In-silico identification of novel DDI2 inhibitor in glioblastoma via repurposing FDA approved drugs using molecular docking and MD simulation study.

Glioblastoma, the most severe form of brain tumor and a leading cause of death within a year of diagnosis, is characterized by excessive protein synthesis and folding in the lumen of the endoplasmic reticulum (ER), leading to increased ER stress in the cells of GBM tissues. To mitigate this stress the cancer cells have intelligently adopted a plethora of response mechanisms and Unfolded Protein Response (UPR) is one of those. To bear with this exhaustive situation cells upregulate a strong protein degradation system in form of 26S proteasome and blocking of proteasomal gene synthesis may be a potential therapeutic action against GBM. Proteasomal gene synthesis is exclusively dependent on the transcription factor Nuclear respiratory factor 1 (NRF1) and its activating enzyme DNA damage inducible 1 homolog 2 (DDI2). Here in this study, we performed molecular docking against DDI2 with the 20 FDA-approved drugs and identified Alvimopan and Levocabastine as the top two compounds with the best binding score along with the standard drug Nelfinavir. MD simulation (100 ns) of these protein-ligand docked complexes reveals that the stability and compactness of Alvimopan are high in comparison with Nelfinavir. Our in-silico (Molecular docking and Molecular dynamics simulation) studies pointed out that Alvimopan may be repurposed as a DDI2 inhibitor and can be used as a potential anticancer agent for the treatment of brain tumors.Communicated by Ramaswamy H. Sarma.

Phenylacetyl pepstatin inhibitors of aspartyl proteases from Streptomyces varsoviensis.

A new pepstatin with a phenylacetyl group, pepstatin Pa (1), and its methyl ester (2) were isolated from Streptomyces varsoviensis DSM 40346. Their structures were determined by high-resolution mass spectrometry and nuclear magnetic resonance techniques. The absolute configuration was determined using the Marfey's method. Both pentapeptide products are inhibitors of pepsin and cathepsin D. Interestingly, the bacterial genome contains no biosynthetic gene cluster for the new pepstatin, suggesting an extrachromosomal origin of the biosynthetic genes.

Aspartic protease-pepstatin A interactions: Structural insights on the thermal inactivation mechanism.

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 ΔGo 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.

Re-emerging Aspartic Protease Targets: Examining Cryptococcus neoformans Major Aspartyl Peptidase 1 as a Target for Antifungal Drug Discovery.

Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.

Targeting adhesion in fungal pathogen Candida albicans.

Fungal infections with increasing resistance to conventional therapies are a growing concern. Candida albicans is a major opportunistic yeast responsible for mucosal and invasive infections. Targeting the initial step of the infection process (i.e., C. albicans adhesion to the host cell) is a promising strategy. A wide variety of molecules can interfere with adhesion processes via an assortment of mechanisms. Herein, we focus on how small molecules disrupt biosynthesis of fungal cell wall components and membrane structure, prevent the localization of GPI-anchor proteins, inhibit production of enzymes involved in adhesion, downregulate genes encoding adhesins and competitively inhibit receptor interactions. As a result, adhesion of C. albicans to host cells is reduced, paving the way to new classes of antifungal agents.

Rapid Discovery of Aspartyl Protease Inhibitors Using an Anchoring Approach.

Pharmacophore searches that include anchors, fragments contributing above average to receptor binding, combined with one-step syntheses are a powerful approach for the fast discovery of novel bioactive molecules. Here, we are presenting a pipeline for the rapid and efficient discovery of aspartyl protease inhibitors. First, we hypothesized that hydrazine could be a multi-valent warhead to interact with the active site Asp carboxylic acids. We incorporated the hydrazine anchor in a multicomponent reaction and created a large virtual library of hydrazine derivatives synthetically accessible in one-step. Next, we performed anchor-based pharmacophore screening of the libraries and resynthesized top-ranked compounds. The inhibitory potency of the molecules was finally assessed by an enzyme activity assay and the binding mode confirmed by several soaked crystal structures supporting the validity of the hypothesis and approach. The herein reported pipeline of tools will be of general value for the rapid generation of receptor binders beyond Asp proteases.

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.

Aspartic protease from Aspergillus niger: Molecular characterization and interaction with pepstatin A.

