In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and potentially MERS, while vinyl sulfone-based inhibitors are excellent lead candidates for Ebola virus therapeutics.

Peptidomimetics of the class of dipeptidyl nitrile analog peptoids were synthesized as inhibitors of mammalian cysteine proteases of the papain superfamily. The dipeptidyl nitrile side chains were attached to the peptide backbone's nitrogen atom, not to the α-carbons. Synthesized nitrile-based peptoid analogs that lack the hydrogen amide at P2-P3 are responsible for many of the secondary structure elements in peptides and proteins, making them resistant to proteolysis. The designed peptoids would lose a hydrogen bond with cruzain Asp161 decreasing the affinity toward the enzyme. A structure-activity relationship and matched molecular pair-based analysis between the dipeptidyl nitrile Neq0409 and its peptoid 4a yielded the following cruzain affinities: pKiNeq0409 = 6.5 and pKi4a = 5.2. respectively. A retrosynthetic matched molecular pair cliff (RMMP-cliff) analysis with a ΔpKiNeq0409-4a of 1.3 log is found for this transformation. These novel peptoids were then optimized, leading to compound 4i, with high cruzain inhibition (pKi = 6.8). Cross-class cathepsin activity was observed for some of these novel compounds against cathepsins K, L and S, while other compounds presented a selective inhibition of cathepsin K (4b, 4c, 4k) over ten times higher than the other enzymes. The putative mode of binding was determined by using covalent docking, which also aided to describe the structure-activity relationship (SAR). Interestingly, none of the peptoids inhibited CatB to any appreciable extent. These results provide guidance to identify novel bioactive nitrile-based peptoids.

Protease substrate profiling has nowadays almost become a routine task for experimentalists, and the knowledge on protease peptide substrates is easily accessible via the MEROPS database. We present a shape-based virtual screening workflow using vROCS that applies the information about the specificity of the proteases to find new small-molecule inhibitors. Peptide substrate sequences for three to four substrate positions of each substrate from the MEROPS database were used to build the training set. Two-dimensional substrate sequences were converted to three-dimensional conformations through mutation of a template peptide substrate. The vROCS query was built from single amino acid queries for each substrate position considering the relative frequencies of the amino acids. The peptide-substrate-based shape-based virtual screening approach gives good performance for the four proteases thrombin, factor Xa, factor VIIa, and caspase-3 with the DUD-E data set. The results show that the method works for protease targets with different specificity profiles as well as for targets with different active-site mechanisms. As no structure of the target and no information on small-molecule inhibitors are required to use our approach, the method has significant advantages in comparison with conventional structure- and ligand-based methods.

Background: The ongoing COVID-19 pandemic has created an alarming situation due to extensive loss of human lives and economy, posing enormous threat to global health security. Till date, no antiviral drug or vaccine against SARS-CoV-2 has reached the market, although a number of clinical trials are under way. The viral 3-chymotrypsin-like cysteine protease (3CLpro), playing pivotal roles in coronavirus replication and polyprotein processing, is essential for its life cycle. In fact, 3CLpro is already a proven drug discovery target for SARS- and MERS-CoVs. This underlines the importance of 3CL protease in the design of potent drugs against COVID-19. Methods: We have collected one hundred twenty-seven relevant literatures to prepare the review article. PubMed, Google Scholar and other scientific search engines were used to collect the literature based on keywords, like "SARS-CoVs-3CL protease," "medicinal plant and anti-SARS-CoVs-3CL protease" published during 2003-2020. However, earlier publications related to this topic are also cited for necessary illustration and discussion. Repetitive articles and non-English studies were excluded. Results: From the literature search, we have enlisted medicinal plants reported to inhibit coronavirus 3CL protease. Some of the plants like Isatis tinctoria L. (syn. Isatis indigotica Fort.), Torreya nucifera (L.) Siebold and Zucc., Psoralea corylifolia L., and Rheum palmatum L. have exhibited strong anti-3CLpro activity. We have also discussed about the phytochemicals with encouraging antiviral activity, such as, bavachinin, psoralidin, betulinic acid, curcumin and hinokinin, isolated from traditional medicinal plants. Conclusion: Currently, searching for a plant-derived novel drug with better therapeutic index is highly desirable due to lack of specific treatment for SARS-CoV-2. It is expected that in-depth evaluation of medicinally important plants would reveal new molecules with significant potential to inhibit coronavirus 3CL protease for development into approved antiviral drug against COVID-19 in future.

