Structural insights into the potential binding sites of Cathepsin D using molecular modelling techniques.

Alzheimer's disease (AD) is the most prevalent type of dementia caused by the accumulation of amyloid beta (Aß) peptides. The extracellular deposition of Aß peptides in human AD brain causes neuronal death. Therefore, it has been found that Aß peptide degradation is a possible therapeutic target for AD. CathD has been known to breakdown amyloid beta peptides. However, the structural role of CathD is not yet clear. Hence, for the purpose of gaining a deeper comprehension of the structure of CathD, the present computational investigation was performed using virtual screening technique to predict CathD's active site residues and substrate binding mode. Ligand-based virtual screening was implemented on small molecules from ZINC database against crystal structure of CathD. Further, molecular docking was utilised to investigate the binding mechanism of CathD with substrates and virtually screened inhibitors. Localised compounds obtained through screening performed by PyRx and AutoDock 4.2 with CathD receptor and the compounds having highest binding affinities were picked as; ZINC00601317, ZINC04214975 and ZINCC12500925 as our top choices. The hydrophobic residues Viz. Gly35, Val31, Thr34, Gly128, Ile124 and Ala13 help stabilising the CathD-ligand complexes, which in turn emphasises substrate and inhibitor selectivity. Further, MM-GBSA approach has been used to calculate binding free energy between CathD and selected compounds. Therefore, it would be beneficial to understand the active site pocket of CathD with the assistance of these discoveries. Thus, the present study would be helpful to identify active site pocket of CathD, which could be beneficial to develop novel therapeutic strategies for the AD.

Myricitrin from bayberry as a potential inhibitor of cathepsin-D: Prospects for squamous lung carcinoma prevention.

Cathepsin-D (CATD) inhibitors' design and development drawn interest due to their potential therapeutic applications in managing different cancer types, including lung cancer. This study investigated myricitrin, a flavonol-3-O-rhamnoside, for its binding affinity to CATD. Molecular docking experiments revealed a strong binding affinity (-7.8 kcal/mol). Molecular dynamics (MD) simulation confirmed the complex's stability, while enzyme activity studies showed inhibitory concentration (IC50) of 35.14 ± 6.08 µM (in cell-free) and 16.00 ± 3.48 µM (in cell-based) test systems. Expression analysis indicated downregulation of CATD with a fold change of 1.35. Myricitrin demonstrated antiproliferative effects on NCIH-520 cells [IC50: 64.11 µM in Sulphorhodamine B (SRB), 24.44 µM in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], but did not affect healthy CHANG cells. It also prolonged the G2/M phase (at 10 µM: 1.19-fold; at 100 µM: 1.13-fold) and increased sub-diploid population by 1.35-fold. Based on the analysis done using SwissADME program, it is predicted that myricitrin is not a cytochrome p450s (CYPs) inhibitor, followed the rule of Ghose and found not permeable to the blood-brain barrier (BBB) which suggests it as a safe molecule. In summary, the experimental findings may establish the foundation for myricitrin and its analogues to be used therapeutically in CATD-mediated lung cancer prevention.

Oxidation of Cathepsin D by Hydroxy Radical: Its Effect on Enzyme Structure and Activity against Myofibrillar Proteins Extracted from Coregonus peled.

In this study, cathepsin D was oxidized in vitro with different concentrations of H2O2, and the activity, structure, and extent of myofibrillar protein degradation by oxidized cathepsin D were evaluated. The sulfhydryl content of cathepsin D decreased to 9.20% after oxidation, while the carbonyl content increased to 100.06%. The ß-sheet in the secondary structure altered due to oxidation as well. The changes in the intrinsic fluorescence and UV absorption spectra indicated that oxidation could cause swelling and aggregation of cathepsin D molecules. The structure of cathepsin D could change its activity, and the activity was highest under 1 mM H2O2. Cathepsin D could degrade myofibrillar proteins in different treatment groups, and the degree of degradation is various. Therefore, this study could provide a scientific basis for the mechanism of interaction among hydroxyl radical oxidation, cathepsin D, and MP degradation.

