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Cathepsin D (CD) is overexpressed in several types of cancer and constitutes an important biological target. Pepstatin A, a pentapeptide incorporating two non-proteinogenic statin residues, is among the most potent inhibitor of CD but lacks selectivity and suffers from poor bioavailability. Eight analogues of Pepstatin A, were synthesized, replacing residues in P3 or P1 position by non-canonical (S)- and (R)-α-Trifluoromethyl Alanine (TfmAla), (S)- and (R)-Trifluoromethionine (TFM) or non-natural d-Valine. The biological activities of those analogues were quantified on isolated CD and Pepsin by fluorescence-based assay (FRET) and cytotoxicity of the best fluorinated inhibitors was evaluated on SKOV3 ovarian cancer cell line. (R)-TFM based analog of Pepstatin A (compound 6) returned a sub-nanomolar IC50 against CD and an increased selectivity. Molecular Docking experiments could partially rationalize these results. Stabilized inhibitor 6 in the catalytic pocket of CD showed strong hydrophobic interactions of the long and flexible TFM side chain with lipophilic residues of S1 and S3 sub-pockets of the catalytic pocket. The newly synthesized inhibitors returned no cytotoxicity at IC50 concentrations on SKOV3 cancer cells, however the compounds derived from (S)-TfmAla and (R)-TFM led to modifications of cells morphologies, associated with altered organization of F-actin and extracellular Fibronectin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Cathepsin D is an aspartic protease and one of the most abundant proteases. It is overexpressed in many cancers and plays an important role in tumor development, progression, and metastasis. While it is a physiologically intracellular protein, cathepsin D is secreted into the extracellular matrix under pathological conditions, making it an appealing target for drug delivery systems. Here, we present the development and evaluation of a new delivery system for tumor targeting based on immunoliposomes functionalized with pepstatin A-a natural peptide inhibitor of cathepsin D. A lipid tail was added to pepstatin A, enabling its incorporation into the liposomal lipid bilayer. The successful targeting of cathepsin D was confirmed using recombinant cathepsin D and in tumor cell lines, showing the feasibility of this targeting approach and its potential for in vivo use in theragnostic applications.

Periodontitis is an inflammatory disease of tooth-supporting tissues caused by oral bacteria. Periodontal ligament loss and alveolar bone destruction occur in progressive periodontitis. Since gingival crevicular fluids (GCF) reflects the inflammatory environment of the periodontal pocket, it is a very important specimen for developing targets for periodontitis diagnosis. An antibody array was performed using GCF collected from healthy participants and patients with periodontitis to identify the proteolytic enzymes involved in periodontitis. Of 21 targets on the antibody array membrane, kallikrein 6 (KLK6), kallikrein 10 (KLK10), cathepsin A (CathA), and cathepsin D (CathD) showed higher levels in periodontitis GCF than in GCF from healthy participants. Lipopolysaccharide stimulation of Porphyromonas gingivalis (PG-LPS) in immortalized gingival fibroblasts only increased CathD protein levels among the four targets. The substrate cleavage activity of CathD was increased in PG-LPS-treated immortalized gingival fibroblast extract. The PG-LPS-induced substrate cleavage effect was abolished by the CathD inhibitor pepstatin A. Osteoclast formation was promoted by treatment with conditioned media from PG-LPS- treated immortalized gingival fibroblasts but inhibited by the CathD inhibitor pepstatin A. These results suggest that PG-LPS affected the osteoclast formation process by increasing CathD expression in cells around the alveolar bone, thereby participating in periodontitis progression., (© 2023 Scandinavian Division of the International Association for Dental Research. Published by John Wiley & Sons Ltd.)

Pepstatin A reversibly inhibits aspartic acid proteases and minimizes the impact of protease-induced degradation in recombinant protein manufacturing process. Pepstatin A is considered as a process-related impurity and must be characterized and controlled during manufacturing. Herein we describe the development and validation of an LC-MS/MS method for the quantitation of pepstatin A to monitor its robust clearance in vaccine purification process. Analyte extraction from process intermediates was carried out using 10% acetonitrile/water extraction method. Acetyl-pepstatin was used as internal standard (IS). Pepstatin A and IS were resolved on a C18 column using 10 mM ammonium acetate in water and methanol/acetonitrile mobile phase system. A triple quadrupole mass spectrometer operating in the positive electrospray ionization mode with multiple reaction monitoring was used to detect Pepstatin A and IS transitions of m/z 686.5 to 229.3 and 644.5 to 229.3, respectively. The method was validated for specificity, linearity, accuracy, repeatability (precision), intermediate precision, and assay robustness. The assay was linear over the range of calibration standards 0.5-100 ng/mL. The Lower-limit-of-quantification (LLOQ) of the method was 0.50 ng/mL., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