The Zika virus (ZIKV) remains a potential threat to the public health due to the lack of both an approved vaccination or a specific treatment. In this work, a series of peptidic inhibitors of the ZIKV protease with boroleucine as P1 residue was synthesized. The highest affinities with Ki values down to 8 nM were observed for compounds with basic residues in both P2 and P3 position and at the N-terminus. The low potency of reference compounds containing leucine, leucine-amide or isopentylamide as P1 residue suggested a covalent binding mode of the boroleucine-derived inhibitors. This was finally proven by crystal structure determination of the most potent inhibitor from this series in complex with the ZIKV protease.

Tumor vascular endothelium is rather resistant to the cytotoxic effects of radiation. The HIV protease inhibitors (HPI) amprenavir, nelfinavir, and saquinavir have previously been shown to sensitize tumor cells to the cytotoxic effects of radiation. Additionally, this class of drug has been shown to inhibit angiogenesis and tumor cell migration. Therefore, in the current study, we wanted to determine whether HPIs could enhance the effect of radiation on endothelial function. Our study shows that HPIs, particularly nelfinavir, significantly enhance radiations effect on human umbilical vein endothelial cells (HUVEC) and tumor vascular endothelium. We show that pretreatment of HUVEC with nelfinavir results in enhanced cytotoxicity, including increased apoptosis, when combined with radiation. Moreover, using several functional assays, we show that combination treatment effectively blocks endothelial cell migration and organization. These findings were accompanied by attenuation of Akt phosphorylation, a known pathway for radioresistance. Last, in vivo analysis of tumor microvasculature destruction showed a more than additive effect for nelfinavir and radiation. This study shows that HPIs can enhance the effect of ionizing radiation on vascular endothelium. Therefore, the Food and Drug Administration-approved drug, nelfinavir, may be an effective radiosensitizer in the clinic.

The Zika virus (ZIKV), a member of the Flaviviridae family, is considered a major health threat causing multiple cases of microcephaly in newborns and Guillain-Barré syndrome in adults. In this study, we targeted a transient, deep, and hydrophobic pocket of the "super-open" conformation of ZIKV NS2B-NS3 protease to overcome the limitations of the active site pocket. After virtual docking screening of approximately seven million compounds against the novel allosteric site, we selected the top six candidates and assessed them in enzymatic assays. Six candidates inhibited ZIKV NS2B-NS3 protease proteolytic activity at low micromolar concentrations. These six compounds, targeting the selected protease pocket conserved in ZIKV, serve as unique drug candidates and open new opportunities for possible treatment against several flavivirus infections.

