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1.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140318, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31740411

RESUMO

Human cathepsin K (hCatK), which is highly expressed in osteoclasts, has the noteworthy ability to cleave type I and II collagens in their helical domain. Its collagenase potency depends strictly on the formation of an oligomeric complex with chondroitin 4-sulfate (C4-S). Accordingly, hCatK is a pivotal protease involved in bone resorption and is an attractive target for the treatment of osteoporosis. As rat is a common animal model for the evaluation of hCatK inhibitors, we conducted a comparative analysis of rat CatK (rCatK) and hCatK, which share a high degree of identity (88%) and similarity (93%). The pH activity profile of both enzymes displayed a similar bell-shaped curve (optimal pH: 6.4). Presence of Ser134 and Val160 in the S2 pocket of rCatK instead of Ala and Leu residues, respectively, in hCatK, led to a weaker peptidase activity, as observed for mouse CatK. Also, regardless of the presence of C4-S, rCatK cleaved in the nonhelical telopeptide regions of both type I (tail) and type II (articular joint) rat collagens. Structure-based computational analyses (electrostatic potential, molecular docking, molecular dynamics, free energy calculations) sustained that the C4-S mediated collagenolytic activity of rCatK obeys distinct molecular interactions from those of hCatK. Additionally, T-kininogen (a.k.a. thiostatin), a unique rat serum acute phase molecule, acted as a tight-binding inhibitor of hCatK (Ki = 0.11 ± 0.05 nM). Taken into account the increase of T-Kininogen level in inflamed rat sera, this may raise the question of the appropriateness to evaluate pharmacological hCatK inhibitors in this peculiar animal model.


Assuntos
Catepsina K/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catepsina K/antagonistas & inibidores , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Alinhamento de Sequência , Especificidade por Substrato , Termodinâmica
2.
Cell Prolif ; 53(1): e12722, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31737959

RESUMO

OBJECTIVES: The mechanisms underlying the effects of Toll-like receptor 9 (TLR9) and autophagy on rheumatoid arthritis (RA)-aggravated periodontitis are unclear. We aimed to explore a novel target, cathepsin K (Ctsk)-mediated TLR9-related autophagy, during the progress of periodontitis with RA. MATERIALS AND METHODS: DBA/J1 mouse model of periodontitis with RA was created by local colonization of Porphyromonas gingivalis (Pg) and injection of collagen. The expression of Ctsk was inhibited by adeno-associated virus (AAV). Micro-CT, immunohistochemistry (IHC), Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of TLR9-related autophagy in periodontitis with RA. Small interfering RNA (siRNA) and CpG oligodeoxynucleotides (CpG ODN) were applied in macrophages. Western blot, immunofluorescence (IF) and qRT-PCR were used to verify the in vivo results. RESULTS: RA can promote periodontitis bone destruction in the lesion area, while inhibiting Ctsk could effectively alleviate this effect. The infiltration of macrophages, TLR9, autophagy proteins (TFEB and LC3) and inflammatory cytokines increased in the periodontitis-with-RA group and was reduced by the inhibition of Ctsk in the periodontal region. Macrophage stimulation confirmed the in vivo results. With the activation of TLR9 by CpG ODN, inhibition of Ctsk could suppress both TLR9 downstream signalling proteins and autophagy-related proteins. CONCLUSIONS: This study advanced a novel role for Ctsk in TLR9 and autophagy to explain the interaction between periodontitis and RA.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Catepsina K/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Periodontite/tratamento farmacológico , Receptor Toll-Like 9/imunologia , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/imunologia , Catepsina K/genética , Catepsina K/imunologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos DBA , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Oligodesoxirribonucleotídeos/genética , Periodontite/genética , Periodontite/imunologia , Periodontite/patologia , Receptor Toll-Like 9/genética
3.
J Cancer Res Clin Oncol ; 145(8): 1999-2012, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31172267

RESUMO

BACKGROUND: The 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. METHODS: CatK 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. RESULTS: We 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). CONCLUSION: The selective CatK inhibitor may prevent the establishment and progression of PCa in bone, thus making it a novel therapeutic approach for advanced PCa.


