Characterization and molecular docking study of cathepsin L inhibitory peptides (SnuCalCpIs) from Calotropis procera R. Br.

Propeptides, released from the autocatalytic activation of its zymogen, are potential inhibitors against proteases involved in cancer cell invasion and migration. Our research team previously obtained novel propeptides (SnuCalCpIs) from transcriptome analysis of the medicinal plant Calotropis procera R. Br. and reported them as promising candidates for cancer therapeutics due to their cathepsin L inhibition activity. In the present study, inhibitory activity among SnuCalCpIs was compared with inhibition efficiency and verified by in silico molecular docking analysis. Only SnuCalCpI03 and SnuCalCpI15, expressed in Escherichia coli, showed inhibitory activity against cathepsin L as competitive inhibitors, and the half-maximal inhibitory concentrations (IC50) values of 2.1 nM and 1.6 nM, respectively. They were stable below 70 °C, maintaining more than 90% inhibitory activity over a wide range of pH (2.0-10.0), except at the isoelectric point (pI). The template-based docking simulation models showed that SnuCalCpI02, SnuCalCpI12, and SnuCalCpI16 could not interact with the substrate-binding cleft of cathepsin L even though they possessed the same conserved domain. In contrast, SnuCalCpI03 and SnuCalCpI15 interacted with cathepsin L along the propeptide binding loop and substrate-binding cleft, resulting in obstruction of substrate access to the active site.

Effects of vitamin C and D on the mRNA expression of angiotensin converting enzyme 2 receptor, cathepsin L, and transmembrane serine protease in the mouse lungs.

Vitamins (Vit) C and D are widely used as immunogenic supplements among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. The SAR-CoV-2 virus enters into the pulmonary endothelial cells through attachment to angiotensin converting enzyme 2 receptor (Ace2) and the proteolytic activity of Cathepsin L (Ctsl) and transmembrane serine protease 2 (Tmprss2) enzymes. This study aimed to determine the influence of Vit C and D on the mRNA expression of Ace2, Tmprss2, and Ctsl genes in the mouse lungs. Vitamins C and D were administrated to different groups of mice through intra-peritoneal route in doses equivalent to human for 30 days. Then, the mRNA expression of SARS-CoV-2 entry gene was analyzed using qRT-PCR. It is found that Vit D, but not C, upregulated significantly (P < 0.05) the mRNA expression of Ace2 by more than six folds, while downregulated the expression of Ctsl and Tmprss2 genes by 2.8 and 2.2 folds, respectively. It can be concluded from this study that Vit D alters the mRNA expression of Ace2, Tmprss, and Ctsl genes in the mouse lungs. This finding can help us in understanding, at least in part, the molecular influence of Vit D on genes involved in the entry of SARS-CoV-2 into the cells.

An Updated Review on Betacoronavirus Viral Entry Inhibitors: Learning from Past Discoveries to Advance COVID-19 Drug Discovery.

Even after one year of its first outbreak reported in China, the coronavirus disease 2019 (COVID-19) pandemic is still sweeping the World, causing serious infections and claiming more fatalities. COVID-19 is caused by the novel coronavirus SARS-CoV-2, which belongs to the genus Betacoronavirus (ß-CoVs), which is of greatest clinical importance since it contains many other viruses that cause respiratory disease in humans, including OC43, HKU1, SARS-CoV, and MERS. The spike (S) glycoprotein of ß-CoVs is a key virulence factor in determining disease pathogenesis and host tropism, and it also mediates virus binding to the host's receptors to allow viral entry into host cells, i.e., the first step in virus lifecycle. Viral entry inhibitors are considered promising putative drugs for COVID-19. Herein, we mined the biomedical literature for viral entry inhibitors of other coronaviruses, with special emphasis on ß-CoVs entry inhibitors. We also outlined the structural features of SARS-CoV-2 S protein and how it differs from other ß-CoVs to better understand the structural determinants of S protein binding to its human receptor (ACE2). This review highlighted several promising viral entry inhibitors as potential treatments for COVID-19.

The Thyroid Hormone Transporter Mct8 Restricts Cathepsin-Mediated Thyroglobulin Processing in Male Mice through Thyroid Auto-Regulatory Mechanisms That Encompass Autophagy.

