Activity Of Cathepsin Research Articles

Effects of ultra high pressure-magnetic field treatment on protein properties and quality characteristics of stored shrimp (LitopenaeusVannamei)

The preservation of ultra high pressure (UHP) and magnetic field (MF) technologies on food have been reported, but the effect of combined treatment (UHP-MF) on aquatic product quality is rarely explored. The purposes of this study were to investigate the effects of UHP-MF treatment on protein properties and quality deterioration of stored Litopenaeus Vannamei. The vacuum-packed shrimps were treated at 200 and 300 MPa for 5 min, then stored in 5 mT MF for 12 days. Results showed the hardness, springiness, chewiness, shear force, L*, a*, b*, and W of muscle were improved by UHP, whereas the texture softening, a* and b* increase, water holding capacity were inhibited during MF storage. On the 12th day, compared with CON group, the cathepsin B, D, H, L activities, TBARS, carbonyl and dityrosine content in UHP300-MF group were reduced by 46.67%, 27.26%, 43.10%, 52.00%, 41.30%, 32.86%, and 30.42%, respectively. Furthermore, UHP-MF treatment slow down the accumulation of surface hydrophobicity, myofibril fragmentation index, and trichloroacetic acid-soluble peptide content. Generally, UHP-MF treatment inhibited lipid oxidation, weakened cathepsin activities, delayed protein oxidation and degradation of stored shrimp, retarded quality deterioration and prolonged shelf life of stored shrimp.

346 (PB126) - Preventing cystatin F proteolytic activation by cathepsin V increases cytotoxicity of effector immune cells

Background: Granule-dependent cytotoxicity of natural killer cells and cytotoxic T lymphocytes is regulated by the proteolytic network. Cysteine cathepsins C, H and L are needed for the activation of granzymes and perforin, which initiate the killing of target cancer cells. Activity of cathepsins C, H and L is inhibited by cystatin F. Therefore, cystatin F can negatively impact the cytotoxicity of immune cells. Activity of cystatin F is regulated by expression levels, N-glycosylation and proteolytic activation.

Effects of Ultra-High Pressure on Endogenous Enzyme Activities, Protein Properties, and Quality Characteristics of Shrimp (Litopenaeus vannamei) during Iced Storage.

The present study aimed to explore the effects of ultra-high pressure (UHP) on the cathepsin (B, D, H, and L) activities, protein oxidation, and degradation properties as well as quality characteristics of iced shrimp (Litopenaeus vannamei). Fresh shrimps were vacuum-packed, treated with UHP (100–500 MPa for 5 min), and stored at 0 °C for 15 days. The results showed that the L* (luminance), b* (yellowness), W (whiteness), ΔE (color difference), hardness, shear force, gumminess, chewiness, and resilience of shrimp were significantly improved by UHP treatment. Moreover, the contents of surface hydrophobicity, myofibril fragmentation index (MFI), trichloroacetic acid (TCA)-soluble peptides, carbonyl, dityrosine, and free sulfhydryl of myofibrillar protein (MP) were significantly promoted by UHP treatment. In addition, UHP (above 300 MPa) treatment enhanced the mitochondrial membrane permeability but inhibited the lysosomal membrane stability, and the cathepsin (B, D, H, and L) activities. UHP treatment notably inhibited the activities of cathepsins, delayed protein oxidation and degradation, as well as texture softening of shrimp during storage. Generally, UHP treatment at 300 MPa for 5 min effectively delayed the protein and quality deterioration caused by endogenous enzymes and prolonged the shelf life of shrimp by 8 days.