In the pursuit of industrial aspartic proteases, aspergillopepsin A-like endopeptidase from the fungi Aspergillus niger, was identified and cultured by solid state fermentation. Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity. The enzyme was found to have single polypeptide chain with a molecular mass of 50 ±â€¯0.5 kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60 °C respectively. The enzyme was stable for 60 min at 50 °C. The purified enzyme had specific activity of 40,000 ±â€¯1800 U/mg. The enzyme had 85% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a Ki value of 0.045 µM. Based on homology modeling and predicted secondary structure, it was inferred that the aspartic protease is rich in ß-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. Degree of hydrolysis of commercial substrates indicated the order of cleaving ability of the enzyme to be hemoglobin > defatted soya flour > gluten > gelatin > skim milk powder. The enzyme also improved the functional characteristics of defatted soya flour. This aspartic protease was found to be an excellent candidate for genetic manipulation for biotechnological application in food and feed industries, due to its high catalytic turn over number and thermostability.

Repositioning of HIV Aspartyl Peptidase Inhibitors for Combating the Neglected Human Pathogen Trypanosoma cruzi.

Chagas disease, caused by the flagellate parasite Trypanosoma cruzi, is a wellknown neglected tropical disease. This parasitic illness affects 6-7 million people and can lead to severe myocarditis and/or complications of the digestive tract. The changes in its epidemiology facilitate co-infection with the Human Immunodeficiency Virus (HIV), making even more difficult the diagnosis and prognosis. The parasitic infection is reactivated in T. cruzi/HIV co-infection, with the appearance of unusual manifestations in the chronic phase and the exacerbation of classical clinical signs. The therapeutic arsenal to treat Chagas disease, in all its clinical forms, is restricted basically to two drugs, benznidazole and nifurtimox. Both drugs are extremely toxic and the therapeutic efficacy is still unclear, making the clinical treatment a huge issue to be solved. Therefore, it seems obvious the necessity of new tangible approaches to combat this illness. In this sense, the repositioning of approved drugs appears as an interesting and viable strategy. The discovery of Human Immunodeficiency Virus Aspartyl Peptidase Inhibitors (HIV-PIs) represented a milestone in the treatment of Acquired Immune Deficiency Syndrome (AIDS) and, concomitantly, a marked reduction in both the incidence and prevalence of important bacterial, fungal and parasitic co-infections was clearly observed. Taking all these findings into consideration, the present review summarizes the promising and beneficial data concerning the effects of HIV-PIs on all the evolutionary forms of T. cruzi and in important steps of the parasite's life cycle, which highlight their possible application as alternative drugs to treat Chagas disease.

Exploiting Structural Dynamics To Design Open-Flap Inhibitors of Malarial Aspartic Proteases.

Malaria is a life-threatening infectious disease caused by Plasmodium parasites. Plasmepsins (proteolytic enzymes of the parasite) have been considered as promising targets for the development of antimalarial drugs. To date, much knowledge has been obtained regarding the interactions of inhibitors with plasmepsins, as well as the structure-activity relationships of the inhibitors. The discovery and characterization of the plasmepsin inhibitors that bind in open flap conformation have led to several inhibitor classes that show high selectivity over other human aspartic proteases. This Perspective addresses the flexibility of the plasmepsins that leads to inhibitor binding to the open flap conformation, summarizes known nonpeptidomimetic plasmepsin inhibitors, and discusses the role of the inhibitor flap pocket substituent.

MicroRNA-3607 inhibits the tumorigenesis of colorectal cancer by targeting DDI2 and regulating the DNA damage repair pathway.

Mutations in the DNA damage repair (DDR) pathway are frequently detected in colorectal cancer (CRC). The dysregulation of miRNAs, such as oncogenes or tumor suppressors, participates in CRC tumorigenesis. A previous study showed that low miR-3607 expression correlated with poor survival in prostate cancer patients, but its role in CRC remains unclear. In this study, we analyzed miR-3607 expression Pan-Cancer data from the NCI's Genomic Data Commons (GDC) and found that miR-3607 was downregulated in lymphatic invasion patients and in recurrent cancer and correlated with Pan-Cancer patient survival. Functional studies indicated that the overexpression of miR-3607 decreased CRC cell proliferation, migration and invasion. Additionally, we used gene set enrichment analysis (GSEA), Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and a protein-protein interaction network to demonstrate that miR-3607 affects the DDR pathway. Luciferase reporter and apoptosis assays confirmed that DNA damage inducible 1 homolog 2 (DDI2) is the functional target of miR-3607. Therefore, miR-3607 inhibits the tumorigenesis of CRC probably by suppressing the oncogene DDI2, and it might serve as a novel target for CRC prediction and therapy.

