as matrix metalloproteinases, serine proteases and cysteine proteases including cathepsin K (CatK). CatK is predominantly secreted by osteoclasts and specifically degrades collagen I leading to bone destruction. PCa and breast cancer preferentially metastasize to the bone. Importantly, CatK expression level is greater in PCa bone metastatic sites compared to primary tumor and normal prostate tissues. However, the underlying mechanism of CatK during PCa metastases into the bone remains to be elucidated. We investigated the functional role of CatK during the PCa establishment and growth process in the murine bone.MethodsCatK mRNA expression was validated by RT-PCR, protein expression by immunoblotting in PCa LNCaP, C4-2B, and PC3 cells as well as in PCa tissues. Its protein production was measured using ELISA assay. The effect of both knockdowns via siRNA and CatK inhibitor was compared in regard to PCa cell invasion. We further studied the dose-dependent CatK inhibitor effect on conditioned media-induced bone resorption. In setting up an animal model, C4-2B cells were injected into the tibiae of SCID mice. The animals treated with either vehicle or CatK inhibitor for 8 weeks at the time of tumor cell injection (tumor establishment model; protocol I) or 4 weeks after tumor cell injection (tumor progression model; protocol II) were applied to histological and histomorphometric analyses.ResultsWe confirmed CatK expression in PCa LNCaP, C4-2B, and PC3 cells as well as in PCa tissues. Furthermore, we observed the inhibitory effects of a selective CatK inhibitor on PCa cell invasion. The CatK inhibitor dose-dependently inhibited PCa-conditioned media-induced bone resorption. Upon injection of C4-2B cells into the tibiae of SCID mice, the selective CatK inhibitor significantly prevented the tumor establishment in protocol I, and reduced the tumor growth in bone in protocol II. It also decreased serum PSA levels in both animal models. The inhibitory effects of the CatK inhibitor were enhanced in combination with zoledronic acid (ZA).ConclusionThe selective CatK inhibitor may prevent the establishment and progression of PCa in bone, thus making it a novel therapeutic approach for advanced PCa.

Clinical and translational pharmacology of the cathepsin K inhibitor odanacatib studied for osteoporosis.

Cathepsin K (CatK) is a cysteine protease abundantly expressed by osteoclasts and localized in the lysosomes and resorption lacunae of these cells. CatK is the principal enzyme responsible for the degradation of bone collagen. Odanacatib is a selective, reversible inhibitor of CatK at subnanomolar potency. The pharmacokinetics of odanacatib have been extensively studied and are similar in young healthy men, postmenopausal women and elderly men, and were qualitatively similar throughout Phase 1 development and in-patient studies. Following 3weeks of 50mg once weekly dosing the geometric mean area under the curve from 0 to 168 hours was 41.1μMh, the concentration at 168 hours was 126nM and the harmonic mean apparent terminal half-life was 84.8hr. Odanacatib exposure increased in a less than dose proportional manner due to solubility limited absorption. It is estimated that approximately 70% of the absorbed dose of odanacatib is eliminated via metabolism, 20% is excreted as unchanged drug in the bile or faeces, and 10% is excreted as unchanged drug in the urine. The systemic clearance was low (approximately 13mL/min). Odanacatib decreases the degradation of bone matrix proteins and reduces the efficiency of bone resorption with target engagement confirmed by a robust decrease in serum C-telopeptides of type 1 collagen (approximately 60%), urinary aminoterminal crosslinked telopeptides of type 1 collagen to creatinine ratio (approximately 50%) and total urine deoxypyridinoline/Cr (approximately 30%), with an increase in serum cross-linked carboxy-terminal telopeptide of type 1 collagen (approximately 55%). The 50-mg weekly dosing regimen evaluated in Phase 3 achieved near maximal reduction in bone resorption throughout the treatment period. The extensive clinical programme for odanacatib, together with more limited clinical experience with other CatK inhibitors (balicatib and ONO-5334), provides important insights into the clinical pharmacology of CatK inhibition and the potential role of CatK in bone turnover and mineral homeostasis. Key findings include the ability of this mechanism to: (i) provide sustained reductions in resorption markers, increases in bone mineral density, and demonstrated fracture risk reduction; (ii) be associated with relative formation-sparing effects such that sustained resorption reduction is achieved without accompanying meaningful reductions in bone formation; and (iii) lead to increases in osteoclast number as well as other osteoclast activity (including build-up of CatK enzyme), which may yield transient increases in resorption following treatment discontinuation and the potential for nonmonotonic responses at subtherapeutic doses.