Inhibitory prodrug mechanism for cysteine cathepsin-targeted self-controlled drug release

Tumour-associated macrophages (TAMs) support tumour development and have emerged as important regulators of therapeutic response to cytostatic agents. To target TAMs, we have developed a novel drug delivery approach which induces drug release as it inhibits cysteine cathepsin activity. This inhibitory prodrug (IPD) approach establishes a self-regulated system where drug release stops after all cysteine cathepsins are inhibited. This could improve the therapeutic window for drugs with severe side effects. We demonstrate and characterise this self-regulation concept with a fluorogenic IPD model. Next, we applied this IPD strategy to deliver cytotoxic drugs, as doxorubicin and monomethyl auristatin E, which are efficiently released and dose-dependently eliminate RAW264.7 macrophages. Lastly, by exploiting the increased cathepsin activity in TAM-like M2-polarised primary macrophages, we show that IPD-Dox selectively eliminates M2 over M1 macrophages. This demonstrates the potential of our IPD strategy for selective drug delivery and modulation of the tumour microenvironment.

Proteolysis and changes in meat quality of chicken pectoralis major and iliotibialis muscles in relation to muscle fiber type distribution

The proteolysis trends and meat quality of the chicken pectoralis major (PM) and iliotibialis (IL) muscles stored at 4°C for 7 d were investigated. After 7 d of storage, the purge loss was higher (P < 0.05) in PM than in IL muscle. The difference in the composition of muscle fibers between PM (100% fast type) and IL (88.85% fast and 11.15% slow types) resulted in differences in proteolysis. Fructose-bisphosphate aldolase, troponin I, myosin heavy chain, and malate dehydrogenase exhibited the same tendencies, but pyruvate kinase, creatine kinase, L-lactate dehydrogenase, and triosephosphate isomerase exhibited different tendencies in the 2 muscles. The activity of cathepsin B was higher in PM than in IL during storage (P < 0.05). These results indicate that the proteolysis trend and changes in meat quality during cold storage are dependent on the different muscle fiber characteristics.

CST6 suppresses osteolytic bone disease in multiple myeloma by blocking osteoclast differentiation.

Osteolytic bone disease is a hallmark of multiple myeloma (MM). A significant fraction (~20%) of MM patients do not develop osteolytic lesions (OLs). The molecular basis for the absence of bone disease in MM is not understood. We combined PET-CT and gene expression profiling (GEP) of purified BM CD138+ MM cells from 512 newly diagnosed MM patients to reveal that elevated expression of cystatin M/E (CST6) was significantly associated with the absence of OL in MM. An enzyme-linked immunosorbent assay revealed a strong correlation between CST6 levels in BM serum/plasma and CST6 mRNA expression. Both recombinant CST6 protein and BM serum from patients with high CST6 significantly inhibited the activity of the osteoclast-specific protease cathepsin K and blocked osteoclast differentiation and function. Recombinant CST6 inhibited bone destruction in ex vivo and in vivo myeloma models. Single-cell RNA-Seq showed that CST6 attenuates polarization of monocytes to osteoclast precursors. Furthermore, CST6 protein blocks osteoclast differentiation by suppressing cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 following RANKL stimulation. Secretion by MM cells of CST6, an inhibitor of osteoclast differentiation and function, suppresses osteolytic bone disease in MM and probably other diseases associated with osteoclast-mediated bone loss.

A LRRK2/dLRRK-mediated lysosomal pathway that contributes to glial cell death and DA neuron survival.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease. A plethora of evidence has indicated a role for LRRK2 in endolysosomal trafficking in neurons, while LRRK2 function in glia, although highly expressed, remains largely unknown. Here, we present evidence that LRRK2/dLRRK mediates a lysosomal pathway that contributes to glial cell death and the survival of dopaminergic (DA) neurons. LRRK2/dLRRK knockdown in the immortalized microglia or flies results in enlarged and swelling lysosomes fewer in number. These lysosomes are less mobile, wrongly acidified, exhibit defective membrane permeability and reduced activity of the lysosome hydrolase cathepsin B. In addition, LRRK2/dLRRK depletion causes glial apoptosis, DA neurodegeneration, and locomotor deficits in an age-dependent manner. Taken together, these findings demonstrate a functional role of LRRK2/dLRRK in regulating the glial lysosomal pathway; deficits in lysosomal biogenesis and function linking to glial apoptosis potentially underlie the mechanism of DA neurodegeneration, providing insights on LRRK2/dLRRK function in normal and pathological brains.