We have previously shown that roflupram (ROF) protects against MPP + -induced neuronal damage in models of Parkinson's disease (PD). Since impaired degradation of α-synuclein (α-syn) is one of the key factors that lead to PD, here we investigated whether and how ROF affects the degradation of α-syn in rotenone (ROT)-induced PD models in vivo and in vitro. We showed that pretreatment with ROF (10 μM) significantly attenuated cell apoptosis and reduced the level of α-syn in ROT-treated SH-SY5Y cells. Furthermore, ROF significantly enhanced the lysosomal function, as evidenced by the increased levels of mature cathepsin D (CTSD) and lysosomal-associated membrane protein 1 (LAMP1) through increasing NAD + /NADH and the expression of sirtuin 1 (SIRT1). Pretreatment with an SIRT1 inhibitor selisistat (SELI, 10 μM) attenuated the neuroprotection of ROF, ROF-reduced expression of α-syn, and ROF-increased expression levels of LAMP1 and mature CTSD. Moreover, inhibition of CTSD by pepstatin A (20 μM) attenuated ROF-reduced expression of α-syn. In vivo study was conducted in mice exposed to ROT (10 mg·kg -1 ·d -1 , i.g.) for 6 weeks; then, ROT-treated mice received ROF (0.5, 1, or 2 mg·kg -1 ·d -1 ; i.g.) for four weeks. ROF significantly ameliorated motor deficits, which was accompanied by increased expression levels of tyrosine hydroxylase, SIRT1, mature CTSD, and LAMP1, and a reduced level of α-syn in the substantia nigra pars compacta. Taken together, these results demonstrate that ROF exerts a neuroprotective action and reduces the α-syn level in PD models. The mechanisms underlying ROF neuroprotective effects appear to be associated with NAD + /SIRT1-dependent activation of lysosomal function., (© 2021. The Author(s), under exclusive licence to CPS and SIMM.)

Aspartic proteases are the targets for structure-based drug design for their role in physiological processes and pharmaceutical applications. Structural insights into the thermal inactivation mechanism of an aspartic protease in presence and absence of bound pepstatin A have been obtained by kinetics of thermal inactivation, CD, fluorescence spectroscopy and molecular dynamic simulations. The irreversible thermal inactivation of the aspartic protease comprised of loss of tertiary and secondary structures succeeded by the loss of activity, autolysis and aggregation The enthalpy and entropy of thermal inactivation of the enzyme in presence of pepstatin A increased from 81.2 to 148.5 kcal mol -1 , and from 179 to 359 kcal mol -1  K -1 respectively. Pepstatin A shifted the mid-point of thermal inactivation of the protease from 58 °C to 77 °C. The association constant (K) for pepstatin A with aspartic protease was 2.5 ± 0.3 × 10 5  M -1 and ΔG o value was -8.3 kcal mol -1 . Molecular dynamic simulation studies were able to delineate the role of pepstatin A in stabilizing backbone conformation and side chain interactions. In the Cα-backbone, the short helical segments and the conserved glycines were part of the most unstable segments of the protein. Understanding the mechanism of thermal inactivation has the potential to develop re-engineered thermostable proteases., Competing Interests: Declaration of competing interest All the authors have seen and approved the manuscript and declare no conflicts of interest with the content of this article., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Finding effective neuroprotective strategies to combat various neurodegenerative disorders still remain a clinically unmet need. Methyl caffeate (MC), a naturally occurring ester of caffeic acid, possesses antioxidant and anti-inflammatory activities; however, its role in neuroprotection is less investigated. In order to better characterize neuroprotective properties of MC, we tested its effectiveness in various models of neuronal cell injury in human neuroblastoma SH-SY5Y cells and in mouse primary neuronal cell cultures. MC at micromolar concentrations attenuated neuronal cell damage induced by hydrogen peroxide (H 2 O 2 ) in undifferentiated and neuronal differentiated SH-SY5Y cells as well as in primary cortical neurons. This effect was associated with inhibition of both caspase-3 and cathepsin D but without involvement of the PI3-K/Akt pathway. MC was neuroprotective when given before and during but not after the induction of cell damage by H 2 O 2 . Moreover, MC was protective against 6-OHDA-evoked neurotoxicity in neuronal differentiated SH-SY5Y cells via inhibition of necrotic and apoptotic processes. On the other hand, MC was ineffective in models of excitotoxicity (induced by glutamate or oxygen-glucose deprivation) and even moderately augmented cytotoxic effects of the classical apoptotic inducer, staurosporine. Finally, in undifferentiated neuroblastoma cells MC at higher concentrations (above 50 microM) induced cell death and when combined with the chemotherapeutic agent, doxorubicin, it increased the cell damaging effects of the latter compound. Thus, neuroprotective properties of MC appear to be limited to certain models of neurotoxicity and depend on its concentrations and time of administration.