Leishmanicidal therapy targeted to parasite proteases.

Leishmaniasis is considered a serious public health problem and the current available therapy has several disadvantages, which makes the search for new therapeutic targets and alternative treatments extremely necessary. In this context, this review focuses on the importance of parasite proteases as target drugs against Leishmania parasites, as a chemotherapy approach. Initially, we discuss about the current scenario for the treatment of leishmaniasis, highlighting the main drugs used and the problems related to their use. Subsequently, we describe the inhibitors of major proteases of Leishmania already discovered, such as Compound s9 (aziridine-2,3-dicarboxylate), Compound 1c (benzophenone derivative), Au2Phen (gold complex), AubipyC (gold complex), MDL 28170 (dipeptidyl aldehyde), K11777, Hirudin, diazo-acetyl norleucine methyl ester, Nelfinavir, Saquinavir, Nelfinavir, Saquinavir, Indinavir, Saquinavir, GNF5343 (azabenzoxazole), GNF6702 (azabenzoxazole), Benzamidine and TPCK. Next, we discuss the importance of the protease gene to parasite survival and the aspects of the validation of proteases as target drugs, with emphasis on gene disruption. Then, we describe novel important strategies that can be used to support the research of new antiparasitic drugs, such as molecular modeling and nanotechnology, whose main targets are parasitic proteases. And finally, we discuss possible perspectives to improve drug development. Based on all findings, proteases could be considered potential targets against leishmaniasis.

Novel non-peptidic small molecule inhibitors of secreted aspartic protease 2 (SAP2) for the treatment of resistant fungal infections.

Targeting secreted aspartic protease 2 (SAP2), a kind of virulence factor, represents a new strategy for antifungal drug discovery. In this report, the first-generation of small molecule SAP2 inhibitors was rationally designed and optimized using a structure-based approach. In particular, inhibitor 23h was highly potent and selective and showed good antifungal potency for the treatment of resistant Candida albicans infections.

Deciphering the targets of retroviral protease inhibitors in Plasmodium berghei.

Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact molecular target(s) for these RPIs in the Plasmodium parasites remains poorly understood. We hypothesised that LP and SQ suppress parasite growth through inhibition of aspartyl proteases. Using reverse genetics approach, we embarked on separately generating knockout (KO) parasite lines lacking Plasmepsin 4 (PM4), PM7, PM8, or DNA damage-inducible protein 1 (Ddi1) in the rodent malaria parasite Plasmodium berghei ANKA. We then tested the suppressive profiles of the LP/Ritonavir (LP/RT) and SQ/RT as well as antimalarials; Amodiaquine (AQ) and Piperaquine (PQ) against the KO parasites in the standard 4-day suppressive test. The Ddi1 gene proved refractory to deletion suggesting that the gene is essential for the growth of the asexual blood stage parasites. Our results revealed that deletion of PM4 significantly reduces normal parasite growth rate phenotype (P = 0.003). Unlike PM4_KO parasites which were less susceptible to LP and SQ (P = 0.036, P = 0.030), the suppressive profiles for PM7_KO and PM8_KO parasites were comparable to those for the WT parasites. This finding suggests a potential role of PM4 in the LP and SQ action. On further analysis, modelling and molecular docking studies revealed that both LP and SQ displayed high binding affinities (-6.3 kcal/mol to -10.3 kcal/mol) towards the Plasmodium aspartyl proteases. We concluded that PM4 plays a vital role in assuring asexual stage parasite fitness and might be mediating LP and SQ action. The essential nature of the Ddi1 gene warrants further studies to evaluate its role in the parasite asexual blood stage growth as well as a possible target for the RPIs.

Modeling and resistant alleles explain the selectivity of antimalarial compound 49c towards apicomplexan aspartyl proteases.

