REVIEW | doi:10.20944/preprints201907.0286.v1
Subject: Biology And Life Sciences, Virology Keywords: HIV-1 Gag; Gag inhibitors; Protease; Protease inhibitors; drug resistance mutations; drug design
Online: 25 July 2019 (10:05:03 CEST)
HIV treatment strategies against viral enzymes are continuously hampered by viral drug resistance. Recent findings show that viral substrate Gag contributes to HIV-1 Protease Inhibitor (PI) resistance, leading to demands for new strategies in HIV treatment where Gag is recognized as a drug target. To successfully target Gag, there is a need of in-depth understanding of the Gag polyprotein and the effects of Gag mutations. Here, we propose new strategies in designing novel Gag inhibitors against existing and novel emerging Gag mutations via a structural understanding of the Gag-Protease relationship in PI resistance. In this review, we discuss the role of both novel and previously reported mutations, revealing insights to how they aid in PI resistance, and how new Gag inhibitors can be designed.
ARTICLE | doi:10.20944/preprints202005.0136.v1
Subject: Biology And Life Sciences, Virology Keywords: deubiquitination; leukemia; ubiquitin-specific protease 2 (USP2); SARS-CoV-2 papain-like protease (PLpro); COVID-19
Online: 8 May 2020 (03:45:22 CEST)
The ubiquitin-specific protease 2 (USP) belongs to the family of deubiquitinases and plays a critical role in tumors cells’ survival and therefore signifies an important therapeutic target. Previous studies have indicated promising efficacies of potent human USP2 inhibitors including, thiopurine analogues against SARS-CoV papain-like proteases (PLpro). The PLpro have significant functional implications in the innate immune response during SARS-CoV-2 infection and considered an important antiviral target. Both proteases share strikingly similar USP fold with right-handed thumb–palm–fingers structural scaffold and conserved catalytic triad Cys-His-Asp/Asn. In this urgency situation of COVID-19 outbreak, there is a lack of in-vitro facilities readily available to test SARS-CoV-2 inhibitors in whole-cell assays. Therefore, we adopted an alternate route to identify potential USP2 inhibitor through integrated structure-based virtual screening efforts. After a subsequent virtual screening protocol, the best compounds were selected and tested. The compound Z93 showed significant IC50 value against Jurkat (9.67 µM) and MOTL-4 cells (11.8 µM). The binding mode of Z93 was extensively analyzed through molecular docking, followed by MD simulations, and molecular interactions were compared with SARS-CoV-2. The relative binding poses of Z93 fitted well in the binding site of both proteases and showed consensus π-π stacking and H-bond interactions with histidine and aspartate/asparagine residues of the catalytic triad. These results led us to speculate that compound Z93 might be the first potential chemical lead against SARS-CoV-2 PLpro, which warrants in-vitro evaluations.
REVIEW | doi:10.20944/preprints202204.0065.v1
Subject: Chemistry And Materials Science, Medicinal Chemistry Keywords: COVID-19; Main protease; Mpro; inhibitors
Online: 8 April 2022 (03:01:37 CEST)
Since the outbreak of COVID-19, one of the strategies used to search for new drugs has been to find inhibitors of the main protease (Mpro) of virus SARS-CoV-2. Initially, previously reported inhibitors of related proteases like the main proteases of SARS-CoV and MERS-CoV were tested. Then a huge effort has been done by the scientific community to design, synthesize and test new small molecules acting as inactivators of SARS-CoV-2 Mpro. From the structure view, these compounds can be classified into two main groups: one corresponds to modified peptides displaying an adequate sequence for high affinity and a reactive warhead, and the second one is a diverse group including chemical compounds which do not have a peptide framework. Although a drug including a SARS-CoV-2 main protease has already been commercialized, denoting the importance of this field, more compounds have been demonstrated to be promising potent inhibitors as potential antiviral drugs.