Acid ceramidase targeting pyruvate kinase affected trypsinogen activation in acute pancreatitis

BackgroundAcute pancreatitis is the sudden inflammation of the pancreas. Severe cases of acute pancreatitis are potentially fatal and have no specific treatment available. Premature trypsinogen activation could initiate acute pancreatitis. However, the mechanism underlying premature trypsinogen activation is not fully understood.MethodsIn this research, a primary pancreatic acinar cell or mouse acute pancreatitis model was constructed. The effect of acid ceramidase (ASAH1), which is responsible for sphingosine production, was investigated in trypsinogen activation in vitro and in vivo. Meanwhile, the proteins regulating ASAH1 or binding to sphingosine were also detected by co-immunoprecipitation followed by mass spectrometry.ResultsThe results showed that ASAH1 increased in acute pancreatitis. Increased ASAH1 promoted the activation of trypsinogen and cathepsin B. On the contrary, ASAH1 downregulation inhibited trypsinogen and cathepsin B. Meanwhile, ASAH1 regulated the activity of trypsin and cathepsin B through sphingosine. Additionally, E3 ligase Mind bomb homolog 1 (MIB1) decreased in acute pancreatitis resulting in the decreased binding between MIB1 and ASAH1. Exogenous MIB1 diminished the elevation in trypsin activity induced by acute pancreatitis inducer. ASAH1 increased owing to the inhibition of the proteasome degradation by MIB1. In acute pancreatitis, sphingosine was found to bind to pyruvate kinase. Pyruvate kinase activation could reduce trypsinogen activation and mitochondrial reactive oxygen species (ROS) production induced by sphingosine.ConclusionsIn conclusion, during the process of acute pancreatitis, MIB1 downregulation led to ASAH1 upregulation, resulting in pyruvate kinase inhibition, followed by trypsinogen activation.

Enzyme-Activatable Chemokine Conjugates for In Vivo Targeting of Tumor-Associated Macrophages.

Increased levels of tumor-associated macrophages (TAMs) are indicators of poor prognosis in most cancers. Although antibodies and small molecules blocking the recruitment of macrophages to tumors are under evaluation as anticancer therapies, these strategies are not specific for macrophage subpopulations. Herein we report the first enzyme-activatable chemokine conjugates for effective targeting of defined macrophage subsets in live tumors. Our constructs exploit the high expression of chemokine receptors (e.g., CCR2) and the activity of cysteine cathepsins in TAMs to target these cells selectively over other macrophages and immune cells (e.g., neutrophils, T cells, B cells). Furthermore, we demonstrate that cathepsin-activatable chemokines are compatible with both fluorescent and therapeutic cargos, opening new avenues in the design of targeted theranostic probes for immune cells in the tumor microenvironment.

Enzyme-Activatable Chemokine Conjugates for In Vivo Targeting of Tumor-Associated Macrophages.

Increased levels of tumor-associated macrophages (TAMs) are indicators of poor prognosis in most cancers. Although antibodies and small molecules blocking the recruitment of macrophages to tumors are under evaluation as anticancer therapies, these strategies are not specific for macrophage subpopulations. Herein we report the first enzyme-activatable chemokine conjugates for effective targeting of defined macrophage subsets in live tumors. Our constructs exploit the high expression of chemokine receptors (e.g., CCR2) and the activity of cysteine cathepsins in TAMs to target these cells selectively over other macrophages and immune cells (e.g., neutrophils, T cells, B cells). Furthermore, we demonstrate that cathepsin-activatable chemokines are compatible with both fluorescent and therapeutic cargos, opening new avenues in the design of targeted theranostic probes for immune cells in the tumor microenvironment.