Assuntos
Neoplasias Ósseas/prevenção & controle , Neoplasias Ósseas/secundário , Catepsina K/antagonistas & inibidores , Terapia de Alvo Molecular/métodos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Inibidores de Proteases/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Neoplasias Ósseas/genética , Catepsina K/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos SCID , Células PC-3 , Neoplasias da Próstata/genética , Inibidores de Proteases/farmacologia , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Bioorg Med Chem ; 27(6): 1034-1042, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30773420

RESUMO

Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC50 values and inhibition constants (Ki) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with Ki amounting to 1.14 and 7.21 nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5 µM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1 µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.


Assuntos
Catepsina K/antagonistas & inibidores , Desdiferenciação Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Nitrilos/farmacologia , Inibidores de Proteases/farmacologia , Animais , Compostos Aza/síntese química , Compostos Aza/química , Compostos Aza/farmacologia , Catepsina K/metabolismo , Linhagem Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/enzimologia , Desenho de Fármacos , Humanos , Camundongos , Nitrilos/síntese química , Nitrilos/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/química
5.
Biochem J ; 476(3): 499-512, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30622151

RESUMO

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.


Assuntos
Catepsina K/antagonistas & inibidores , Peptídeos/química , Inibidores de Proteases/química , Animais , Catepsina K/química , Catepsina K/metabolismo , Bovinos , Humanos , Osteoclastos/enzimologia , Especificidade por Substrato
6.
Br J Clin Pharmacol ; 85(6): 1072-1083, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30663085

RESUMO

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 3 weeks of 50 mg once weekly dosing the geometric mean area under the curve from 0 to 168 hours was 41.1 µM h, the concentration at 168 hours was 126 nM and the harmonic mean apparent terminal half-life was 84.8 hr. 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 13 mL/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.


Assuntos
Compostos de Bifenilo/uso terapêutico , Conservadores da Densidade Óssea/uso terapêutico , Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Catepsina K/antagonistas & inibidores , Inibidores de Cisteína Proteinase/uso terapêutico , Osteoporose/tratamento farmacológico , Animais , Compostos de Bifenilo/efeitos adversos , Compostos de Bifenilo/farmacocinética , Conservadores da Densidade Óssea/efeitos adversos , Conservadores da Densidade Óssea/farmacocinética , Osso e Ossos/enzimologia , Osso e Ossos/fisiopatologia , Catepsina K/metabolismo , Inibidores de Cisteína Proteinase/efeitos adversos , Inibidores de Cisteína Proteinase/farmacocinética , Feminino , Humanos , Masculino , Osteoporose/enzimologia , Osteoporose/patologia , Transdução de Sinais , Pesquisa Médica Translacional , Resultado do Tratamento
7.
J Bone Miner Metab ; 37(4): 636-647, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30357565

RESUMO

This study evaluated the long-term effects of the cathepsin K inhibitor ONO-5334 on bone mass and strength in ovariectomised (OVX) cynomolgus monkeys. Animals were assigned to one of the following six groups: Sham (non-OVX), OVX control treated with vehicle, ONO-5334 1.2, 6 or 30 mg/kg/day, p.o., or alendronate (ALN) 0.05 mg/kg/2 weeks, i.v. for 16 months. Peripheral quantitative computed tomography (pQCT) analysis revealed that ONO-5334 increased not only trabecular bone mineral density (BMD) but also cortical BMD in the distal radius and the lumbar vertebra. ONO-5334 and ALN suppressed the deterioration of trabecular architecture by micro-CT analysis in the distal radius. Assessments of bone strength showed that ONO-5334 increased maximum load at the distal and midshaft radius. The linear regression lines between bone mass and strength in the lumbar vertebra were tended to be shifted towards increasing bone strength in the ONO-5334 6 and 30 mg/kg groups compared with the ALN groups. This indicated that bone strength was higher in the ONO-5334 groups than the ALN group, even though bone mineral content (BMC) and BMD were comparable. Subpopulation analysis revealed that, at similar integral BMC or BMD level, cortical bone mass for ONO-5334 was higher than for ALN; the opposite effects were observed for trabecular bone. In conclusion, ONO-5334 preferentially increased cortical bone, which may provide a greater contribution to bone strength. Since these results support a different mode of action for ONO-5334 compared with that of ALN, ONO-5334 may offer new therapeutic options to patients with osteoporosis.