The thyroid gland is both a thyroid hormone (TH) generating as well as a TH responsive organ. It is hence crucial that cathepsin-mediated proteolytic cleavage of the precursor thyroglobulin is regulated and integrated with the subsequent export of TH into the blood circulation, which is enabled by TH transporters such as monocarboxylate transporters Mct8 and Mct10. Previously, we showed that cathepsin K-deficient mice exhibit the phenomenon of functional compensation through cathepsin L upregulation, which is independent of the canonical hypothalamus-pituitary-thyroid axis, thus, due to auto-regulation. Since these animals also feature enhanced Mct8 expression, we aimed to understand if TH transporters are part of the thyroid auto-regulatory mechanisms. Therefore, we analyzed phenotypic differences in thyroid function arising from combined cathepsin K and TH transporter deficiencies, i.e., in Ctsk-/-/Mct10-/-, Ctsk-/-/Mct8-/y, and Ctsk-/-/Mct8-/y/Mct10-/-. Despite the impaired TH export, thyroglobulin degradation was enhanced in the mice lacking Mct8, particularly in the triple-deficient genotype, due to increased cathepsin amounts and enhanced cysteine peptidase activities, leading to ongoing thyroglobulin proteolysis for TH liberation, eventually causing self-thyrotoxic thyroid states. The increased cathepsin amounts were a consequence of autophagy-mediated lysosomal biogenesis that is possibly triggered due to the stress accompanying intrathyroidal TH accumulation, in particular in the Ctsk-/-/Mct8-/y/Mct10-/- animals. Collectively, our data points to the notion that the absence of cathepsin K and Mct8 leads to excessive thyroglobulin degradation and TH liberation in a non-classical pathway of thyroid auto-regulation.

In Silico Identification of Potential Natural Product Inhibitors of Human Proteases Key to SARS-CoV-2 Infection.

Presently, there are no approved drugs or vaccines to treat COVID-19, which has spread to over 200 countries and at the time of writing was responsible for over 650,000 deaths worldwide. Recent studies have shown that two human proteases, TMPRSS2 and cathepsin L, play a key role in host cell entry of SARS-CoV-2. Importantly, inhibitors of these proteases were shown to block SARS-CoV-2 infection. Here, we perform virtual screening of 14,011 phytochemicals produced by Indian medicinal plants to identify natural product inhibitors of TMPRSS2 and cathepsin L. AutoDock Vina was used to perform molecular docking of phytochemicals against TMPRSS2 and cathepsin L. Potential phytochemical inhibitors were filtered by comparing their docked binding energies with those of known inhibitors of TMPRSS2 and cathepsin L. Further, the ligand binding site residues and non-covalent interactions between protein and ligand were used as an additional filter to identify phytochemical inhibitors that either bind to or form interactions with residues important for the specificity of the target proteases. This led to the identification of 96 inhibitors of TMPRSS2 and 9 inhibitors of cathepsin L among phytochemicals of Indian medicinal plants. Further, we have performed molecular dynamics (MD) simulations to analyze the stability of the protein-ligand complexes for the three top inhibitors of TMPRSS2 namely, qingdainone, edgeworoside C and adlumidine, and of cathepsin L namely, ararobinol, (+)-oxoturkiyenine and 3α,17α-cinchophylline. Interestingly, several herbal sources of identified phytochemical inhibitors have antiviral or anti-inflammatory use in traditional medicine. Further in vitro and in vivo testing is needed before clinical trials of the promising phytochemical inhibitors identified here.

A novel zebrafish model to emulate lung injury by folate deficiency-induced swim bladder defectiveness and protease/antiprotease expression imbalance.

Lung injury is one of the pathological hallmarks of most respiratory tract diseases including asthma, acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). It involves progressive pulmonary tissue damages which are usually irreversible and incurable. Therefore, strategies to facilitate drug development against lung injury are needed. Here, we characterized the zebrafish folate-deficiency (FD) transgenic line that lacks a fully-developed swim bladder. Whole-mount in-situ hybridization revealed comparable distribution patterns of swim bladder tissue markers between wild-type and FD larvae, suggesting a proper development of swim bladder in early embryonic stages. Unexpectedly, neutrophils infiltration was not observed in the defective swim bladder. Microarray analysis revealed a significant increase and decrease of the transcripts for cathepsin L and a cystatin B (CSTB)-like (zCSTB-like) proteins, respectively, in FD larvae. The distribution of cathepsin L and the zCSTB-like transcripts was spatio-temporally specific in developing wild-type embryos and, in appropriate measure, correlated with their potential roles in maintaining swim bladder integrity. Supplementing with 5-formyltetrahydrofolate successfully prevented the swim bladder anomaly and the imbalanced expression of cathepsin L and the zCSTB-like protein induced by folate deficiency. Injecting the purified recombinant zebrafish zCSTB-like protein alleviated FD-induced swim bladder anomaly. We concluded that the imbalanced expression of cathepsin L and the zCSTB-like protein contributed to the swim bladder malformation induced by FD and suggested the potential application of this transgenic line to model the lung injury and ECM remodeling associated with protease/protease inhibitor imbalance.