Cathepsin H: Molecular characteristics and clues to function and mechanism.

Cathepsin H (CatH) is a lysosomal cysteine protease with a unique aminopeptidase activity that is extensively expressed in the lung, pancreas, thymus, kidney, liver, skin, and brain. Owing to its specific enzymatic activity, CatH has critical effects on the regulation of biological behaviours of cancer cells and pathological processes in brain diseases. Moreover, a neutral pH level is optimal for CatH activity, so it is expected to be active in the extra-lysosomal and extracellular space. In the present review, we describe the expression, maturation, and enzymatic properties of CatH, and summarize the available experimental evidence that mechanistically links CatH to various physiological and pathological processes. Finally, we discuss the challenges and potentials of CatH inhibitors in CatH-induced disease therapy.

Pepstatin-Based Probes for Photoaffinity Labeling of Aspartic Proteases and Application to Target Identification.

Aspartic proteases are a small class of proteases implicated in a wide variety of human diseases. Covalent chemical probes for photoaffinity labeling (PAL) of these proteases are underdeveloped. We here report a full on-resin synthesis of clickable PAL probes based on the natural product inhibitor pepstatin incorporating a minimal diazirine reactive group. The position of this group in the inhibitor determines the labeling efficiency. The most effective probes sensitively detect cathepsin D, a biomarker for breast cancer, in cell lysates. Moreover, through chemical proteomics experiments and deep learning algorithms, we identified sequestosome-1, an important player in autophagy, as a direct interaction partner and substrate of cathepsin D.

Morin hydrate attenuates adenine-induced renal fibrosis via targeting cathepsin D signaling.

Lysosomal proteases such as cathepsins B, D, L, and K can regulate the process of fibrosis in most of the organs. However, the role of cathepsin D (CATD) in kidney fibrosis and corresponding chronic kidney disease (CKD) is still unknown. We investigated whether CATD immunomodulation using morin hydrate (MH) can attenuate kidney fibrosis in CKD. Here, CKD was developed by an oral dosage of adenine (AD) in the mice model. Histopathological detection using H & E and Oil-Red-O staining revealed tissue deposition. An escalation in serum creatinine, albumin, and blood urea nitrogen (BUN) revealed a failure in kidney function. An increase in fibrosis was determined using protein analysis and mRNA analysis of MMP-9 and MMP-2 respectively. Both immunoblot analysis and histological analysis indicated that MH immunomudulated CATD expression in AD treated kidneys. With docking analysis, we found MH can bind with the catalytic core of CATD with binding efficiency of -6.83 kcal/mol. Further, MH prevented AD mediated fibrosis by reducing collagen fragmentation as evidenced by the decrease in MMP-2 (P < 0.05) and MMP-9 (P < 0.001) protein levels. MH lowered the levels of inflammation by reducing the AD enhanced expression of MCP-1 and COX-2 nearly threefold. MH treatment increased body weight, enhance kidney function, and improved survival by nearly 150% compared to AD treated mice. CATD inactivation by MH after AD treatment resulted in decreased ECM degradation, fibrosis, and inflammation which resulted in improved renal function and survival.

Biomimetic Macrocyclic Inhibitors of Human Cathepsin D: Structure-Activity Relationship and Binding Mode Analysis.

Human cathepsin D (CatD), a pepsin-family aspartic protease, plays an important role in tumor progression and metastasis. Here, we report the development of biomimetic inhibitors of CatD as novel tools for regulation of this therapeutic target. We designed a macrocyclic scaffold to mimic the spatial conformation of the minimal pseudo-dipeptide binding motif of pepstatin A, a microbial oligopeptide inhibitor, in the CatD active site. A library of more than 30 macrocyclic peptidomimetic inhibitors was employed for scaffold optimization, mapping of subsite interactions, and profiling of inhibitor selectivity. Furthermore, we solved high-resolution crystal structures of three macrocyclic inhibitors with low nanomolar or subnanomolar potency in complex with CatD and determined their binding mode using quantum chemical calculations. The study provides a new structural template and functional profile that can be exploited for design of potential chemotherapeutics that specifically inhibit CatD and related aspartic proteases.

