Aspartic peptidase of Phialophora verrucosa as target of HIV peptidase inhibitors: blockage of its enzymatic activity and interference with fungal growth and macrophage interaction.

Phialophora verrucosa causes several fungal human diseases, mainly chromoblastomycosis, which is extremely difficult to treat. Several studies have shown that human immunodeficiency virus peptidase inhibitors (HIV-PIs) are attractive candidates for antifungal therapies. This work focused on studying the action of HIV-PIs on peptidase activity secreted by P. verrucosa and their effects on fungal proliferation and macrophage interaction. We detected a peptidase activity from P. verrucosa able to cleave albumin, sensitive to pepstatin A and HIV-PIs, especially lopinavir, ritonavir and amprenavir, showing for the first time that this fungus secretes aspartic-type peptidase. Furthermore, lopinavir, ritonavir and nelfinavir reduced the fungal growth, causing remarkable ultrastructural alterations. Lopinavir and ritonavir also affected the conidia-macrophage adhesion and macrophage killing. Interestingly, P. verrucosa had its growth inhibited by ritonavir combined with either itraconazole or ketoconazole. Collectively, our results support the antifungal action of HIV-PIs and their relevance as a possible alternative therapy for fungal infections.

Docking simulation between HIV peptidase inhibitors and Trypanosoma cruzi aspartyl peptidase.

OBJECTIVE: The low investment in research, diagnosis and treatment are factors that contribute to the continuity of Chagas' disease as a neglected tropical diseases (NTDs). In this context, the repositioning of drugs represents a useful strategy, in the search for new chemotherapeutic approaches for NTDs. HIV aspartic peptidase inhibitors (HIV IPs) are good candidates for drug repurposing. Here, we modeled the three dimensional structure of an aspartyl peptidase of Trypanosoma cruzi, the causative agent of Chagas' disease, aligned it to the HIV aspartyl peptidase and performed docking binding assays with the HIV PIs. RESULTS: The 3D structure confirmed the presence of acid aspartic residues, which are critical to enzyme activity. The docking experiment revealed that HIV IPs bind to the active site of the enzyme, being ritonavir and lopinavir the ones with greater affinity. Benznidazole presented the worst binding affinity, this drug is currently used in Chagas' disease treatment and was included as negative control. These results together with previous data on the trypanocidal effect of the HIV PIs support the hypothesis that a T. cruzi aspartyl peptidase can be the intracellular target of these inhibitors. However, the direct demonstration of the inhibition of T. cruzi aspartyl peptidase activity by HIV PIs is still a goal to be persuaded.

Miniature cheeses made with blends of chymosin and a vegetable rennet from flowers of Silybum marianum: Enzymatic characterization of the flower-coagulant peptidase.

Binary blends of S. marianum-flower extract and chymosin, as coagulant preparations, enabled the manufacture of miniature cheeses with distinctive characteristics compared to those of chymosin-renneted cheeses. The physicochemical parameters, sensory attributes of the cheeses, and in-vitro water-soluble antioxidant activity were analyzed and compared to those properties obtained from control chymosin-renneted cheeses. The preponderant proteolytic constituent in the flower extract was isolated in a two-step-purification protocol. The thus purified aspartic peptidase was maximally active at acidic pHs and exhibited a preference for peptide bonds between hydrophobic residues. Enzymologic characterization revealed differences in the kinetic parameters and specificity compared to other enzymes employed, such as rennet. S. marianum-flower extract, as a source of peptidase with distinctive characteristics, is a suitable substitute for chymosin in miniature-cheese production. The addition of vegetable rennet contributed to the development of an intense aroma and conferred antioxidant activity to the cheeses and wheys.

Fonsecaea pedrosoi Sclerotic Cells: Secretion of Aspartic-Type Peptidase and Susceptibility to Peptidase Inhibitors.