An optimized method of extracting and quantifying active Neutrophil serine proteases from human whole blood cells.

PurposeNeutrophil serine proteases (NSPs) are implicated in numerous inflammatory diseases. Thus, a robust methodology to monitor and quantify NSPs is important to study disease progression and evaluate the effect of pharmacological interventions. A comparison of the various methods used to extract NSPs from neutrophil granulocytes has not been published, providing the impetus to conduct this method optimization and comparison study.MethodsTwo NSP recovery methodologies were evaluated on samples from five human donors: zymosan stimulation and cell pellet extraction. For the zymosan stimulation method, 1 mL donor blood was added to zymosan and samples were incubated at 37°C for 30 min while shaking. Samples were then centrifuged, and the plasma was collected for quantitation of NSP activity. For the cell pellet extraction procedure, 2 mL whole blood samples were centrifuged into white blood cell pellets following red blood cell lysis. To each pellet, three sequential lysis steps were performed using either 0.05% Nonidet P-40 Substitute (NP40) or 0.02% Triton X-100 lysis buffers under agitation followed by centrifugation. NSP activities were quantified using an exogenous peptide substrate specific to each of the three NSPs being analyzed: neutrophil elastase, cathepsin G, and proteinase 3.Results and discussionThe zymosan stimulation method resulted in lower recovery of active NSPs and was unable to stimulate significant release of active cathepsin G. In contrast, the NP40 pellet extraction method showed consistent inter-donor NSP release with greater recoveries of active NSPs than the Triton method or the zymosan stimulation method. Overall, the pellet extraction procedure provided 13.3-fold greater recovery of active neutrophil elastase, 283-fold greater recovery of active cathepsin G, and 2.9-fold greater recovery of active proteinase 3 than the zymosan method.ConclusionThe NP40 cell pellet extraction method resulted in greater extraction of active NSPs compared to the other methods investigated here, which may allow for a more accurate and complete biomarker profile when evaluating human clinical samples.

Effect of ultra-high pressure on the relationship between endogenous proteases and protein degradation of Yesso scallop (Mizuhopecten yessoensis) adductor muscle during iced storage.

This study aimed to explore the effect of ultra-high pressure (UHP) treatment (100-500MPa, 5min, 15±1℃) on the relationship between endogenous proteases and protein degradation of Yesso scallop (Mizuhopecten yessoensis) adductor muscle during iced storage for 28days. Our findings showed that the UHP treatment kept the water holding capacity stable, increased the hardness and decreased the springiness of scallop adductor muscle during iced storage. 400 and 500MPa UHP treatments caused protein denaturation and oxidation significantly, decreased protein degradation rate and inhibited the activities of endogenous proteases. According to the correlation analysis, the activities of cathepsin B, D, H, L, calpain and serine protease were positively correlated with TCA-soluble peptides. The activities of endogenous proteases were significantly correlated with protein degradation. Therefore, the effect of UHP on endogenous protease caused the protein degradation rate to slow down and prevented the texture deterioration in scallops.

Identification of a Novel Mutation in the Cathepsin K (CTSK) Gene in Iranian Patients with Pycnodysostosis

Background: Pycnodysostosis is a rare autosomal recessive skeletal dysplasia, which is characterized by various skeletal malformations and distinctive facial characteristics. The disorder is caused by mutations in CTSK gene at chromosomal region 1q21, which encodes a lysosomal cysteine protease, cathepsin K.&#x0D; Purpose: To describe the clinical, radiological, and molecular findings in a consanguineous Iranian family with 2 affected individuals demonstrating pycnodysostosis.&#x0D; Methods: Whole exome sequencing (WES) was utilized to detect disease-causing variants. To prove the impact of the identified variants, in silico analysis using SIFT and PROVEAN web servers were carried out. The effects of the novel mutation on the protein structure was evaluated by Chimera software using normal and mutated structures derived from SWISS model web server.&#x0D; Results: We identified the novel homozygous mutation, c.830C&gt;A, in exon 7 of the CTSK gene. This missense mutation leads to the substitution of the amino acid alanine at position 277 by glutamic acid (p.A277E) in cathepsin K protease. Sanger sequencing analysis confirmed this mutation in the affected individuals and revealed that the parents were heterozygous for this gene mutation. Bioinformatics databases and the variant’s protein structure proved the deleterious effect of this novel mutation on the enzymatic activity of cathepsin K protease.