Toxoplasma gondii aspartyl protease 3 (TgASP3) phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X (PfPMIX, PfPMX). These proteases are essential for parasite survival, acting as key maturases for secreted proteins implicated in invasion and egress. A potent antimalarial peptidomimetic inhibitor (49c) originally developed against Plasmepsin II selectively targets TgASP3, PfPMIX, and PfPMX To unravel the molecular basis for the selectivity of 49c, we constructed homology models of PfPMIX, PfPMX, and TgASP3 that were first validated by identifying the determinants of microneme and rhoptry substrate recognition. The flap and flap-like structures of several reported Plasmepsins are highly flexible and critically modulate the access to the binding cavity. Molecular docking of 49c to TgASP3, PfPMIX, and PfPMX models predicted that the conserved phenylalanine residues in the flap, F344, F291, and F305, respectively, account for the sensitivity toward 49c. Concordantly, phenylalanine mutations in the flap of the three proteases increase twofold to 15-fold the IC50 values of 49c. Compellingly the selection of mutagenized T. gondii resistant strains to 49c reproducibly converted F344 to a cysteine residue.

Engineering improved variants of the antifungal peptide histatin 5 with reduced susceptibility to Candida albicans secreted aspartic proteases and enhanced antimicrobial potency.

Candida albicans is an opportunistic fungal pathogen and a commensal organism that commonly colonizes mucosal surfaces, including those inside the human mouth. To help control C. albicans, human saliva contains the antifungal peptide histatin 5 (Hst-5), which has strong antifungal activity against C. albicans. However, the pathogen produces secreted aspartic proteases (Saps) that cleave Hst-5 at lysine residues and eliminate its antifungal properties. We designed variants of Hst-5 with its lysine residues substituted with arginine or leucine to evaluate the effect on proteolysis by Saps. We found site-, residue-, and Sap-dependent effects from single amino acid substitutions. The K17R and K17L modifications led to dramatic results, with over 77% and 100% intact peptide remaining after incubation with Sap9 and Sap2, respectively, compared to 47% and 61% of Hst-5. This decrease in proteolysis was accompanied by a reduction in cleavage on the C-terminal side of K17, suggesting the Saps prefer lysine at K17 for cleavage. Incubation with C. albicans cells and culture supernatant corroborated the results with purified Saps and highlighted their biological relevance. The modifications to Hst-5 do not diminish the antifungal activity of Hst-5, and, in fact, the K17R, K17L, and K11R peptides retained significantly more antifungal activity after treatment with Saps than Hst-5. Our results indicate that single amino acid modifications drastically impact both proteolysis at the modification sites and the overall level of proteolysis of the peptide, demonstrating the potential of designing peptides for resistance to proteolysis as a means for improving therapeutic efficacy.

Characterization of a novel aspartyl protease inhibitor from Haemonchus contortus.

BACKGROUND: Aspartyl protease inhibitor (API) was thought to protect intestinal parasitic nematodes from their hostile proteolytic environment. Studies on Ostertagia ostertagi, Ascaris suum and Brugia malayi indicated that aspins might play roles in nematode infection. In a recent study, proteins differentially expressed between free-living third-stage larvae (L3) and activated L3 (xL3) of Haemonchus contortus were identified by 2D-DIGE. API was found downregulated in xL3 when compared with L3. However, there was no report about the functions of H. contortus API in the parasite-host interaction. In this study, the gene encoding API from H. contortus was cloned, expressed, and part of its biological characteristics were studied. RESULTS: A DNA fragment of 681 bp was amplified by RT-PCR. Ninety one percent of the amino acid sequence was similar with that for aspin from O. ostertagi. The recombinant API protein was fusion-expressed with a molecular weight of 48 × 103. Results of Western blot showed that the recombinant API could be recognized by serum from goat infected with H. contortus. It was found that API was localized exclusively in the subcutaneous tissue and epithelial cells of the gastrointestinal tract in adult H. contortus. qRT-PCR suggested that the API gene was differentially transcribed in different life-cycle stages, with the lowest level in female adults and the highest in free-living L3 larvae. Enzyme inhibition assay indicated that the recombinant API can inhibit the activity of pepsin significantly, and the optimal reaction pH and temperature were 4.0 and 37-50 °C respectively. In vitro study showed that the recombinant API could induce goat PBMCs to express IFN-γ, IL-4 and IL-10. CONCLUSIONS: A new aspartyl protease inhibitor was cloned from H. contortus and its characteristics were studied for the first time. The results indicate that API may regulate the immune response of the host and play roles in the infection.

Partial Characterization of the Proteolytic Properties of an Enzymatic Extract From "Aguama" Bromelia pinguin L. Fruit Grown in Mexico.