Although antiretroviral treatment decreases HIV-AIDS morbidity/mortality, long-term side effects may include the onset of insulin resistance and cardiovascular diseases. However, the underlying molecular mechanisms responsible for highly active antiretroviral therapy (HAART)-induced cardio-metabolic effects are poorly understood. In light of this, we hypothesized that HIV protease inhibitor (PI) treatment (Lopinavir/Ritonavir) elevates myocardial oxidative stress and concomitantly inhibits the ubiquitin proteasome system (UPS), thereby attenuating cardiac function. Lopinavir/Ritonavir was dissolved in 1% ethanol (vehicle) and injected into mini-osmotic pumps that were surgically implanted into Wistar rats for 8 weeks vs. vehicle and sham controls. We subsequently evaluated metabolic parameters, gene/protein markers and heart function (ex vivo Langendorff perfusions). PI-treated rats exhibited increased serum LDL-cholesterol, higher tissue triglycerides (heart, liver), but no evidence of insulin resistance. In parallel, there was upregulation of hepatic gene expression, i.e. acetyl-CoA carboxylase b and 3-hydroxy-3-methylglutaryl-CoA-reductase, key regulators of fatty acid oxidation and cholesterol synthesis, respectively. PI-treated hearts displayed impaired cardiac contractile function together with attenuated UPS activity. However, there was no significant remodeling of hearts exposed to PIs, i.e. lack of ultrastructural changes, fibrosis, cardiac hypertrophic response, and oxidative stress. Western blot analysis of PI-treated hearts revealed that perturbed calcium handling may contribute to the PI-mediated contractile dysfunction. Here chronic PI administration led to elevated myocardial calcineurin, nuclear factor of activated T-cells 3 (NFAT3), connexin 43, and phosphorylated phospholamban, together with decreased calmodulin expression levels. This study demonstrates that early changes triggered by PI treatment include increased serum LDL-cholesterol levels together with attenuated cardiac function. Furthermore, PI exposure inhibits the myocardial UPS and leads to elevated calcineurin and connexin 43 expression that may be associated with the future onset of cardiac contractile dysfunction.

Proteolytic enzymes play essential metabolic and regulatory functions in many biological processes and also offer a wide range of biotechnological applications. Because of their essential roles, their proteolytic activity needs to be tightly regulated. Therefore, small molecules and proteins that inhibit proteases can be versatile tools in the fields of medicine, agriculture and biotechnology. In medicine, protease inhibitors can be used as diagnostic or therapeutic agents for viral, bacterial, fungal and parasitic diseases as well as for treating cancer and immunological, neurodegenerative and cardiovascular diseases. They can be involved in crop protection against plant pathogens and herbivorous pests as well as against abiotic stress such as drought. Furthermore, protease inhibitors are indispensable in protein purification procedures to prevent undesired proteolysis during heterologous expression or protein extraction. They are also valuable tools for simple and effective purification of proteases, using affinity chromatography. Because there are such a large number and diversity of proteases in prokaryotes, yeasts, filamentous fungi and mushrooms, we can expect them to be a rich source of protease inhibitors as well.

The South African national combination antiretroviral therapy (cART) roll-out program started in 2006, with over 4.4 million people accessing treatment since it was first introduced. HIV-1 drug resistance can hamper the success of cART. This study determined the patterns of HIV-1 drug-resistance associated mutations (RAMs) in People Living with HIV-1 (PLHIV-1). Receiving first (for children below 3 years of age) and second-line (for adults) cART regimens in South Africa. During 2017 and 2018, 110 patients plasma samples were selected, 96 samples including those of 17 children and infants were successfully analyzed. All patients were receiving a boosted protease inhibitor (bPI) as part of their cART regimen. The viral sequences were analyzed for RAMs through genotypic resistance testing. We performed genotypic resistance testing (GRT) for Protease inhibitors (PIs), Reverse transcriptase inhibitors (RTIs) and Integrase strand transfer inhibitors (InSTIs). Viral sequences were subtyped using REGAv3 and COMET. Based on the PR/RT sequences, HIV-1 subtypes were classified as 95 (99%) HIV-1 subtype C (HIV-1C) while one sample as 02_AG. Integrase sequencing was successful for 89 sequences, and all the sequences were classified as HIV-1C (99%, 88/89) except one sequence classified CRF02_AG, as observed in PR/RT. Of the 96 PR/RT sequences analyzed, M184V/I (52/96; 54%) had the most frequent RAM nucleoside reverse transcriptase inhibitor (NRTI). The most frequent non-nucleoside reverse transcriptase inhibitor (NNRTI) RAM was K103N/S (40/96, 42%). Protease inhibitor (PI) RAMs M46I and V82A were present in 12 (13%) of the sequences analyzed. Among the InSTI major RAM two (2.2%) sequences have Y143R and T97A mutations while one sample had T66I. The accessory RAM E157Q was identified in two (2.2%). The data indicates that the majority of the patients failed on bPIs didn't have any mutation; therefore adherence could be major issue in these groups of individuals. We propose continued viral load monitoring for better management of infected PLHIV.