Modulation of Cathepsin L Expression in the Coronary Arteries of Atherosclerotic Swine.

INTRODUCTION: Neointimal hyperplasia (NIH) and restenosis after percutaneous transluminal coronary angioplasty (PTCA) and intravascular stenting remain a problem on a long-term basis by causing endothelial denudation and damage to the intima and media. Vascular sterile inflammation has been attributed to the formation of NIH. Cathepsin L (CTSL), a lysosome protease, is associated with diet-induced atherogenesis. Vitamin D regulates the actions and regulatory effects of proteases and protease inhibitors in different cell types. Objectives of this study are to evaluate the modulatory effect of vitamin D on CTSL activity in post-PTCA coronary arteries of atherosclerotic swine. METHODS: Yucatan microswine were fed with high-cholesterol atherosclerotic diets. The swine were stratified to receive three diets: (1) vitamin D-deficient diet, (2) vitamin D-sufficient diet, and (3) vitamin D-supplement diet. After 6 mo, PTCA was performed in the left circumflex coronary artery (LCx). After 1 y, angiography and optical coherence tomography imaging were performed, and swine was euthanized. Coronary arteries were embedded in paraffin. Tissue sections were stained with hematoxylin and eosin. Expression of Ki67 and CTSL were evaluated by immunofluorescence. RESULTS: Increased number of Ki67 + cells were observed in the postangioplasty LCx in vitamin D-deficient compared with vitamin D-sufficient or vitamin D-supplemented swine. Notably, the expression of CTSL was significantly increased in postangioplasty LCx of vitamin D-deficient swine compared with the vitamin D-sufficient or vitamin D-supplemented animal groups. CONCLUSIONS: Increased expression of CTSL correlates with the formation of NIH in the PTCA-injured coronary arteries. However, in the presence of sufficient or supplemented levels of vitamin D in the blood, CTSL expression was significantly reduced.

Discovery of covalent enzyme inhibitors using virtual docking of covalent fragments.

Here we present a virtual docking screen of 1648 commercially available covalent fragments, and identified covalent inhibitors of cysteine protease cathepsin L. These inhibitors did not inhibit closely related protease cathepsin B. Thus, we have established virtual docking of covalent fragments as an approach to discover covalent enzyme inhibitors.

Changes in Activity of Cysteine Cathepsins B and L in Brain Structures of Mice with Aggressive and Depressive-Like Behavior Formed under Conditions of Social Stress.

We studied activity of lysosomal cysteine proteases, cathepsins B and L, in brain structures (frontal cortex, caudate nucleus, hippocampus, and hypothalamus) of C57Bl/6J mice with aggressive and depressive-like behavior formed under conditions of chronic social stress (repeated experience of victories and defeats within 20 days). Mice with depressive-like behavior showed increased activity of cathepsin В in the hypothalamus and nucleus caudatus and increased activity of cathepsin L in the hippocampus compared to control animals not subjected to agonistic confrontations. In mice with aggressive behavior, protease activity in the studied brain structures was not changed. In 4 h after immune system activation with LPS (250 µg/kg), cathepsin L activity in the hippocampus of control mice increased in comparison with mice receiving saline. In contrast to control animals, LPS caused a decrease in activity of the enzyme in the caudate nucleus and frontal cortex of aggressive mice and in the hippocampus of mice with depressive-like behavior.

Highly Conserved Arg Residue of ERFNIN Motif of Pro-Domain is Important for pH-Induced Zymogen Activation Process in Cysteine Cathepsins K and L.