Assuntos
Densidade Óssea/fisiologia , Catepsina K/antagonistas & inibidores , Osso Cortical/fisiologia , Ovariectomia , Tiazolidinas/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Osso Cortical/diagnóstico por imagem , Osso Cortical/efeitos dos fármacos , Feminino , Humanos , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/efeitos dos fármacos , Macaca fascicularis , Tamanho do Órgão , Rádio (Anatomia)/diagnóstico por imagem , Rádio (Anatomia)/efeitos dos fármacos , Tomografia Computadorizada por Raios X , Microtomografia por Raio-X
8.
Bioorg Chem ; 84: 226-238, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30502634

RESUMO

A series of novel 7-aminoalkyl substituted pyrazolo[1,5-a]pyrimidine 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 pyrazolo[1,5-a]pyrimidine scaffold at various positions. Molecular docking studies with pyrazolo[1,5-a]pyrimidine 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).


Assuntos
Catepsina K/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Pirazóis/química , Pirimidinas/química , Sítios de Ligação , Domínio Catalítico , Catepsina K/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Pirazóis/metabolismo , Pirimidinas/metabolismo , Relação Estrutura-Atividade
9.
Calcif Tissue Int ; 104(1): 92-101, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30194476

RESUMO

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.


Assuntos
Compostos de Bifenilo/farmacologia , Reabsorção Óssea/metabolismo , Catepsina K/antagonistas & inibidores , Osteoclastos/metabolismo , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/tratamento farmacológico , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Catepsina K/metabolismo , Humanos , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo
10.
Eur J Immunol ; 48(12): 1944-1957, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30365157

RESUMO

This study sought to identify novel CD8+ T cell homing markers by studying acute graft versus host disease (aGvHD), typically involving increased T cell homing to the skin and gut. FACS-sorted skin-homing (CD8ß+ /CLA+ ), gut-homing (CD8ß+ /integrinß7+ ), and reference (CD8ß+ /CLA- /integrinß7- ) T cells were compared in patients affected by cutaneous and/or gastrointestinal aGVHD. Microarray analysis, qPCR, and flow cytometry revealed increased expression of peptidase inhibitor 16 (PI16) in skin-homing CD8+ T cells. Robust association of PI16 with skin homing was confirmed in all types of aGvHD and in healthy controls, too. PI16 was not observed on CLA+ leukocytes other than T cells. Induction of PI16 expression on skin-homing T cells occurred independently of vitamin D3. Among skin-homing T cells, PI16 expression was most pronounced in memory-like CD45RO+ /CD127+ /CD25+ /CD69- /granzyme B- cells. PI16 was confined to the plasma membrane, was GPI-anchored, and was lost upon restimulation of memory CD8+ T cells. Loss of PI16 occurred by downregulation of PI16 transcription, and not by Phospholipase C (PLC)- or Angiotensin-converting enzyme (ACE)-mediated shedding, or by protein recycling. Inhibitor screening and pull-down experiments confirmed that PI16 inhibits cathepsin K, but may not bind to other skin proteases. These data link PI16 to skin-homing CD8+ T cells, and raise the possibility that PI16 may regulate cutaneous cathepsin K.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Pele/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Catepsina K/antagonistas & inibidores , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Citometria de Fluxo , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Receptores de Retorno de Linfócitos/metabolismo
11.
Br J Oral Maxillofac Surg ; 56(8): 732-738, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30131193

RESUMO

Cathepsin K inhibitors are new drugs with the potential for the treatment of osteoporosis because they sustain bony remodelling better than bone resorption inhibitors such as bisphosphonates. The treatment of osteoporosis with inhibitors of bony resorption is associated with osteonecrosis of the jaw, as the deterioration in bony quality that they induce is thought to be one of its causes. The quality of bone is delineated by structural and material characteristics (which include the degree and quality of mineralisation, and depends on the content of proteoglycan and the structural integrity of the bony collagen).1,2 Animal and clinical studies have shown that cathepsin K inhibitors improve the mineral density and structural characteristics of bone, but their effect on the rest remains unknown. We therefore hypothesised that these inhibitors will affect the material characteristics of newly-formed mandibular bone. To verify our hypothesis, we used Raman microspectroscopy to examine such bone in rats that were given a cathepsin K inhibitor, and found unusual crystallinity and an increased substitution of carbonate (CO32-) in its crystal structure.


