scholarly journals Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures

2021 ◽  
Author(s):  
Zhengnan Shen ◽  
Kiira Ratia ◽  
Laura Cooper ◽  
Deyu Kong ◽  
Hyun Lee ◽  
...  
Keyword(s):  
Viral Replication ◽  
Cell Cultures ◽  
Cysteine Protease ◽  
Antiviral Agents ◽  
Cysteine Proteases ◽  
Positive Control ◽  
Host Protein ◽  
Structure Based Drug Design ◽  
Active Site Cysteine ◽  
Starting Point

ABSTRACTAntiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 µM). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a “BL2 groove” that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

scholarly journals A Novel Cysteine Protease Inhibitor of Naegleria fowleri That Is Specifically Expressed during Encystation and at Mature Cysts

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 388
Author(s):  
Hương Giang Lê ◽  
A-Jeong Ham ◽  
Jung-Mi Kang ◽  
Tuấn Cường Võ ◽  
Haung Naw ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Expression Profiles ◽  
Critical Role ◽  
Cyst Formation ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Naegleria Fowleri ◽  
Nægleria Fowleri ◽  
Cystatin Family

Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.

Hepatoma Cell Cultures as In Vitro Models for the Hepatotoxicity of Xenobiotics

1988 ◽  
Vol 16 (1) ◽  
pp. 32-37
Author(s):  
Margherita Ferro ◽  
Anna Maria Bassi ◽  
Giorgio Nanni
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Membrane Lipids ◽  
Hepatoma Cell ◽  
Positive Control ◽  
In Vitro Models ◽  
Low Concentrations ◽  
Formation Efficiency ◽  
Dose Dependent

Two hepatoma cell cultures were examined as in vitro models to be used in genotoxicity and cytotoxicity tests without the addition of bioactivating enzymes. The MH1C1, and HTC hepatoma lines were used in this study to establish their sensitivity to a number of xenobiotics, namely, cyclophosphamide (CP), the classical positive control in bioactivation tests; benzaldehyde (BA), a short-chain aldehyde; and 4-hydroxynonenal (HNE), a major toxic end-product of the peroxidative degradation of cell membrane lipids. As a first approach, we compared the following cytotoxicity tests: release of lactate dehydrogenase (LDH), and colony formation efficiency (CF). Colony-forming cells were exposed to the drugs according to different procedures, before or after the anchorage phase. The leakage of LDH into the medium following exposure of both cell lines to HNE, CP and BA for up to 24 hours was found not to be a good index of cytotoxicity. A better indicator of cytotoxicity was CF, as evaluated by exposure of the cells 24 hours after seeding. The effects were detectable at very low concentrations, corresponding to 10, 90 and 100μM for HNE, CP and BA, respectively. The impairment of CF efficiency was dose-dependent and time-dependent, and several differences between the two cell lines were observed.

Expression of Cysteine Protease Protein 32 in Prostatic Adenocarcinoma Correlates With Tumor Grade

2004 ◽  
Vol 128 (6) ◽  
pp. 649-652
Author(s):  
Shahgul Anwar ◽  
Robert A. Ambros ◽  
Timothy A. Jennings ◽  
Jeffrey S. Ross ◽  
Anton Beza ◽  
...  
Keyword(s):  
Gleason Score ◽  
Cysteine Protease ◽  
Tumor Grade ◽  
Cysteine Proteases ◽  
Prostatic Adenocarcinoma ◽  
Gleason Grade ◽  
High Grade ◽  
Cytoplasmic Staining ◽  
Apoptosis Pathway ◽  
High Gleason Score