Pro-domain of a cysteine cathepsin contains a highly conserved Ex2Rx2Fx2Nx3Ix3N (ERFNIN) motif. The zymogen structure of cathepsins revealed that the Arg(R) residue of the motif is a central residue of a salt-bridge/H-bond network, stabilizing the scaffold of the pro-domain. Importance of the arginine is also demonstrated in studies where a single mutation (Arg → Trp) in human lysosomal cathepsin K (hCTSK) is linked to a bone-related genetic disorder "Pycnodysostosis". In the present study, we have characterized in vitro Arg → Trp mutant of hCTSK and the same mutant of hCTSL. The R → W mutant of hCTSK revealed that this mutation leads to an unstable zymogen that is spontaneously activated and auto-proteolytically degraded rapidly. In contrast, the same mutant of hCTSL is sufficiently stable and has proteolytic activity almost like its wild-type counterpart; however it shows an altered zymogen activation condition in terms of pH, temperature and time. Far and near UV circular dichroism and intrinsic tryptophan fluorescence experiments have revealed that the mutation has minimal effect on structure of the protease hCTSL. Molecular modeling studies shows that the mutated Trp31 in hCTSL forms an aromatic cluster with Tyr23 and Trp30 leading to a local stabilization of pro-domain and supplements the loss of salt-bridge interaction mediated by Arg31 in wild-type. In hCTSK-R31W mutant, due to presence of a non-aromatic Ser30 residue such interaction is not possible and may be responsible for local instability. These differences may cause detrimental effects of R31W mutation on the regulation of hCTSK auto-activation process compared to altered activation process in hCTSL.

Prenatal alcohol-induced neuroapoptosis in rat brain cerebral cortex: protective effect of folic acid and betaine.

PURPOSE: Alcohol consumption in pregnancy may cause fetal alcohol syndrome (FAS) in the infant. This study aims to investigate prenatal alcohol exposure related neuroapoptosis on the cerebral cortex tissues of newborn rats and possible neuroprotective effects of betaine, folic acid, and combined therapy. METHODS: Pregnant rats were divided into five experimental groups: control, ethanol, ethanol + betaine, ethanol + folic acid, and ethanol + betaine + folic acid combined therapy groups. We measured cytochrome c release, caspase-3, calpain and cathepsin B and L. enzyme activities. In order to observe apoptotic cells in the early stages, TUNEL method was chosen together with histologic methods such as assessing the diameters of the apoptotic cells, their distribution in unit volume and volume proportion of cortical intact neuron nuclei. RESULTS: Calpain, caspase-3 activities, and cytochrome c levels were significantly increased in alcohol group while cathepsin B and L. activities were also found to be elevated albeit not statistically significant. These increases were significantly reversed by folic acid and betaine + folic acid treatments. While ethanol increased the number of apoptotic cells, this increase was prevented in ethanol + betaine and ethanol + betaine + folic acid groups. Morphometric examination showed that the mean diameter of apoptotic cells was increased with ethanol administration while this increase was reduced by betaine and betaine + folic acid treatments. CONCLUSION: We observed that ethanol is capable of triggering apoptotic cell death in the newborn rat brains. Furthermore, folic acid, betaine, and combined therapy of these supplements may reduce neuroapoptosis related to prenatal alcohol consumption, and might be effective on preventing fetal alcohol syndrome in infants.

[Effect of Wenyang Huoxue Lishul Recipe Containing Serum on Expression of Cathepsin L in Puromycin Aminonucleoside-induced Injury of Mouse Glomerular Podocytes].