Cathepsin B Dipeptidyl Carboxypeptidase and Endopeptidase Activities Demonstrated across a Broad pH Range.

Cathepsin B is a lysosomal protease that participates in protein degradation. However, cathepsin B is also active under neutral pH conditions of the cytosol, nuclei, and extracellular locations. The dipeptidyl carboxypeptidase (DPCP) activity of cathepsin B, assayed with the Abz-GIVR↓AK(Dnp)-OH substrate, has been reported to display an acidic pH optimum. In contrast, the endopeptidase activity, monitored with Z-RR-↓AMC, has a neutral pH optimum. These observations raise the question of whether other substrates can demonstrate cathepsin B DPCP activity at neutral pH and endopeptidase activity at acidic pH. To address this question, global cleavage profiling of cathepsin B with a diverse peptide library was conducted under acidic and neutral pH conditions. Results revealed that cathepsin B has (1) major DPCP activity and modest endopeptidase activity under both acidic and neutral pH conditions and (2) distinct pH-dependent amino acid preferences adjacent to cleavage sites for both DPCP and endopeptidase activities. The pH-dependent cleavage preferences were utilized to design a new Abz-GnVR↓AK(Dnp)-OH DPCP substrate, with norleucine (n) at the P3 position, having improved DPCP activity of cathepsin B at neutral pH compared to the original Abz-GIVR↓AK(Dnp)-OH substrate. The new Z-VR-AMC and Z-ER-AMC substrates displayed improved endopeptidase activity at acidic pH compared to the original Z-RR-AMC. These findings illustrate the new concept that cathepsin B possesses DPCP and endopeptidase activities at both acidic and neutral pH values. These results advance understanding of the pH-dependent cleavage properties of the dual DPCP and endopeptidase activities of cathepsin B that function under different cellular pH conditions.

An extracellular matrix biosensing mimetic for evaluating cathepsin as a host target for COVID-19

To combat the new virus currently ravaging the whole world, every possible anti-virus strategy should be explored. As the main strategy of targeting the virus itself is being frustrated by the rapid mutation of the virus, people are seeking an alternative “host targeting” strategy: neutralizing proteins in the human body that cooperate with the virus. The cathepsin family is such a group of promising host targets, the main biological function of which is to digest the extracellular matrix (ECM) to clear a path for virus spreading. To evaluate the potential of cathepsin as a host target, we have constructed a biosensing interface mimicking the ECM, which can detect cathepsin from 3.3 pM to 33 nM with the limit of detection of 1 pM. Based on our quantitative analysis enabled by this biosensing interface, it is clear that patients with background diseases such as chronic inflammation and tumor, tend to have higher cathepsin activity, confirming the potential of cathepsin to serve as a host target for combating COVID-19 virus.

The Key Role of Lysosomal Protease Cathepsins in Viral Infections.