Plant proteases are capable of performing several functions in biological systems, and their use is attractive for biotechnological process due to their interesting catalytic properties. Bromelia pinguin (aguama) is a wild abundant natural resource in several regions of Central America and the Caribbean Islands but is underutilized. Their fruits are rich in proteases with properties that are still unknown, but they represent an attractive source of enzymes for biotechnological applications. Thus, the proteolytic activity in enzymatic crude extracts (CEs) from wild B. pinguin fruits was partially characterized. Enzymes in CEs showed high proteolytic activity at acid (pH 2.0-4.0) and neutral alkaline (pH 7.0-9.0) conditions, indicating that different types of active proteases are present. Proteolytic activity inhibition by the use of specific protease inhibitors indicated that aspartic, cysteine, and serine proteases are the main types of proteases present in CEs. Activity at pH 3.0 was stable in a broad range of temperatures (25-50 °C) and retained its activity in the presence of surfactants (SDS, Tween-80), reducing agents (DTT, 2-mercapoethanol), and organic solvents (methanol, ethanol, acetone, 2-propanol), which suggests that B. pinguin proteases are potential candidates for their application in brewing, detergent, and pharmaceutical industries.

Therapeutic activity of a Saccharomyces cerevisiae-based probiotic and inactivated whole yeast on vaginal candidiasis.

Vulvovaginal candidiasis is the most prevalent vaginal infection worldwide and Candida albicans is its major agent. Vulvovaginal candidiasis is characterized by disruption of the vaginal microbiota composition, as happens following large spectrum antibiotic usage. Recent studies support the effectiveness of oral and local probiotic treatment for prevention of recurrent vulvovaginal candidiasis. Saccharomyces cerevisiae is a safe yeast used as, or for, the production of ingredients for human nutrition and health. Here, we demonstrate that vaginal administration of probiotic Saccharomyces cerevisiae live yeast (GI) and, in part, inactivated whole yeast Saccharomyces cerevisiae (IY), used as post-challenge therapeutics, was able to positively influence the course of vaginal candidiasis by accelerating the clearance of the fungus. This effect was likely due to multiple interactions of Saccharomyces cerevisiae with Candida albicans. Both live and inactivated yeasts induced coaggregation of Candida and consequently inhibited its adherence to epithelial cells. However, only the probiotic yeast was able to suppress some major virulence factors of Candida albicans such as the ability to switch from yeast to mycelial form and the capacity to express several aspartyl proteases. The effectiveness of live yeast was higher than that of inactivated whole yeast suggesting that the synergy between mechanical effects and biological effects were dominant over purely mechanical effects. The protection of epithelial cells to Candida-induced damage was also observed. Overall, our data show for the first time that Saccharomyces cerevisiae-based ingredients, particularly the living cells, can exert beneficial therapeutic effects on a widespread vaginal mucosal infection.

Nelfinavir and lopinavir impair Trypanosoma cruzi trypomastigote infection in mammalian host cells and show anti-amastigote activity.

There is an urgent need to implement new strategies and to search for new chemotherapeutic targets to combat Chagas' disease. In this context, repositioning of clinically approved drugs appears as a viable tool to combat this and several other neglected pathologies. An example is the use of aspartic peptidase inhibitors (PIs) currently applied in human immunodeficiency virus (HIV) treatment against different infectious agents. Therefore, the main objective of this work was to verify the effects of the HIV-PIs nelfinavir and lopinavir against Trypanosoma cruzi using in vitro models of infection. Cytotoxicity assays with LLC-MK2 epithelial cells and RAW macrophages allowed an evaluation of the effects of HIV-PIs on the interaction between trypomastigotes and these cells as well as the survival of intracellular amastigotes. Pre-treatment of trypomastigotes with nelfinavir and lopinavir inhibited the association index with LLC-MK2 cells and RAW macrophages in a dose- and time-dependent manner. In addition, nelfinavir and lopinavir also significantly reduced the number of intracellular amastigotes in both mammalian cell lineages, particularly when administered in daily doses. Both compounds had no effect on nitric oxide production in infected RAW macrophages. These results open the possibility for the use of HIV-PIs as a tangible alternative in the treatment of Chagas' disease. However, the main mechanism of action of nelfinavir and lopinavir has yet to be elucidated, and more studies using in vivo models must be conducted.