Assuntos
Compostos de Bifenilo/farmacologia , Densidade Óssea/efeitos dos fármacos , Catepsina K/antagonistas & inibidores , Mandíbula/efeitos dos fármacos , Animais , Feminino , Mandíbula/diagnóstico por imagem , Ratos , Ratos Wistar , Tomografia Computadorizada por Raios X
12.
J Transl Med ; 16(1): 125, 2018 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-29743078

RESUMO

BACKGROUND: Cathepsin K is an attractive therapeutic target for diseases in which bone resorption is excessive such as osteoporosis and osteoarthritis (OA). The current paper characterized the pharmacological profile of the potent and selective cathepsin K inhibitor, MIV-711, in vitro and in cynomolgus monkeys, and assessed translation to human based on a single dose clinical study in man. METHODS: The potency and selectivity of MIV-711 were assessed in vitro using recombinant enzyme assays and differentiated human osteoclasts. MIV-711 was administered to healthy cynomolgus monkeys (3-30 µmol/kg, p.o.). Plasma levels of MIV-711 and the bone resorption biomarker CTX-I were measured after single dose experiments, and urine levels of CTX-I, NTX-I and CTX-II biomarkers were measured after repeat dose experiments. The safety, pharmacokinetics and pharmacodynamics (serum CTX-I) of MIV-711 were assessed in human healthy subjects after single ascending doses from 20 to 600 mg. RESULTS: MIV-711 was a potent inhibitor of human cathepsin K (Ki: 0.98 nmol/L) with > 1300-fold selectivity towards other human cathepsins. MIV-711 inhibited human osteoclast-mediated bone resorption with an IC50 value of 43 nmol/L. Single oral doses of MIV-711 to monkeys reduced plasma levels of CTX-I in a dose-dependent fashion by up to 57% at trough. The effect on CTX-I was linearly correlated to the plasma exposure of MIV-711, while the efficacy duration outlasted plasma exposure. Repeat oral dosing with MIV-711 also reduced urinary levels of the bone resorption biomarkers CTX-I (by 93%) and NTX-I (by 71%) and the cartilage degradation biomarker CTX-II (by 71%). MIV-711 was safe and well-tolerated when given as single ascending doses to healthy subjects. MIV-711 reduced serum CTX-I levels in a dose-dependent manner by up to 79% at trough. The relationship between MIV-711 exposure and effects on these biomarkers in humans was virtually identical when compared to the corresponding monkey data. CONCLUSIONS: MIV-711 is a potent and selective cathepsin K inhibitor with dose-dependent effects on biomarkers of bone and cartilage degradation in monkey and human. Taken together, MIV-711 shows promise for the treatment of bone and cartilage related disorders in humans, such as OA. Trial Registration EudraCT number 2011-003024-12, registered on June 22nd 2011.


Assuntos
Catepsina K/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Compostos Orgânicos/farmacologia , Administração Oral , Adulto , Animais , Biomarcadores/urina , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Inibidores de Cisteína Proteinase/administração & dosagem , Inibidores de Cisteína Proteinase/sangue , Inibidores de Cisteína Proteinase/farmacocinética , Feminino , Humanos , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Compostos Orgânicos/administração & dosagem , Compostos Orgânicos/sangue , Compostos Orgânicos/farmacocinética , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Adulto Jovem
13.
J Enzyme Inhib Med Chem ; 33(1): 890-904, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29723068