OBJECTIVE: To observe the effect of Wenyang Huoxue Lishui Recipe (WHLR) containing serum on the expression of cathepsin L (CatL) in puromycin aminonucleoside-induced injury of mouse glomerular podocytes. METHODS: Mouse podocyte cells (MPCs) in vitro cultured were divided into the normal control group, the model group, the dexamethasone (DEX) group, 10% WHLR containing serum group, 20% WHLR containing serum group, the vehicle serum control group. MPCs in the normal control group were cultured at 37 degrees C culture solution for 24 h. 45 mg/L puromycin was acted on MPCs in the model group for 24 h. On the basis of puromycin intervention, 1 limol/L DEX was co-incubated in MPCs of the DEX group for 24 h; 10% or 20% WHLR containing serum was co-incubated in MPCs of the 10% WHLR containing serum group and 20% WHLR containing serum group for 24 h. The vehicle serum control group was also set up by incubating with WHLR containing serum alone for 24 h. The expression of CatL and its substrate Synaptopodin in podocytes were detected by cell immunofluorescence staining. FITC-conjugated phalloidin was used to stain F-actin. A cortical F-actin score index (CFS index) was designed to quantify the degree of cytoskeletal reorganization in cultured podocytes. RESULTS: Compared with the normal control group, the expression of synaptopodin significantly decreased and the expression of CatL significantly-increased in the model group. F-actin arranged in disorder, gradually forming pericellular F-actin ring. CFS index was obviously elevated (P < 0.01). Compared with the model group, the epression of synaptopodin increased, the expression of CatL decreased, and CFS index also decreased in the DEX group, 10% WHLR containing serum group, and 20% WHLR containing serum group (P < 0.05, P < 0.01). Compared with the DEX group, the expression of synaptopodin decreased in 10% WHLR containing serum group, CFS index also decreased in 20% WHLR containing serum group (P < 0.05). CONCLUSIONS: WHLR could up-regulate the expression of synaptopodin, down-regulate the expression of CatL, and alleviate cytoskeletal reorganization of F-actin. It was helpful to stabilize the cytoskeleton of F-actin and improve the merging of podocytes.

Synthesis and Preclinical Evaluation of a Highly Improved Anticancer Prodrug Activated by Histone Deacetylases and Cathepsin L.

Lack of absolute selectivity against cancer cells is a major limitation for current cancer therapies. In the previous study, we developed a prodrug strategy for selective cancer therapy using a masked cytotoxic agent puromycin [Boc-Lys(Ac)-Puromycin], which can be sequentially activated by histone deacetylases (HDACs) and cathepsin L (CTSL) to kill cancer cells expressing high levels of both enzymes. Despite the promise as a selective cancer therapy, its requirement of relatively high dosage could be a potential issue in the clinical setting. To address this issue, we aimed to further improve the overall efficacy of our prodrug strategy. Since the proteolytic cleavage by CTSL is the rate-limiting step for the drug activation, we sought to improve the substrate structure for CTSL activity by modifying the α-amino protecting group of lysine. Here we show that protection with Fmoc [Fmoc-Lys(Ac)-Puromycin] exhibits a marked improvement in overall anticancer efficacy compared to the original Boc-Lys(Ac)-Puromycin and this is mainly due to the highly efficient cellular uptake besides its improved substrate structure. Furthermore, to address a concern that the improved drug efficacy might direct high toxicity to the normal cells, we confirmed that Fmoc-Lys(Ac)-Puromycin still retains excellent cancer selectivity in vitro and no obvious systemic off-target toxicity in vivo. Thus our preclinical evaluation data presented here demonstrate that the Fmoc-Lys(Ac)-Puromycin exhibits substantially improved anticancer efficacy, further supporting our approach for the selective cancer therapy.

Hyperthermia exacerbates the effects of cathepsin L on claudin-1 in a blood-brain barrier model in vitro.

PURPOSE: The effects of cathepsin L on claudin-1 expression were investigated under hyperthermic condition in a blood-brain barrier (BBB) model in vitro, in order to estimate the potential effects of hyperthermia on BBB dysfunction. MATERIALS AND METHODS: Brain microvascular endothelial cells (BMECs) and astrocytes were obtained from rat brain. The BBB models were randomly divided into a sham (37°C) group, a 39°C group, a 37°C+cathepsin L group and a 39°C+cathepsin L group. The permeability of BBB was judged. The expressions of cathepsin L in astrocytes and claudin-1 in BMECs were detected using immunohistochemistry method and western blot assay. RESULTS: The permeability of BBB models was higher in the 39°C group than in the sham group. The cathepsin L expression in astrocytes was higher in the 39°C group than in the sham group (P<0.01), whereas the claudin-1 expression in BMECs was lower in the 39°C group than in the sham group (P<0.01). The claudin-1 expression in BMECs was significantly lower in the 37°C+cathepsin L group than in the sham group (P<0.01). At the same time point, the claudin-1 expression in BMECs was significantly lower in the 39°C+cathepsin L group than in the 37°C+cathepsin L group (P<0.01). CONCLUSION: Hyperthermia can probably decrease claudin-1 expression in BMECs by upregulating cathepsin L expression in astrocytes in a BBB model in vitro.