Necroptosis, a recently discovered form of non-apoptotic programmed cell death, can be implicated in many pathological conditions including neuronal cell death. Moreover, an inhibition of this process by necrostatin-1 (Nec-1) has been shown to be neuroprotective in in vitro and in vivo models of cerebral ischemia. However, the involvement of this type of cell death in oxidative stress-induced neuronal cell damage is less recognized. Therefore, we tested the effects of Nec-1, an inhibitor of necroptosis, in the model of hydrogen peroxide (H 2 O 2 )-induced cell damage in human neuroblastoma SH-SY5Y and murine hippocampal HT-22 cell lines. The data showed that Nec-1 (10-40 μM) attenuated the cell death induced by H 2 O 2 in undifferentiated (UN-) and neuronal differentiated (RA-) SH-SY5Y cells with a higher efficacy in the former cell type. Moreover, Nec-1 partially reduced cell damage induced by 6-hydroxydopamine in UN- and RA-SH-SY5Y cells. The protective effect of Nec-1 was of similar magnitude as the effect of a caspase-3 inhibitor in both cell phenotypes and this effect were not potentiated after combined treatment. Furthermore, the non-specific apoptosis and necroptosis inhibitor curcumin augmented the beneficial effect of Nec-1 against H 2 O 2 -evoked cell damage albeit only in RA-SH-SY5Y cells. Next, it was found that the mechanisms of neuroprotective effect of Nec-1 against H 2 O 2 -induced cell damage in SH-SY5Y cells involved the inhibition of lysosomal protease, cathepsin D, but not caspase-3 or calpain activities. In HT-22 cells, Nec-1 was protective in two models of oxidative stress (H 2 O 2 and glutamate) and that effect was blocked by a caspase inhibitor. Our data showed neuroprotective effects of the necroptosis inhibitor, Nec-1, against oxidative stress-induced cell damage and pointed to involvement of cathepsin D inhibition in the mechanism of its action. Moreover, a cell type-specific interplay between necroptosis and apoptosis has been demonstrated.

Napsin A is an intracellular aspartic protease and biomarker of various malignancies like lung adenocarcinoma and ovarian clear cell carcinoma, but its detection is usually limited to immunohistochemical techniques gaining excellent information on its distribution but missing information about posttranslational modifications (e.g. maturation state) of the protein. We present a protocol for specific enrichment of napsin A from clinical or biological specimens, that facilitates detailed analysis of the protein. By using the exceptionally broad pH range under which napsin A binds to its inhibitor pepstatin A we achieve highly selective binding of napsin A while other aspartic proteases have negligible affinity. Using this method we demonstrate that lung napsin A in many mammals is a heterogeneous enzyme with a characteristic ladder-like appearance in SDS-PAGE that might be caused by proteolytically processed N- and/or C-termini, in contrast to the more homogeneous form found in kidneys and primary lung adenocarcinoma., (Copyright © 2019 Elsevier Inc. All rights reserved.)

(1) Background: Nanomedicine has recently emerged as a new area of research, particularly to fight cancer. In this field, we were interested in the vectorization of pepstatin A, a peptide which does not cross cell membranes, but which is a potent inhibitor of cathepsin D, an aspartic protease particularly overexpressed in breast cancer. (2) Methods: We studied two kinds of nanoparticles. For pepstatin A delivery, mesoporous silica nanoparticles with large pores (LPMSNs) and hollow organosilica nanoparticles (HOSNPs) obtained through the sol⁻gel procedure were used. The nanoparticles were loaded with pepstatin A, and then the nanoparticles were incubated with cancer cells. (3) Results: LPMSNs were monodisperse with 100 nm diameter. HOSNPs were more polydisperse with diameters below 100 nm. Good loading capacities were obtained for both types of nanoparticles. The nanoparticles were endocytosed in cancer cells, and HOSNPs led to the best results for cancer cell killing. (4) Conclusions: Mesoporous silica-based nanoparticles with large pores or cavities are promising for nanomedicine applications with peptides., Competing Interests: The authors declare no conflict of interest.