RESUMO

Cathepsin K (Cat K), highly expressed in osteoclasts, is a cysteine protease member of the cathepsin lysosomal protease family and has been of increasing interest as a target of medicinal chemistry efforts for its role in bone matrix degradation. Inhibition of the Cat K enzyme reduces bone resorption and thus, has rendered the enzyme as an attractive target for anti-resorptive osteoporosis therapy. Over the past decades, considerable efforts have been made to design and develop highly potent, excellently selective and orally applicable Cat K inhibitors. These inhibitors are derived from synthetic compounds or natural products, some of which have passed preclinical studies and are presently in clinical trials at different stages of advancement. In this review, we briefly summarised the historic development of Cat K inhibitors and discussed the relationship between structures of inhibitors and active sites in Cat K for the purpose of guiding future development of inhibitors.


Assuntos
Reabsorção Óssea/tratamento farmacológico , Catepsina K/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Descoberta de Drogas , Animais , Reabsorção Óssea/metabolismo , Catepsina K/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
14.
Dent Traumatol ; 34(3): 201-207, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29533502

RESUMO

BACKGROUND/AIM: Continuous research efforts have been focusing on promoting healing of delayed replantation of avulsed teeth. The aim of this in vivo study was to assess the effect of cathepsin K inhibitor surface treatment on delayed tooth replantation. MATERIALS AND METHODS: Thirty-four premolar roots of 4 beagle dogs were extracted, and conventional root canal treatment was performed. The canals were filled with gutta-percha and cement. All extracted teeth were air-dried for 1 hour. Teeth were divided into 2 groups according to the root surface treatment prior to replantation: control (n = 13) and cathepsin K inhibitor (n = 21). Treated teeth were replanted in their original sockets and stabilized with a resin-wire splint for 1 week. After 12 weeks, the dogs were euthanized and micro-computed tomography was performed. Tissues were then further processed as resin-embedded specimens stained with hematoxylin and eosin. In each evaluation, data were analyzed using the Mann-Whitney U test (P < .05). RESULTS: In the micro-computed tomography evaluation, inflammation scores of both groups were not statistically different (P > .05). In the histological evaluation, the mean proportions of inflammatory resorption and replacement resorption in the control group were similar to those in the cathepsin K inhibitor group (P > .05). CONCLUSIONS: Cathepsin K inhibitor did not demonstrate significant inhibitory effects on root resorption after delayed replantation in vivo.


Assuntos
Catepsina K/antagonistas & inibidores , Reimplante Dentário/métodos , Animais , Dente Pré-Molar/diagnóstico por imagem , Dente Pré-Molar/cirurgia , Modelos Animais de Doenças , Cães , Tratamento do Canal Radicular , Reabsorção da Raiz/diagnóstico por imagem , Propriedades de Superfície , Fatores de Tempo , Microtomografia por Raio-X
15.
J Transl Med ; 16(1): 56, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29523155

RESUMO

BACKGROUND: MIV-711 is a highly potent and selective cathepsin K inhibitor. The current article summarizes the therapeutic effects of MIV-711 on joint pathology in rabbits subjected to anterior cruciate ligament transection (ACLT), and the prophylactic effects on joint pathology in dogs subjected to partial medial meniscectomy, two surgical models of osteoarthritis (OA). METHODS: Starting 1 week after surgery, rabbits were dosed daily via oral gavage with either MIV-711 or vehicle (n = 7/group) for 7 weeks. The four treatment groups were: (1) sham + vehicle; (2) ACLT + vehicle; (3) ACLT + MIV-711, 30 µmol/kg and (4) ACLT + MIV-711, 100 µmol/kg. Subchondral bone and articular cartilage structures were assessed by µCT, histomorphometry, and scoring. Dogs subjected to partial medial meniscectomy received either MIV-711 (30 µmol/kg) or vehicle (n = 15/group) via oral gavage once daily, starting 1 day before meniscectomy, for 28 days. Cartilage degradation was assessed at the macroscopic and microscopic levels. The exposures of MIV-711 were assessed in both studies and biomarkers reflecting bone resorption (HP-1 in rabbits, CTX-I in dogs) and cartilage degradation (CTX-II) were measured. RESULTS: In ACLT rabbits, MIV-711 decreased HP-1 levels by up to 72% (p < 0.001) and CTX-II levels by up to 74% (p < 0.001) compared to ACLT vehicle controls. ACLT surgery significantly reduced the total thickness of the subchondral bone plate and reduced trabecular bone volume in the femur and tibia. These effects were reversed by MIV-711. ACLT resulted in cartilage thickening, which was attenuated by MIV-711. MIV-711 did not affect osteophyte formation or Mankin scores. In dogs, MIV-711 reduced CTX-I and CTX-II levels by 86% (p < 0.001) and 80% (p < 0.001), respectively. Synovial CTX-II levels were reduced by 55-57% (p < 0.001) compared to baseline. MIV-711-treated animals had 25-37% lower macroscopic scores in the femur condyles and 13-33% lower macroscopic scores in the tibial plateaus. CONCLUSIONS: MIV-711 prevents subchondral bone loss and partially attenuates cartilage pathology in two animal models of OA. These beneficial effects of MIV-711 on joint pathology are observed in conjunction with decreases in bone and cartilage biomarkers that have been shown to be clinically attainable in human. The data support the further development of MIV-711 for the treatment of OA.