Fonsecaea pedrosoi is a dematiaceous fungus and the main causative agent of chromoblastomycosis that is a chronic disease usually affecting the human skin and subcutaneous tissues, which causes deformations and incapacities, being frequently refractory to available therapies. A typical globe-shaped, multiseptated and pigmented cells, known as sclerotic cells, are found in the lesions of infected individuals. In the present work, we have investigated the production of aspartic-type peptidase in F. pedrosoi sclerotic cells as well as the effect of peptidase inhibitors (PIs) on its enzymatic activity and viability. Our data showed that sclerotic cells are able to secrete pepstatin A-sensible aspartic peptidase when grown under chemically defined conditions. In addition, aspartic PIs (ritonavir, nelfinavir, indinavir, and saquinavir), which are clinically used in the HIV chemotherapy, significantly decreased the fungal peptidase activity, varying from 55 to 99%. Moreover, sclerotic cell-derived aspartic peptidase hydrolyzed human albumin, an important serum protein, as well as laminin, an extracellular matrix component, but not immunoglobulin G and fibronectin. It is well-known that aspartic peptidases play important physiological roles in fungal cells. With this task in mind, the effect of pepstatin A, a classical aspartic peptidase inhibitor, on the F. pedrosoi proliferation was evaluated. Pepstatin A inhibited the fungal viability in both cellular density- and drug-concentration manners. Moreover, HIV-PIs at 10 µM powerfully inhibited the viability (>65%) of F. pedrosoi sclerotic cells. The detection of aspartic peptidase produced by sclerotic cells, the parasitic form of F. pedrosoi, may contribute to reveal new virulence markers and potential targets for chromoblastomycosis therapy.

Trichosporon asahii secretes a 30-kDa aspartic peptidase.

Trichosporon asahii is a fungal opportunistic pathogen that causes superficial and deep-seated infections presenting high mortality. Very little is known about the virulence attributes produced by this fungus. Herein, aspartic peptidase production was identified in Brazilian clinical isolates of T. asahii by different methodologies. Initially, T. asahii strain 250 (from skin lesion) was inoculated in both liquid and solid culture media containing bovine serum albumin (BSA) as the sole nitrogenous source. A translucent halo around the fungal colony was observed from the 5th day of culture. The cell-free culture supernatant revealed that soluble BSA was hydrolyzed along the growth, generating low molecular mass polypeptides as observed by electrophoresis. Subsequently, the secretions from four clinical strains of T. asahii were analyzed by BSA-SDS-PAGE and a single proteolytic band of 30-kDa was detected under acidic pH at 37°C. The secreted aspartic peptidase of T. asahii efficiently cleaved the cathepsin D peptide substrate, but not the substrates with specificity to HIV-1 peptidase and rennin. The capability to cleave either cathepsin D substrate in a fluorogenic assay or BSA immobilized within a gel matrix varied according to the T. asahii isolate. T. asahii extracellular peptidase activity was strongly inhibited by pepstatin A and HIV peptidase inhibitors, classifying it as an aspartic-type peptidase. Human serum albumin, mucin, non-immune immunoglobulin G and gelatin induced, in different levels, the secretion of this aspartic peptidase. With these results, T. asahii must be included in the list of many human fungal opportunistic pathogens able to secrete an aspartic-type peptidase.

HIV Aspartic Peptidase Inhibitors Modulate Surface Molecules and Enzyme Activities Involved with Physiopathological Events in Fonsecaea pedrosoi.

Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis, a recalcitrant disease that is extremely difficult to treat. Therefore, new chemotherapeutics to combat this fungal infection are urgently needed. Although aspartic peptidase inhibitors (PIs) currently used in the treatment of human immunodeficiency virus (HIV) have shown anti-F. pedrosoi activity their exact mechanisms of action have not been elucidated. In the present study, we have investigated the effects of four HIV-PIs on crucial virulence attributes expressed by F. pedrosoi conidial cells, including surface molecules and secreted enzymes, both of which are directly involved in the disease development. In all the experiments, conidia were treated with indinavir, nelfinavir, ritonavir and saquinavir (100 µM) for 24 h, and then fungal cells were used to evaluate the effects of HIV-PIs on different virulence attributes expressed by F. pedrosoi. In comparison to untreated controls, exposure of F. pedrosoi cells to HIV-PIs caused (i) reduction on the conidial granularity; (ii) irreversible surface ultrastructural alterations, such as shedding of electron dense and amorphous material from the cell wall, undulations/invaginations of the plasma membrane with and withdrawal of this membrane from the cell wall; (iii) a decrease in both mannose-rich glycoconjugates and melanin molecules and an increase in glucosylceramides on the conidial surface; (iv) inhibition of ergosterol and lanosterol production; (v) reduction in the secretion of aspartic peptidase, esterase and phospholipase; (vi) significant reduction in the viability of non-pigmented conidia compared to pigmented ones. In summary, HIV-PIs are efficient drugs with an ability to block crucial biological processes of F. pedrosoi and can be seriously considered as potential compounds for the development of new chromoblastomycosis chemotherapeutics.

Role of cathepsins D in the midgut of Dysdercus peruvianus.

Hemipteran ancestors probably lost their digestive serine peptidases on adapting to a plant sap diet. On returning to protein ingestion, these insects start using cathepsin (lysosomal) peptidases as digestive enzymes, from which the less known is cathepsin D. Nine of the ten cathepsin D transcribing genes found in Dysdercus peruvianus midgut are expressed exclusively in this tissue and only DpCatD10 is also expressed in other tissues. The main action of cathepsins D is in the first (V1) (from three, V1-3) midgut regions, where 40% of the total proteolytic activity was assigned to aspartic peptidases with an optimum pH of 3.5. The most expressed cathepsins D were identified in the midgut luminal contents by proteomics. The data indicate that D. peruvianus have kept a lysosomal gene expressed in all tissues and evolved another set of genes with a digestive function restricted to midgut. Digestive cathepsins D apparently complement the action of digestive cathepsin L and they are arguably responsible for the hydrolysis of cysteine peptidase inhibitors known to be present in the cotton seeds eaten by the insect, before they meet cathepsin L.

HIV aspartic peptidase inhibitors are effective drugs against the trypomastigote form of the human pathogen Trypanosoma cruzi.

There is a general lack of effective and non-toxic chemotherapeutic agents against Chagas' disease despite more than a century of research. In this regard, we have verified the impact of human immunodeficiency virus aspartic peptidase inhibitors (HIV-PIs) on the viability and morphology of infective trypomastigote forms of Trypanosoma cruzi as well as on the aspartic peptidase and proteasome activities produced by this parasite. The effects of HIV-PIs on viability were assessed by counting motile parasites in a Neubauer chamber. Morphological alterations were detected by light microscopy of Giemsa-stained smears and scanning electron microscopy. Modulation of aspartic peptidase and proteasome activities by the HIV-PIs was measured by cleavage of fluorogenic peptide substrates. The majority of the HIV-PIs (6/9) were able to drastically decrease the viability of trypomastigotes after 4 h of treatment, with nelfinavir and lopinavir being the most effective compounds presenting LD50 values of 8.6 µM and 10.6 µM, respectively. Additionally, both HIV-PIs were demonstrated to be effective in a time- and cell density-dependent manner. Treatment with nelfinavir and lopinavir caused many morphological/ultrastructural alterations in trypomastigotes; parasites became round in shape, with reduced cell size and flagellar shortening. Nelfinavir and lopinavir were also capable of significantly inhibiting the aspartic peptidase and proteasome activities measured in trypomastigote extracts. These results strengthen the data on the positive effects of HIV-PIs on parasitic infections, possibly by targeting the parasite aspartic peptidase(s) and proteasome(s), opening a new possibility for the use of these clinically approved drugs as an alternative chemotherapy to treat Chagas' disease.