Muscadine grape skin extract can antagonize Snail-cathepsin L-mediated invasion, migration and osteoclastogenesis in prostate and breast cancer cells.

To develop new and effective chemopreventive agents against bone metastasis, we assessed the effects of muscadine grape skin extract (MSKE), whose main bioactive component is anthocyanin, on bone turnover, using prostate and breast cancer cell models overexpressing Snail transcription factor. MSKE has been shown previously to promote apoptosis in prostate cancer cells without affecting normal prostate epithelial cells. Snail is overexpressed in prostate and breast cancer, and is associated with increased invasion, migration and bone turnover/osteoclastogenesis. Cathepsin L (CatL) is a cysteine cathepsin protease that is overexpressed in cancer and involved in bone turnover. Snail overexpression in prostate (LNCaP, ARCaP-E) and breast (MCF-7) cancer cells led to increased CatL expression/activity and phosphorylated STAT-3 (pSTAT-3), compared to Neo vector controls, while the reverse was observed in C4-2 (the aggressive subline of LNCaP) cells with Snail knockdown. Moreover, CatL expression was higher in prostate and breast tumor tissue compared to normal tissue. MSKE decreased Snail and pSTAT3 expression, and abrogated Snail-mediated CatL activity, migration and invasion. Additionally, Snail overexpression promoted osteoclastogenesis, which was significantly inhibited by the MSKE as effectively as Z-FY-CHO, a CatL-specific inhibitor, or osteoprotegerin, a receptor activator of nuclear factor kappa B ligand (RANKL) antagonist. Overall, these novel findings suggest that Snail regulation of CatL may occur via STAT-3 signaling and can be antagonized by MSKE, leading to decreased cell invasion, migration and bone turnover. Therefore, inhibition using a natural product such as MSKE could potentially be a promising bioactive compound for bone metastatic cancer.

Identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and Ebola, Hendra, and Nipah viruses by using a novel high-throughput screening assay.

UNLABELLED: Severe acute respiratory syndrome coronavirus (SARS-CoV) and Ebola, Hendra, and Nipah viruses are members of different viral families and are known causative agents of fatal viral diseases. These viruses depend on cathepsin L for entry into their target cells. The viral glycoproteins need to be primed by protease cleavage, rendering them active for fusion with the host cell membrane. In this study, we developed a novel high-throughput screening assay based on peptides, derived from the glycoproteins of the aforementioned viruses, which contain the cathepsin L cleavage site. We screened a library of 5,000 small molecules and discovered a small molecule that can inhibit the cathepsin L cleavage of all viral peptides with minimal inhibition of cleavage of a host protein-derived peptide (pro-neuropeptide Y). The small molecule inhibited the entry of all pseudotyped viruses in vitro and the cleavage of SARS-CoV spike glycoprotein in an in vitro cleavage assay. In addition, the Hendra and Nipah virus fusion glycoproteins were not cleaved in the presence of the small molecule in a cell-based cleavage assay. Furthermore, we demonstrate that the small molecule is a mixed inhibitor of cathepsin L. Our broad-spectrum antiviral small molecule appears to be an ideal candidate for future optimization and development into a potent antiviral against SARS-CoV and Ebola, Hendra, and Nipah viruses. IMPORTANCE: We developed a novel high-throughput screening assay to identify small molecules that can prevent cathepsin L cleavage of viral glycoproteins derived from SARS-CoV and Ebola, Hendra, and Nipah viruses that are required for their entry into the host cell. We identified a novel broad-spectrum small molecule that could block cathepsin L-mediated cleavage and thus inhibit the entry of pseudotypes bearing the glycoprotein derived from SARS-CoV or Ebola, Hendra, or Nipah virus. The small molecule can be further optimized and developed into a potent broad-spectrum antiviral drug.

Initial evaluation of the antitumour activity of KGP94, a functionalized benzophenone thiosemicarbazone inhibitor of cathepsin L.