Assuntos
Lesões do Ligamento Cruzado Anterior/tratamento farmacológico , Catepsina K/antagonistas & inibidores , Inibidores de Cisteína Proteinase/uso terapêutico , Articulações/patologia , Osteoartrite/tratamento farmacológico , Animais , Ligamento Cruzado Anterior , Biomarcadores/sangue , Biomarcadores/urina , Reabsorção Óssea/patologia , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Inibidores de Cisteína Proteinase/sangue , Inibidores de Cisteína Proteinase/farmacocinética , Inibidores de Cisteína Proteinase/farmacologia , Modelos Animais de Doenças , Cães , Feminino , Articulações/diagnóstico por imagem , Articulações/efeitos dos fármacos , Masculino , Compostos Orgânicos , Osteoartrite/sangue , Osteoartrite/diagnóstico por imagem , Osteoartrite/patologia , Análise de Componente Principal , Coelhos
16.
J Biomol Struct Dyn ; 36(3): 634-655, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28150528

RESUMO

Computational studies on the interaction of novel inhibitor compounds with the Cathepsin K protease have been performed to study the inhibition properties of the inhibitor compounds. The quantum chemical calculations have been performed to analyze the molecular geometries, structural stability, reactivity, nature of interaction, and the charge transfer properties using B3LYP level of theory by implementing 6-311g(d,p) basis set. The calculated C-S and N-H…N bond lengths of the inhibitor-triad complexes are found to agree well with the previous literature results. The chemical reactivity of the inhibitors and catalytic triad are analyzed through frontier molecular orbital analysis and found that the inhibitors are subjected to nucleophilic attack by the catalytic triad. The nature of inhibition of the inhibitor compounds is examined using the quantum theory of Atoms in Molecules analysis and found to be partially covalent. The NBO stabilization energy for the Cys - inhibitor are found to be most stable than the other interactions. The molecular dynamic simulations were performed to study the influence of dynamic of the active site on the QM results. The many body decomposition interaction energy calculated for the final results of MD simulation reveals that the dynamic of the active site induces significant changes in the interaction energy and occupancy of H-bonds plays a major role in the stabilizing the active site inhibitor interactions. The present study reveals that the inhibitor compounds can inhibit the proteolytic activity of the proteases on binding with the catalytic active site.


Assuntos
Catepsina K/química , Inibidores Enzimáticos/química , Modelos Teóricos , Nitrilos/química , Catálise , Domínio Catalítico , Catepsina K/antagonistas & inibidores , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Teoria Quântica
17.
Br J Pharmacol ; 175(6): 902-923, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29278432

RESUMO

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.


Assuntos
Abietanos/farmacologia , Conservadores da Densidade Óssea/farmacologia , Reabsorção Óssea/prevenção & controle , Catepsina K/antagonistas & inibidores , Abietanos/administração & dosagem , Abietanos/isolamento & purificação , Animais , Sítios de Ligação , Conservadores da Densidade Óssea/administração & dosagem , Conservadores da Densidade Óssea/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colagenases/metabolismo , Humanos , Concentração Inibidora 50 , Camundongos , Simulação de Acoplamento Molecular , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Salvia miltiorrhiza/química
18.
Eur J Med Chem ; 144: 201-210, 2018 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-29272750

RESUMO

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.