The pep4um gene (um04926) of Ustilago maydis encodes a protein related to either vacuolar or lysosomal aspartic proteases. Bioinformatic analysis of the Pep4um protein revealed that it is a soluble protein with a signal peptide suggesting that it likely passes through the secretory pathway, and it has two probable self-activation sites, which are similar to those in Saccharomyces cerevisiae PrA. Moreover, the active site of the Pep4um has the two characteristic aspartic acid residues of aspartyl proteases. The pep4um gene was cloned, expressed in Pichia pastoris and a 54 kDa recombinant protein was observed. Pep4um-rec was confirmed to be an aspartic protease by specifically inhibiting its enzymatic activity with pepstatin A. Pep4um-rec enzymatic activity on acidic hemoglobin was optimal at pH 4.0 and at 40 °C. To the best of our knowledge this is the first report about the heterologous expression of an aspartic protease from a basidiomycete. An in-depth in silico analysis suggests that Pep4um is homolog of the human cathepsin D protein. Thus, the Pep4um-rec protein may be used to test inhibitors of human cathepsin D, an important breast cancer therapeutic target.

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., (© 2017 American Institute of Chemical Engineers.)

The role of the kinase ataxia-telangiectasia mutated (ATM), a well-known protein engaged in DNA damage repair, in the regulation of neuronal responses to oxidative stress remains unexplored. Thus, the neuroprotective efficacy of KU-55933, a potent inhibitor of ATM, against cell damage evoked by oxidative stress (hydrogen peroxide, H 2 O 2 ) has been studied in human neuroblastoma SH-SY5Y cells and compared with the efficacy of this agent in models of doxorubicin (Dox)- and staurosporine (St)-evoked cell death. KU-55933 inhibited the cell death induced by H 2 O 2 or Dox but not by St in undifferentiated (UN-) and retinoic acid-differentiated (RA)-SH-SY5Y cells, with a more pronounced effect in the latter cell phenotype. Furthermore, this ATM inhibitor attenuated the Dox- but not H 2 O 2 -induced caspase-3 activity in both UN- and RA-SH-SY5Y cells. Although KU-55933 inhibited the H 2 O 2 - and Dox-induced activation of ATM, it attenuated the toxin-induced phosphorylation of the proteins H2AX and p53 only in the latter model of cell damage. Moreover, the ATM inhibitor prevented the H 2 O 2 -evoked increases in calpain and cathepsin D activity and attenuated cell damage to a similar degree as inhibitors of calpain (MDL28170) and cathepsin D (pepstatin A). Finally, we confirmed the neuroprotective potential of KU-55933 against the H 2 O 2 - and Dox-evoked cell damage in primary mouse cerebellar granule cells and in the mouse hippocampal HT-22 cell line. Altogether, our results extend the neuroprotective portfolio of KU-55933 to a model of oxidative stress, with this effect not involving inhibition of the γH2AX/p-p53/caspase-3 pathway and instead associated with the attenuation of calpain and cathepsin D activity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Plant aspartic proteases are generally divided into three categories: typical, nucellin-like, and atypical aspartic proteases based on their gene and protein structures. In this report, a full-length cDNA SPAP1 was cloned from sweet potato leaves, which contained 1515 nucleotides (504 amino acids) and exhibited high amino acid sequence identity (ca. 51-72%) with plant typical aspartic proteases, including tomato LeAspP, potato StAsp, and wheat WAP2. SPAP1 also contained conserved DTG and DSG amino acid residues within its catalytic domain and plant specific insert (PSI) at the C-terminus. The cDNA corresponding to the mature protein (starting from the 66th to 311th amino acid residues) without PSI domain was constructed with pET30a expression vector for fusion protein and antibody production. RT-PCR and protein blot hybridization showed that SPAP1 expression level was the highest in L3 mature leaves, then gradually declined until L5 completely yellow leaves. Ethephon, an ethylene-releasing compound, also enhanced SPAP1 expression at the time much earlier than the onset of leaf senescence. Exogenous application of SPAP1 fusion protein promoted ethephon-induced leaf senescence, which could be abolished by pre-treatment of SPAP1 fusion protein with (a) 95 °C for 5 min, (b) aspartic protease inhibitor pepstatin A, and (c) anti-SPAP1 antibody, respectively. Exogenous SPAP1 fusion protein, whereas, did not significantly affect leaf senescence under dark. These data conclude that sweet potato SPAP1 is a functional typical aspartic protease and participates in ethephon-mediated leaf senescence. The SPAP1-promoted leaf senescence and its activity are likely not associated with the PSI domain. Interaction of ethephon-inducible components for effective SPAP1 promotion on leaf senescence is also suggested., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5-genes essential for autophagosome formation-was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)