A Novel Dual Fluorochrome Near-Infrared Imaging Probe for Potential Alzheimer's Enzyme Biomarkers-BACE1 and Cathepsin D.

A molecular imaging probe to fluorescently image the ß-site of the amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) and cathepsin D (CatD) enzymes associated with Alzheimer's disease (AD) was designed and synthesized. This imaging probe was built upon iron oxide nanoparticles (cross-linked dextran iron oxide nanoparticles, or CLIO). Peptide substrates containing a terminal near-infrared fluorochrome (fluorophore emitting at 775 nm for CatD or fluorophore emitting at 669 nm for BACE1) were conjugated to the CLIO nanoparticles. The CatD substrate contained a phenylalanine-phenylalanine cleavage site more specific to CatD than BACE1. The BACE1 substrate contained the sequence surrounding the leucine-asparagine cleavage site of the BACE1 found in the Swedish mutation of APP, which is more specific to BACE1 than CatD. These fluorescently-labeled peptide substrates were then conjugated to the nanoparticle. The nanoparticle probes were purified by gel filtration, and their fluorescence intensities were determined using a fluorescence plate reader. The CatD peptide substrate demonstrated a 15.5-fold increase in fluorescence when incubated with purified CatD enzyme, and the BACE1 substrate exhibited a 31.5-fold increase in fluorescence when incubated with purified BACE1 enzyme. Probe specificity was also demonstrated in the human H4 neuroglioma cells and the H4 cells stably transfected with BACE1 in which the probe monitored enzymatic cleavage. In the H4 and H4-BACE1 cells, BACE1 and active CatD activity increased, an occurrence that was reflected in enzyme expression levels as determined by immunoblotting. These results demonstrate the applicability of this probe for detecting potential Alzheimer's enzyme biomarkers.

Comprehensive Exploration of Target-specific Ligands Using a Graph Convolution Neural Network.

Machine learning approaches are widely used to evaluate ligand activities of chemical compounds toward potential target proteins. Especially, exploration of highly selective ligands is important for the development of new drugs with higher safety. One difficulty in constructing well-performing model predicting such a ligand activity is the absence of data on true negative ligand-protein interactions. In other words, in many cases we can access to plenty of information on ligands that bind to specific protein, but less or almost no information showing that compounds don't bind to proteins of interest. In this paper, we suggested an approach to comprehensively explore candidates for ligands specifically targeting toward proteins without using information on the true negative interaction. The approach consists of 4 steps: 1) constructing a model that distinguishes ligands for the target proteins of interest from those targeting proteins that cause off-target effects, by using graph convolution neural network (GCNN); 2) extracting feature vectors after convolution/pooling processes and mapping their principal components in two dimensions; 3) specifying regions with higher density for two ligand groups through kernel density estimation; and 4) investigating the distribution of compounds for exploration on the density map using the same classifier and decomposer. If compounds for exploration are located in higher-density regions of ligand compounds, these compounds can be regarded as having relatively high binding affinity to the major target or off-target proteins compared with other compounds. We applied the approach to the exploration of ligands for ß-site amyloid precursor protein [APP]-cleaving enzyme 1 (BACE1), a major target for Alzheimer Disease (AD), with less off-target effect toward cathepsin D. We demonstrated that the density region of BACE1 and cathepsin D ligands are well-divided, and a group of natural compounds as a target for exploration of new drug candidates also has significantly different distribution on the density map.

Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells.

Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield (16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties.