Abstract Context.—Controlled cell death is mediated by apoptosis-specific genes, tumor suppressor genes, and oncogenes. The caspase family is a group of at least 15 known cysteine proteases that serve as initiator and effector molecules of the apoptosis pathway. On activation, caspases cause cell shrinkage, condensation of chromatin, fragmentation of DNA, and the formation of blebs in the cytoplasmic membrane. Objectives.—The patterns of cysteine protease protein (CCP) 32 (caspase-3) expression have been determined in normal human tissues and a variety of tumors, and have been shown to correlate with the outcome in breast cancer and linked to resistance to chemotherapy in other tumors. This study was performed to determine whether CPP32 is expressed in prostatic adenocarcinoma and to define its relationship with outcome variables. Design.—Formalin-fixed, paraffin-embedded radical prostatectomy specimens from 211 patients with prostatic adenocarcinoma were evaluated for CPP32 expression by immunohistochemistry. Hematoxylin-eosin–stained slides were reviewed, and tumors were graded based on the Gleason grading system. Tumors were scored for CPP32 expression semiquantitatively, based on the staining intensity and distribution patterns. These results were compared with Gleason grade and clinical and pathologic stages. Results.—One hundred thirty-three (63%) of 211 cases showed high expression of CPP32, whereas expression was low in 78 (37%) cases. One hundred three (49%) of 211 cases had a high Gleason score (7 and above). Of 103 cases with a high Gleason score, 74 (72%) showed high CPP32 expression. Strong cytoplasmic staining for CPP32 in high-grade tumors was statistically significant (P = .01). Also, by linear regression analysis a significant correlation was seen between the Gleason score and the cytoplasmic CPP32 expression (P = .001). Expression of CPP32 did not correlate with either clinical stage (P = .28) or pathologic stage (P = .60); however, this study included very few patients with stage IV disease. Conclusion.—The correlation between CPP32 and high tumor grade suggests a CPP32-related high turnover rate in high-grade prostatic adenocarcinoma. Moreover, strong correlation with Gleason grade, a powerful predictor of disease progression and overall survival, suggests potential usefulness of CPP32 as a prognostic factor, especially in limited biopsy samples.

scholarly journals Structure-Based Drug Design and Characterization of Sulfonyl-Piperazine Benzothiazinone Inhibitors of DprE1 from Mycobacterium tuberculosis

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Jérémie Piton ◽  
Anthony Vocat ◽  
Andréanne Lupien ◽  
Caroline S. Foo ◽  
Olga Riabova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Design ◽  
Antitubercular Activity ◽  
Cysteine Residue ◽  
Drug Candidate ◽  
Structure Based Drug Design ◽  
Content Type ◽  
Active Site Cysteine ◽  
Covalent Adducts

ABSTRACT Macozinone (MCZ) is a tuberculosis (TB) drug candidate that specifically targets the essential flavoenzyme DprE1, thereby blocking synthesis of the cell wall precursor decaprenyl phosphoarabinose (DPA) and provoking lysis of Mycobacterium tuberculosis. As part of the MCZ backup program, we exploited structure-guided drug design to produce a new series of sulfone-containing derivatives, 2-sulfonylpiperazin 8-nitro 6-trifluoromethyl 1,3-benzothiazin-4-one, or sPBTZ. These compounds are less active than MCZ but have a better solubility profile, and some derivatives display enhanced stability in microsomal assays. DprE1 was efficiently inhibited by sPBTZ, and covalent adducts with the active-site cysteine residue (C387) were formed. However, despite the H-bonding potential of the sulfone group, no additional bonds were seen in the crystal structure of the sPBTZ-DprE1 complex with compound 11326127 compared to MCZ. Compound 11626091, the most advanced sPBTZ, displayed good antitubercular activity in the murine model of chronic TB but was less effective than MCZ. Nonetheless, further testing of this MCZ backup compound is warranted as part of combination treatment with other TB drugs.

scholarly journals A Parasite Cysteine Protease Is Key to Host Protein Degradation and Iron Acquisition

2008 ◽  
Vol 283 (43) ◽  
pp. 28934-28943 ◽  
Author(s):  
Theresa C. O'Brien ◽  
Zachary B. Mackey ◽  
Richard D. Fetter ◽  
Youngchool Choe ◽  
Anthony J. O'Donoghue ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Cysteine Protease ◽  
Iron Acquisition ◽  
Host Protein

scholarly journals 2-Acryloyl-4,5-methylenedioxyphenol: A Small Molecule Endowed with Antidermatophytic Activity

2019 ◽  
Vol 16 (4) ◽  
pp. 461-466
Author(s):  
Marco Zuccolo ◽  
Sabrina Dallavalle ◽  
Raffaella Cincinelli ◽  
Luce Mattio ◽  
Stefania Mazzini ◽  
...  
Keyword(s):  
Small Molecule ◽  
Natural Compound ◽  
Fungal Infections ◽  
Positive Control ◽  
Fungal Species ◽  
Fungal Diseases ◽  
Antifungal Compounds ◽  
Starting Point ◽  
Antidermatophytic Activity

Background: Superficial fungal infections are the most common fungal diseases in humans, affecting more than 25% of the population worldwide. Methods: In the present study, we have investigated the activity of kakuol, a natural compound isolated from the rhizomes of Asarum sieboldii, and some analogues, against various dermatophytes and pharmacologically relevant yeasts. Results: One of the tested compounds, 2-acryloyl-4,5-methylenedioxyphenol, showed a broadspectrum activity against most of the fungal species assayed, resulting particularly effective against dermatophyte strains (MIC values in the range of 0.25-0.5 &#181;g/mL, two/four-fold lower than the positive control miconazole). Conclusion: The results suggest that this molecule can be considered a promising starting point for the development of new antifungal compounds.