Cathepsins encompass a family of lysosomal proteases that mediate protein degradation and turnover. Although mainly localized in the endolysosomal compartment, cathepsins are also found in the cytoplasm, nucleus, and extracellular space, where they are involved in cell signaling, extracellular matrix assembly/disassembly, and protein processing and trafficking through the plasma and nuclear membrane and between intracellular organelles. Ubiquitously expressed in the body, cathepsins play regulatory roles in a wide range of physiological processes including coagulation, hormone secretion, immune responses, and others. A dysregulation of cathepsin expression and/or activity has been associated with many human diseases, including cancer, diabetes, obesity, cardiovascular and inflammatory diseases, kidney dysfunctions, and neurodegenerative disorders, as well as infectious diseases. In viral infections, cathepsins may promote (1) activation of the viral attachment glycoproteins and entry of the virus into target cells; (2) antigen processing and presentation, enabling the virus to replicate in infected cells; (3) up-regulation and processing of heparanase that facilitates the release of viral progeny and the spread of infection; and (4) activation of cell death that may either favor viral clearance or assist viral propagation. In this review, we report the most relevant findings on the molecular mechanisms underlying cathepsin involvement in viral infection physiopathology, and we discuss the potential of cathepsin inhibitors for therapeutical applications in viral infectious diseases.

Doenitin-1: A novel Kunitz family protein with versatile functions during feeding and reproduction of the tick Haemaphysalis doenitzi.

As obligate blood-feeding ectoparasites, ticks secrete a great diversity of antithrombin molecules during feeding. In this study, a novel antithrombin gene named Doenitin-1 was characterized from the tick Haemaphysalis doenitzi. It has an open reading frame size of 426 bp; it encodes 141 amino acids and has a predicted molecular weight of 15.8 kDa. The fibrinogen coagulation test showed that the time of coagulation was increased significantly with increase in rDoenitin-1 protein concentration, and the activated partial thromboplastin time (APTT) and prothrombin time (PT) assays showed that rDoenitin-1 significantly prolonged the coagulation time of APTT, indicating that rDoenitin-1 has an anticoagulant activity in vitro. In addition, rDoenitin-1 presents a significant inhibitory activity in thrombin and cathepsin G. The hemolysis rate of rDoenitin-1 in healthy human blood cells was 4.25%, and no obvious hemolysis activity was observed. The comparison with other life stages shows that the higher expression occurs in adults, and tissue comparison indicated a higher expression in the midgut. The RNAi results indicated that interference of Doenitin-1 significantly reduced the engorgement rate and egg hatchability of H. doenitzi, and that the engorged body weight was slightly reduced. In conclusion, the results suggested that the novel gene Doenitin-1 functions in blood-feeding of H. doenitzi and performs various functions during feeding and reproduction of H. doenitzi. Doenitin-1 may be a potential vaccine candidate for tick control and for developing new antithrombotic drugs in the future.

Reactive oxygen species-dependent upregulation of death receptor, tumor necrosis factor receptor 1, is responsible for theophylline-mediated cytotoxicity in MDA-MB-231 breast cancer cells.

Theophylline, a methylxanthine drug, has been used as a therapy for respiratory diseases. Recently, it has also been shown to have a potential in treating different cancers. Also, it has shown promising results in clinical trials for AML in combination therapy. Subsequently, studies have shown theophylline to kill breast cancer cells but not normal breast cells. Therefore, in this study, we have explored the molecular mechanism underlying the cytotoxic effect of theophylline on breast cancer cells. Theophylline-treated cancer cells were analyzed for the transcript and protein expression of candidate apoptotic genes such as TNFR1, caspase-8, -9, -3 using qPCR and immunoblotting, respectively. Cell viability and apoptosis was measured in the presence or absence of TNFR1 inhibitor, R7050, using AO/EtBr staining and MTT assay, respectively. Similarly, oxidative stress was studied by analyzing ROS in the presence or absence of ROS inhibitor, NAC, using DCFDA assay. Theophylline caused reduced cell viability in cancer but not normal cells. Theophylline-treated breast cancer cells showed increased expression of death receptor, TNFR1, along with elevated levels of active caspase-8, -9 and -3. Inhibition of TNFR1 reduced caspase-dependent apoptosis even in the presence of theophylline. Theophylline further caused increased ROS generation, inhibition of which resulted in reduced TNFR1-mediated apoptosis. Theophylline also increased cathepsin activity, which was reduced on exposure of cells to TNFR1 inhibitor, R7050. We conclude that ROS-mediated activation of TNFR1 is responsible for caspase-3 and cathepsin-dependent cell death in breast cancer cells on exposure to theophylline.