Selective Proteolysis of α-Lactalbumin by Endogenous Enzymes of Human Milk at Acidic pH.

SCOPE: The use of human milk products is increasing for high-risk infants. Human milk contains endogenous enzymes that comprise a dynamic proteolytic system, yet biological properties of these enzymes and their activities in response to variations including pH within infants are unclear. Human milk has a neutral pH around 7, while infant gastric pH varies from 2 to 6 depending on individual conditions. This study is designed to determine the specificity of enzyme-substrate interactions in human milk as a function of pH. METHODS AND RESULTS: Endogenous proteolysis is characterized by incubating freshly expressed human milk at physiologically relevant pH ranging from 2 to 7 without the addition of exogenous enzymes. Results show that the effects of pH on endogenous proteolysis in human milk are protein-specific. Further, specific interactions between cathepsin D and α-lactalbumin are confirmed. The endogenous enzyme cathepsin D in human milk cleaves α-lactalbumin as the milk pH shifts from 7 to 3. CONCLUSIONS: This study documents that selective proteolysis activated by pH shift is a mechanism for dynamic interactions between human milk and the infant. Controlled proteolysis can guide the use of human milk products based on individual circumstance.

Extraction of Cathepsin D-Like Protease from Neon Flying Squid (Ommastrephes bartramii) Viscera and Application in Antioxidant Hydrolysate Production.

A protease from neon flying squid (Ommastrephes bartramii) viscera (SVCE3(f)) was partially purified by isoelectric solubilization/precipitation combined with ultra-membrane filtration (ISP-UMF). Two protein bands of 45 and 27 KDa were determined by SDS-PAGE assay. The protease characteristic of the protein band of 45 KDa was confirmed using casein zymography analysis. The result of UPLC-ESI-MS/MS suggested that the band of 45 KDa could be a cathepsin D-like protease. This cathepsin D-like protease showed an optimum pH of 3.0 and optimum temperature of 60 °C when casein was used as s substrate. Furthermore, its protease activity was stable at 30-50 °C and under a pH range of 1.0-5.0, maintaining about 60% of its initial activity. SVCE3(f) can digest half-fin anchovy (Setipinna taty) to generate antioxidant hydrolysates (HAHp-SEs). The degree of hydrolysis (DH) of HAHp-SEs increased along with the hydrolysis time and reached stability after 60 min of digestion. HAHp-SEs(30) with relatively lower DH exhibited the highest DPPH radical scavenging activity as compared with other HAHp-SEs. However, a stronger hydroxyl radical scavenging activity and greater reducing power were observed for HAHp-SEs that underwent higher DH. Accordingly, the partially purified cathepsin D-like protease of neon flying squid viscera using ISP-UMF could have potential application in antioxidant hydrolysates production.

Anxa2- and Ctsd-knockout CHO cell lines to diminish the risk of contamination with host cell proteins.

Chinese hamster ovary (CHO) cells have been used as host cells in the production of a range of recombinant therapeutic proteins, including monoclonal antibodies and Fc-fusion proteins. Host cell proteins (HCP) represent impurities that must be removed from therapeutic formulations because of their potential risks for immunogenicity. While the majority of HCP impurities are effectively removed in typical downstream purification processes, clearance of a small population of HCP remains challenging. In this study, we knocked out the Anxa2 and Ctsd genes to assess the feasibility of knockout approaches for diminishing the risk of contamination with HCP. Using the CRISPR/Cas9 system, Anxa2-, and Ctsd-knockout CHO cell lines were successfully established, and we confirmed the complete elimination of the corresponding HCP in cell lysates. Importantly, all knockout cell lines showed similar growth and viability to those of the wild-type control during 8 days of cultivation. Thus, knockout of unrequired genes can reduce contamination with HCP in the production of recombinant therapeutic proteins.

Effect of Temperature and pH on the Secondary Structure and Denaturation Process of Jumbo Squid Hepatopancreas Cathepsin D.