Influenza A viruses balance ER stress with host protein synthesis shutoff

2021 ◽  
Vol 118 (36) ◽  
pp. e2024681118
Author(s):  
Beryl Mazel-Sanchez ◽  
Justyna Iwaszkiewicz ◽  
Joao P. P. Bonifacio ◽  
Filo Silva ◽  
Chengyue Niu ◽  
...  
Keyword(s):  
Viral Replication ◽  
Er Stress ◽  
Host Cell ◽  
Messenger Rna ◽  
Influenza A ◽  
Nonstructural Protein ◽  
Host Protein ◽  
Stress Induction ◽  
Nonstructural Protein 1

Excessive production of viral glycoproteins during infections poses a tremendous stress potential on the endoplasmic reticulum (ER) protein folding machinery of the host cell. The host cell balances this by providing more ER resident chaperones and reducing translation. For viruses, this unfolded protein response (UPR) offers the potential to fold more glycoproteins. We postulated that viruses could have developed means to limit the inevitable ER stress to a beneficial level for viral replication. Using a relevant human pathogen, influenza A virus (IAV), we first established the determinant for ER stress and UPR induction during infection. In contrast to a panel of previous reports, we identified neuraminidase to be the determinant for ER stress induction, and not hemagglutinin. IAV relieves ER stress by expression of its nonstructural protein 1 (NS1). NS1 interferes with the host messenger RNA processing factor CPSF30 and suppresses ER stress response factors, such as XBP1. In vivo viral replication is increased when NS1 antagonizes ER stress induction. Our results reveal how IAV optimizes glycoprotein expression by balancing folding capacity.

Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor inTrypanosoma cruzi

2001 ◽  
Vol 114 (21) ◽  
pp. 3933-3942 ◽  
Author(s):  
Ana C. S. Monteiro ◽  
Magnus Abrahamson ◽  
Ana P. C. A. Lima ◽  
Marcos A. Vannier-Santos ◽  
Julio Scharfstein
Keyword(s):  
Cell Surface ◽  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Mammalian Cells ◽  
Tight Binding ◽  
Cysteine Proteases ◽  
Host Cells ◽  
Cysteine Protease Inhibitor ◽  
Reversible Inhibitor ◽  
Mammalian Host

Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas’ heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.

scholarly journals Benzimidazole inhibitors of the major cysteine protease of Trypanosoma brucei

2019 ◽  
Vol 11 (13) ◽  
pp. 1537-1551 ◽  
Author(s):  
Glaécia AN Pereira ◽  
Lucianna H Santos ◽  
Steven C Wang ◽  
Luan C Martins ◽  
Filipe S Villela ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Cysteine Proteases ◽  
African Trypanosomiasis ◽  
High Similarity ◽  
Trypanosoma Brucei Brucei ◽  
Structure Activity ◽  
Starting Point ◽  
Competitive Inhibitors ◽  
Enzyme Selectivity

Aim: Limitations in available therapies for trypanosomiases indicate the need for improved medicines. Cysteine proteases cruzain and rhodesain are validated targets for treatment of Chagas disease and human African trypanosomiasis. Previous studies reported a benzimidazole series as potent cruzain inhibitors. Results & methodology: Considering the high similarity between these proteases, we evaluated 40 benzimidazoles against rhodesain. We describe their structure-activity relationships (SAR), revealing trends similar to those observed for cruzain and features that lead to enzyme selectivity. This series comprises noncovalent competitive inhibitors (best Ki = 0.21 μM against rhodesain) and micromolar activity against Trypanosoma brucei brucei. A cheminformatics analysis confirms scaffold novelty, and the inhibitors described have favorable predicted physicochemical properties. Conclusion: Our results support this series as a starting point for new human African trypanosomiasis medicines.

scholarly journals Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly

2020 ◽  
Vol 117 (2) ◽  
pp. 1049-1058 ◽  
Author(s):  
Xingjian Xu ◽  
Raquel Godoy-Ruiz ◽  
Kaylin A. Adipietro ◽  
Christopher Peralta ◽  
Danya Ben-Hail ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Receptor Binding ◽  
Treatment Options ◽  
Binding Domain ◽  
Binary Toxin ◽  
Receptor Binding Domain ◽  
Design Strategies ◽  
Recurrence Rates ◽  
Structure Based Drug Design ◽  
Starting Point

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.

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