Effect of sodium replacement on the quality characteristics of pastırma (a dry-cured meat product)

This study aims to investigate the effect of sodium replacement on the quality characteristics of pastırma. For this purpose, pastırma production with four different salt mixtures (I. 100 % NaCl; II. 50 % NaCl + 50 % KCl; III. 40 % NaCl + 40 % KCl + 20 % CaCl2; IV. 30 % NaCl + 40 % KCl + 20 % CaCl2 + 10 % MgCl2) were carried out using traditional method. The use of different salt mixtures for pastırma had no statistically significant effect on the microbial counts and residual nitrite of the final product. The a* and b* values were affected by this treatment. The salt mixture containing CaCl2 or CaCl2 + MgCl2 significantly decreased the pH values and sensory scores. The eighteen volatile compounds were affected by the salt mixtures. Na+, K+, Ca2+, and Mg2+ had a certain increase depending on their ratio in the salt mixtures. The highest Cathepsin activity in all pastırma samples was observed in Cathepsin B + L. The salt mixture with NaCl + KCl + CaCl2 + MgCl2 increased acid lipase activity. However, this mixture had no significant effect on neutral lipase activity.

Preclinical evaluation of a novel cathepsin-activated fluorescent probe VGT-309 for identification of ovarian cancer in a mouse model (248)

<b>Objectives:</b> Cathepsin activity is upregulated in many solid cancers. VGT-309 is a novel near-infrared cathepsin-activated fluorescent probe that covalently binds to cysteine cathepsins, localizing the fluorescent signal to sites of high cathepsin activity. The goal was to determine the utility of VGT-309 in a mouse model of ovarian cancer. <b>Methods:</b> NCG mice with luciferase-labeled ovarian cancer cell lines (OVCAR3-ffLuc, OVCAR8-ffLuc), implanted subcutaneously (SQ) (<i>n</i>=6) or intraperitoneally (IP) (<i>n</i>=6), were injected with intravenous VGT-309 (2 mg/kg) and sacrificed after 24 hours for fluorescence imaging and anatomical dissection. Near-infrared (NIR) and luciferase imaging were performed on tumors and internal organs with the Pearl Imaging system. Image Studio Lite was used to quantitate fluorescence signals. Background muscle signal was compared with specimen signal from various sized tumors and organs to establish signal-to-background ratio (SBR) and an average was taken. <b>Results:</b> Twenty-four hours after mice were injected with VGT-309, we were able to visualize NIR fluorescence signal in SQ mouse models for OVCAR3 and OVCAR8 <i>in situ</i>. After dissecting tumors and organs, we compared NIR fluorescence activity with luciferase activity. The fluorescence signal from VGT-309 overlapped with the luciferase signal, indicating that VGT-309 was able to specifically image cancerous tissue in both SQ and IP models. There was high SBR noted for the tumors from the SQ tumor models (SBR 14 for OVCAR8 and 14.3 for OVCAR3), as compared to the peritoneum (SBR 0.7 for OVCAR8 and 0.6 for OVCAR3). Similar findings were also noted for the IP tumors (SBR 10.1 for OVCAR8 and 12.6 for OVCAR3) as compared to the peritoneum (SBR 1.4 for OVCAR8 and 0.6 for OVCAR3). <b>Conclusions:</b> VGT-309 is a cathepsin-based activatable fluorescence probe that can visualize ovarian cancer cells in an animal model with a high signal-to-background ratio as compared to the peritoneum. VGT-309 is currently in early phase clinical trials for lung cancer. These data would encourage clinical testing of VGT-309 as a tool for the intraoperative identification of ovarian cancers.