HIV-1 can develop resistance to antiretroviral drugs, mainly through mutations within the target regions of the drugs. In HIV-1 protease, a majority of resistance-associated mutations that develop in response to therapy with protease inhibitors are found in the protease's active site that serves also as a binding pocket for the protease inhibitors, thus directly impacting the protease-inhibitor interactions. Some resistance-associated mutations, however, are found in more distant regions, and the exact mechanisms how these mutations affect protease-inhibitor interactions are unclear. Furthermore, some of these mutations, e.g. N88S and L76V, do not only induce resistance to the currently administered drugs, but contrarily induce sensitivity towards other drugs. In this study, mutations N88S and L76V, along with three other resistance-associated mutations, M46I, I50L, and I84V, are analysed by means of molecular dynamics simulations to investigate their role in complexes of the protease with different inhibitors and in different background sequence contexts.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in millions of infections worldwide. While the search for an effective antiviral is still ongoing, experimental therapies based on repurposing of available antivirals is being attempted, of which HIV protease inhibitors (PIs) have gained considerable interest. Inhibition profiling of the PIs directly against the viral protease has never been attempted in vitro, and while few studies reported an efficacy of lopinavir and ritonavir in SARS-CoV-2 context, the mechanism of action of the drugs remains to be validated.

The use of protease inhibitors in human immunodeficiency virus type 1 (HIV-1) treatment is limited by adverse effects, including metabolic complications. To address these challenges, efforts are underway in the pursuit of more potent and less toxic HIV-1 protease inhibitors. Repurposing existing drugs offers a promising avenue to expedite the drug discovery process, saving both time and costs compared to conventional de novo drug development. This study screened FDA-approved and investigational drugs in the DrugBank database for their potential as HIV-1 protease inhibitors. Molecular docking studies and cell-based assays, including anti-HIV-1 in vitro assays and XTT cell viability tests, were conducted to evaluate their efficacy. The study findings revealed that CBR003PS, an antibiotic currently in clinical use, and CBR013PS, an investigational drug for treating endometriosis and uterine fibroids, exhibited significant binding affinity to the HIV-1 protease with high stability. Their EC50 values, measured at 100% cell viability, were 9.4 nM and 36.6 nM, respectively. Furthermore, cell-based assays demonstrated that these two compounds showed promising results, with therapeutic indexes higher than 32. In summary, based on their favorable therapeutic indexes, CBR003PS and CBR013PS show potential for repurposing as HIV-1 protease inhibitors.

Mice deficient for the fibulin-5 gene (Fbln5(-/-)) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/-) mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/-) mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5(-/-) and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5(-/-) epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.

The COVID-19 pandemic has urged for the repurposing of existing drugs for rapid management and treatment. Renin inhibitors down regulation of ACE2, which is an essential receptor for SARS-CoV-2 infection that is responsible for COVID-19, in addition to their ability to act as protease inhibitors were encouraging aspects for their investigation as possible inhibitors of main protease of SARS-CoV-2 via computational studies. A Pharmacophore model was generated using the newly released SARS-COV-2 main protease inhibitors. Virtual screening was performed on renin inhibitors, and Drug likeness filter identified remikiren and 0IU as hits. Molecular docking for both compounds showed that the orally active renin inhibitor remikiren (Ro 42-5892) of Hoffmann-La Roche exhibited good molecular interaction with Cys145 and His41 in the catalytic site of SARS-CoV-2 main protease. Molecular dynamics simulation suggested that the drug is stable in the active site of the enzyme.