Kinetic analysis of the mode of inhibition of cathepsin L by KGP94, a lead compound from a privileged library of functionalized benzophenone thiosemicarbazone derivatives, demonstrated that it is a time-dependent, reversible, and competitive inhibitor of the enzyme. These results are consistent with the formation of a transient covalent bond, and are supported by molecular modeling that places the thiocarbonyl of the inhibitor in proximity to the thiolate moiety of the enzyme active site Cys25. KGP94 significantly decreased the activity of cathepsin L toward human type I collagen, and impeded both migration and invasion of MDA-MB-231 human breast cancer cells. Growth retardation was achieved in vivo against both recently implanted and established tumours using a C3H mouse mammary carcinoma model.

Modulation of blood cell gene expression by DHA supplementation in hypertriglyceridemic men.

Our previous study with docosahexaenoic acid (DHA) supplementation to hypertriglyceridemic men showed that DHA reduced several risk factors for cardiovascular disease, including the plasma concentration of inflammatory markers. To determine the effect of DHA supplementation on the global gene expression pattern, we performed Affymetrix GeneChip microarray analysis of blood cells [treated with lipopolysaccharide (LPS) or vehicle] drawn before and after the supplementation of DHA from the hypertriglyceridemic men who participated in that study. Genes that were significantly differentially regulated by the LPS treatment and DHA supplementation were identified. Differential regulation of 18 genes was then verified by quantitative real-time polymerase chain reaction (qRT-PCR). Both microarray and qRT-PCR data showed that DHA supplementation significantly suppressed the expression of low-density lipoprotein (LDL) receptor and cathepsin L1, both of which were also up-regulated by LPS. DHA supplementation also suppressed oxidized LDL (lectin-like) receptor 1 (OLR1). However, LPS did not induce OLR1 mRNA expression. Enrichment with Gene Ontology categories demonstrated that the genes related to transcription factor activity, immunity, host defense and inflammatory responses were inversely regulated by LPS and DHA. These results provide supporting evidence for the anti-inflammatory effects of DHA supplementation, and reveal previously unrecognized genes that are regulated by DHA and are associated with risk factors of cardiovascular diseases.

The combined effect of electrical stimulation and high-load isometric contraction on protein degradation pathways in muscle atrophy induced by hindlimb unloading.

High-load isometric exercise is considered an effective countermeasure against muscle atrophy, but therapeutic electrical stimulation for muscle atrophy is often performed without loading. In the present study, we investigated the combined effectiveness of electrical stimulation and high-load isometric contraction in preventing muscle atrophy induced by hindlimb unloading. Electrical stimulation without loading resulted in slight attenuation of muscle atrophy. Moreover, combining electrical stimulation with high-load isometric contraction enhanced this effect. In electrical stimulation without loading, inhibition of the overexpression of calpain 1, calpain 2, and MuRF-1 mRNA was confirmed. On the other hand, in electrical stimulation with high-load isometric contraction, inhibition of the overexpression of cathepsin L and atrogin-1 mRNA in addition to calpain 1, calpain 2, and MuRF-1 mRNA was confirmed. These findings suggest that the combination of electrical stimulation and high-load isometric contraction is effective as a countermeasure against muscle atrophy.

[Effect of bushen tiaojing recipe and xiaoyao pill on expression of cathepsin-L mRNA in gonadotropin-primed mice].

OBJECTIVE: To study the effect of Bushen Tiaojing Recipe (BTR) and Xiaoyao Pill (XP) on cathepsin-L (Cat-L) mRNA in mice. METHODS: Immature mice were randomly divided into the normal group, the control group, the BTR group and the XP group, three in each group. Cat-L mRNA expression in mice was detected using reverse transcription polymerase chain reaction (RT-PCR) at 0, 4, 8 and 12 h after injecting 5 IU (human chorionic gonadotropin, HCG). RESULTS: Cat-L mRNA expression increased gradually after HCG injection, the relative levels in the control group at 0, 4, 8 and 12 h were 0.066 +/- 0.005, 0.383 +/- 0.045, 0.737 +/- 0.024 and 1.036 +/- 0.073 respectively, comparisons between different time-points showed significant difference (P < 0.01). Compared with the control group, the Cat L mRNA expression was higher at 4 h in both BTR and XP groups (P < 0.01), at 8 h in the XP group (P < 0.05), and at 12 h in BTR group after injecting HCG (P < 0.05). Compared with the control group, cat L mRNA expression showed no statistic difference at 8 h in BTR group and at 12 h in XC group. CONCLUSIONS: BTR promoted the ovulation by enhancing the expression of CatL gene, and that of XP by advancing the peak of CatL gene expression.