BACKGROUND: Cathepsin D is a lysosomal enzyme that is found in all organisms acting in protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D of some marine organisms has a low thermostability and that it has the ability to have activity at very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and partially characterized. The secondary structure of these enzymes is highly conserved so the role of temperature and pH in the secondary structure and in protein denaturation is of great importance in the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas was determined by means of circular dichroism spectroscopy. OBJECTIVE: In this article, our purpose was to determine the secondary structure of the enzyme and how it is affected by subjecting it to different temperature and pH conditions. METHODS: Circular dichroism technique was used to measure the modifications of the secondary structure of cathepsin D when subjected to different treatments. The methodology consisted in dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step, a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation from the far-UV CD spectra was calculated using K2D Dichroweb software. RESULTS: It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating the enzyme above 70°C maintains a low percentage of α helix and increases ß sheet. Far-UV CD measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly dependent on the heating rate, accompanied by a process of oligomerization of the protein that appears when the sample is heated, and maintained a certain time at this temperature. An amount typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified. CONCLUSION: In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to 4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and follows an irreversible model.

Investigation of cathepsin D-mAb interactions using a combined experimental and computational tool set.

Cathepsin D has been identified as a challenge to remove in downstream bioprocessing of monoclonal antibodies (mAbs) due to interactions with some mAbs. This study focused on investigating the mechanisms of interaction between cathepsin D and two industrial mAbs using a combined experimental and computational approach. Surface plasmon resonance was used to study the impact of pH and salt concentration on these protein-protein interactions. While salt had a moderate effect on the interactions with one of the mAbs, the other mAb demonstrated highly salt-dependent association behavior. Cathepsin D binding to the mAbs was also seen to be highly pH dependent, with operation at pH 9 resulting in a significant decrease in the binding affinity. Protein-protein docking simulations identified three interaction sites on both mAbs; near the complementarity determining region (CDR), in the hinge, and in the CH 3 domain. In contrast, only one face of cathepsin D was identified to interact with all the three sites on the mAbs. Surface property analysis revealed that the binding regions on the mAbs contained strong hydrophobic clusters and were predominantly negatively charged. In contrast, the binding site on cathepsin D was determined to be highly positively charged and hydrophobic, indicating that these protein-protein interactions were likely due to a combination of hydrophobic and electrostatic interactions. Finally, covalent crosslinking coupled with mass spectrometry was used to validate the docking predictions and to further investigate the regions of interaction involved in mAb-cathepsin D binding. A strong agreement was observed between the two approaches, and the CDR loops were identified to be important for cathepsin D interactions. This study establishes a combined experimental and computational platform that can be used to probe mAb-host cell protein (HCP) interactions of importance in biomanufacturing.

Partial Characterization of Two Cathepsin D Family Aspartic Peptidases of Clonorchis sinensis.

Cathepsin D (CatD, EC 3.4.23.5) is a member belonging to the subfamily of aspartic endopeptidases, which are classified into the MEROPS clan AA, family A1. Helminth parasites express a large set of different peptidases that play pivotal roles in parasite biology and pathophysiology. However, CatD is less well known than the other classes of peptidases in terms of biochemical properties and biological functions. In this study, we identified 2 novel CatDs (CsCatD1 and CsCatD2) of Clonorchis sinensis and partially characterized their properties. Both CsCatDs represent typical enzymes sharing amino acid residues and motifs that are tightly conserved in the CatD superfamily of proteins. Both CsCatDs showed similar patterns of expression in different developmental stages of C. sinensis, but CsCatD2 was also expressed in metacercariae. CsCatD2 was mainly expressed in the intestines and eggs of C. sinensis. Sera obtained from rats experimentally infected with C. sinensis reacted with recombinant CsCatD2 beginning 2 weeks after infection and the antibody titers were gradually increased by maturation of the parasite. Structural analysis of CsCatD2 revealed a bilobed enzyme structure consisting of 2 antiparallel ß-sheet domains packed against each other forming a homodimeric structure. These results suggested a plausible biological role of CsCatD2 in the nutrition and reproduction of parasite and its potential utility as a serodiagnostic antigen in clonorchiasis.