Small-molecule peptidomimetic inhibitors that already showed activity towards Secreted aspartic protease 2 as anti-Candida agents are herein presented as candidate HIV protease inhibitors. A library of 6,8-dioxa-3-azabicyclo[3.2.1]-octane peptidomimetic scaffolds was screened towards HIV protease, resulting in the identification of hit compounds possessing IC50 in the sub-micromolar range, and showing the bicyclic acetal portion as a potential transition state analogue in the interaction with catalytic aspartic acid residues.

Protease inhibitor (PI) may cause abnormal glucose metabolism, abnormal lipid metabolism, and metabolic syndrome in HIV-infected adults but less well studied in Asian adolescents. This cross-sectional study evaluated anthropometric factors, oral glucose tolerance test, and lipid profiles of perinatally HIV-infected Thai adolescents who had received PI-based antiretroviral therapy for at least 6 months. Eighty adolescents were enrolled [median (IQR) age 16.7 (14.6-18.0) years, 42 males]. Metabolic syndrome, prediabetes, and type 2 diabetes mellitus (T2DM) were found in 8 (10%), 17 (22.1%), and 3 (3.8%) adolescents, respectively. Dyslipidemia was found in 56 (70%) adolescents, with hypertriglyceridemia being the most common type. In multivariate analysis, presence of lipohypertrophy (OR: 25.7, 95% CI: 3.2-202.8; p = 0.002) and longer duration of PI use (OR: 1.04, 95% CI: 1.00-1.08; p = 0.023) were associated with metabolic syndrome. Obesity (OR: 7.71, 95% CI: 1.36-43.7; p = 0.021), presence of lipohypertrophy (OR: 62.9, 95% CI: 4.97-795.6; p = 0.001), and exposure to stavudine for ≥6 months (OR: 8.18, 95% CI: 1.37-48.7; p = 0.021) were associated with prediabetes/T2DM, while exposure to tenofovir for ≥6 months reduced the risk (OR: 0.17, 95% CI: 0.04-0.78; p = 0.022). Metabolic disorders were commonly found in adolescents receiving PI. Careful monitoring and early intervention to modify cardiovascular risk should be systematically implemented in this population particularly those with exposure to stavudine.

In this paper we investigate 10 different HIV protease inhibitors (HPIs) as possible repurposed-drugs candidates against SARS-CoV-2. To this end, we execute molecular docking and molecular dynamics simulations. The in silico data demonstrated that, despite their molecular differences, all HPIs presented a similar behavior for the parameters analyzed, with the exception of Nelfinavir that showed better results for most of the molecular dynamics parameters in comparison with the N3 inhibitor.

Based on previous large-scale in silico screening several factor Xa inhibitors were proposed to potentially inhibit SARS-CoV-2 Mpro. In addition to their known anticoagulants activity this potential inhibition could have an additional therapeutic effect on patients with COVID-19 disease. In this study we examined the binding of the Apixaban, Betrixaban and Rivaroxaban to the SARS-CoV-2 Mpro with the use of the MicroScale Thermophoresis technique. Our results indicate that the experimentally measured binding affinity is weak and the therapeutic effect due to the SARS-CoV-2 Mpro inhibition is rather negligible.

Chikungunya fever results from an infection with Chikungunya virus (CHIKV, genus Alphavirus) that is prevalent in tropical regions and is spreading fast to temperate climates with documented outbreaks in Europe and the Americas. Currently, there are no available vaccines or antiviral drugs for prevention or treatment of Chikungunya fever. The nonstructural proteins (nsPs) of CHIKV responsible for virus replication are promising targets for the development of new antivirals. This study was attempted to find out new potential inhibitors of CHIKV nsP2 protease using the ligand-based drug design. Two compounds 10 and 10c, identified by molecular docking, showed antiviral activity against CHIKV with IC50 of 13.1 and 8.3 μM, respectively. Both compounds demonstrated the ability to inhibit the activity of nsP2 in a cell-free assay, and the impact of compound 10 on virus replication was confirmed by western blot. The molecular dynamics study of the interactions of compounds 10 and 10c with CHIKV nsP2 showed that a possible mechanism of action of these compounds is the blocking of the active site and the catalytic dyad of nsP2.