Assuntos
Catepsina K/antagonistas & inibidores , Catepsinas/antagonistas & inibidores , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Triazóis/química , Triazóis/farmacologia , Sequência de Aminoácidos , Animais , Catepsina K/metabolismo , Catepsinas/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Peptídeos/química , Peptídeos/farmacologia , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Relação Estrutura-Atividade , Especificidade por Substrato
19.
Mol Med Rep ; 17(1): 1633-1641, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29257214

RESUMO

The present study aimed to investigate bone deterioration in glucocorticoid­induced osteoporosis (GIOP) mice, and the anti­osteoporosis effect and underlying molecular mechanism of icariin. Dexamethasone (DSM) treatment was demonstrated to facilitate the induction of hypercalciuria in GIOP mice. Icariin treatment reversed the dexamethasone (DXM)­induced disequilibrium of calcium homeostasis and bone resorption, and increased serum alkaline phosphatase, tartrate resistant acid phosphatase, osteocalcin and deoxypyridinoline. Haematoxylin and eosin staining revealed an increase in disconnections and separation in the trabecular bone network of the tibial proximal metaphysis, in the GIOP group. Icariin treatment reversed the DXM­induced trabecular deleterious effects, and stimulated bone remodeling in GIOP mice. Furthermore, the results demonstrated that the mRNA and protein expression of cathepsin K were significantly increased in GIOP mice, compared with the control group. Icariin treatment may suppress the expression of cathepsin K in the tibia of GIOP mice. The levels of microRNA (miR)­186 were markedly reduced in the tibia of GIOP mice compared with control group; however, this was inhibited by icariin treatment. Bioinformatics analysis demonstrated that miR­186 regulates cathepsin K via binding to the upstream 3'­untranslated region. Furthermore, transfection with miR­186 mimics resulted in inhibition of cathepsin K expression, whereas miR­186 inhibitors facilitated cathepsin K expression in osteoclasts. In conclusion, the present study demonstrated the protective effects of icariin against bone deteriorations in the experimental GIOP mice, and the underlying mechanism was mediated, at least partially, via activation of miR­186­mediated suppression of cathepsin K. These results provide evidence to support the use of icariin as a therapeutic approach in the management of glucocorticoid­induced bone loss, and the disequilibrium of calcium homeostasis.


Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Osso e Ossos/efeitos dos fármacos , Catepsina K/antagonistas & inibidores , Medicamentos de Ervas Chinesas/uso terapêutico , Flavonoides/uso terapêutico , MicroRNAs/metabolismo , Osteoporose/tratamento farmacológico , Animais , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Catepsina K/genética , Catepsina K/metabolismo , Dexametasona , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Osteoporose/induzido quimicamente , Osteoporose/metabolismo , Osteoporose/patologia , Células RAW 264.7
20.
Bioorg Med Chem ; 26(1): 8-16, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29223717

RESUMO

A series of chalcone derivatives bearing benzamide or benzenesulfonamide moieties were synthesized and evaluated for their anti-tumor effect on HCT116, MCF7 and 143B cell lines in vitro. SAR analysis showed that compounds bearing a benzenesulfonamide group had greater potency than those bearing a benzamide group. It was also shown that compounds with a mono-methyl or mono-halogen group at the 3-position on the terminal phenyl ring were more effective than those with trifluoromethyl or methoxy groups. Compound 8e exhibited the most potent anti-tumor activities against HCT116, MCF7 and 143B cell lines, with IC50 values of 0.597, 0.886 and 0.791µM, respectively. Molecular docking studies and enzymatic assays demonstrated that the anti-tumor activity of compound 8e might be regulated by Cat L and Cat K.


Assuntos
Antineoplásicos/farmacologia , Catepsina K/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Chalcona/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chalcona/síntese química , Chalcona/química , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade
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