2-Aminoquinazolin-4(3H)-one based plasmepsin inhibitors with improved hydrophilicity and selectivity.

2-Aminoquinazolin-4(3H)-ones were previously discovered as perspective leads for antimalarial drug development targeting the plasmepsins. Here we report the lead optimization studies with the aim to reduce inhibitor lipophilicity and increase selectivity versus the human aspartic protease Cathepsin D. Exploiting the solvent exposed area of the enzyme provides an option to install polar groups (R1) the 5-position of 2-aminoquinazolin-4(3H)-one to inhibitors such as carboxylic acid without scarifying enzymatic potency. Moreover, introduction of R1 substituents increased selectivity factors of compounds in this series up to 100-fold for Plm II, IV vs CatD inhibition. The introduction of flap pocket substituent (R2) at 7-postion of 2-aminoquinazolin-4(3H)-one allows to remove Ph group from THF ring without notably impairing Plm inhibitory potency. Based on these findings, inhibitors were developed, which show Plm II and IV inhibitory potency in low nanomolar range and remarkable selectivity against Cathepsin D along with decreased lipophilicity and increased solubility.

Novel Structural Mechanism of Allosteric Regulation of Aspartic Peptidases via an Evolutionarily Conserved Exosite.

Pepsin-family aspartic peptidases are biosynthesized as inactive zymogens in which the propeptide blocks the active site until its proteolytic removal upon enzyme activation. Here, we describe a novel dual regulatory function for the propeptide using a set of crystal structures of the parasite cathepsin D IrCD1. In the IrCD1 zymogen, intramolecular autoinhibition by the intact propeptide is mediated by an evolutionarily conserved exosite on the enzyme core. After activation, the mature enzyme employs the same exosite to rebind a small fragment derived from the cleaved propeptide. This fragment functions as an effective natural inhibitor of mature IrCD1 that operates in a pH-dependent manner through a unique allosteric inhibition mechanism. The study uncovers the propeptide-binding exosite as a target for the regulation of pepsin-family aspartic peptidases and defines the structural requirements for exosite inhibition.

Cathepsin D immobilized capillary reactors for on-flow screening assays.

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (KM = 81.9 ±â€¯7.49 µmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts.

Characterization of a cathepsin D protease from CHO cell-free medium and mitigation of its impact on the stability of a recombinant therapeutic protein.

During purification process development of a recombinant therapeutic protein, an endoproteolytic activity endogenous to the Chinese hamster ovary (CHO) cells and leading to degradation at particular hydrophobic amino acid residues (e.g., Phe and Trp) was observed when processing at acidic pH. The presence of residual levels of protease activity in purified protein batches affected the inherent activity of the product when stored as a solution. To develop a robust purification strategy to minimize this undesirable impact, identification and characterization of this protease was essential to ultimately ensure that a solution formulation was stable for many years. A protease was isolated from CHO cell-free medium (CFM) using a combination of immobilized pepstatin-A agarose chromatography and size exclusion chromatography (SEC). The isolated protease has significant proteolytic activity at pH ∼ 3 to neutral pH and was identified as cathepsin D by mass spectrometry. Analytical SEC, chip-based capillary gel electrophoresis, imaged capillary isoelectric focusing (cIEF), and circular dichroism (CD) spectropolarimetry analyses were performed for additional characterization of the protease. The identification and characterization of this protease enabled the development of a robust purification process by implementation of a controlled temperature inactivation unit operation (heat inactivation) that enabled essentially complete inactivation of the protease, resulting in the production of a stable drug product that had not been possible using column chromatography alone. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:120-129, 2018.