ARTICLE | doi:10.20944/preprints202009.0390.v2
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Recombinant protein; Protease; DPP4; SARS-CoV-2; MERS-CoV
Online: 17 November 2020 (11:38:02 CET)
Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS-CoV, SARS-CoV-2 does not bind human DPP4.
ARTICLE | doi:10.20944/preprints201801.0106.v2
Subject: Chemistry And Materials Science, Analytical Chemistry Keywords: catalytide; serine protease-like peptide; Tob1
Online: 17 January 2018 (07:27:30 CET)
After screening nearly 1000 synthetic peptides, a synthetic peptide termed JAL-AK22 (KYEGHWYPEKPYKGSGFRCIHI) derived from the BoxA domain in Tob1 protein was found to activate both unfolded and folded proMMP-7. In addition, JAL-AK22 showed auto-proteolytic activity. Interestingly, the smaller derivative of JAL-AK22 termed JAL-TA9 (YKGSGFRMI) also possessed auto-proteolytic activity and cleaved 2 fragment peptides (MMP18-33 and MMP18-40) derived from the prodomain of proMMP-7 under physiological conditions. These proteolytic activities were inhibited by AEBSF, a serine protease inhibitor. Our results demonstrate that a small synthetic peptide consisting of only 9 amino acids has serine protease-like activity and activates proMMP-7 by cleaving the prodomain region. We thus propose calling small peptides possessing with protease-like activity Catalytides (catalytic peptides). We expect that our findings will stimulate the development of novel Catalytides and related applications.
REVIEW | doi:10.20944/preprints202205.0316.v1
Subject: Biology And Life Sciences, Virology Keywords: HIV; protease; CARD8; NNRTI; Inflammasome; Latent reservoir
Online: 24 May 2022 (03:54:52 CEST)
HIV-1 protease (PR) is a viral enzyme that cleaves viral polyprotein precursors to convert them into functional forms, a process essential to generate infectious viral particles. Due to its broad substrate specificity, HIV-1 PR can also cleave certain host cell proteins. Several studies have identified host cell substrates of HIV-1 PR and described the potential impact of their cleavage on HIV-1-infected cells. Of particular interest is the interaction between PR and the caspase recruitment domain-containing protein 8 (CARD8) inflammasome. While PR typically has low levels of intracellular activity prior to viral budding, induction of premature PR activation to trigger CARD8-mediated cell killing may help eliminate latent reservoirs in people living with HIV. In this review, we discuss the viral and host substrates of HIV-1 protease and highlight potential applications and advantages of targeting CARD8 sensing of HIV-1 PR.
ARTICLE | doi:10.20944/preprints202004.0015.v2
Subject: Medicine And Pharmacology, Pharmacology And Toxicology Keywords: virtual screening; COVID-19; Protease 3CL pro
Online: 3 April 2020 (15:08:50 CEST)
Ligand and structure based virtual screening approaches were applied to clinical stage drugs as well as those approved for human use in an attempt to repurpose drugs for potential use against COVID-19. This approach involved ligand-based shape similarity searches, structure-based docking and pharmacophore searches with the help of pharmacophore queries derived from available ligands and receptor structures. Several compounds appeared as hits in pharmacophore and shape similarity searches and those docking to the SARS-CoV-2 viral 3CL protease were then ranked on the basis of docking scores.
ARTICLE | doi:10.20944/preprints202305.0957.v1
Subject: Environmental And Earth Sciences, Soil Science Keywords: enzyme degradation; nitrogen; phosphorus; protease; forest soil; tropics
Online: 12 May 2023 (13:45:17 CEST)
Soil enzyme activity can be affected by both production and degradation, as enzymes can be degraded by proteases. However, the impact of nutrient addition on enzyme activity is often solely attributed to changes in enzyme production without fully considering degradation. Here, we demonstrated that the activities of β-1,4-glucosidase (BG), β-D-cellobiohydrolase (CBH), β-1,4-Xylosidase (BX), and β-1,4-N-acetyl-glucosaminnidase (NAG) were comparable in nitrogen (N) and phosphorus (P) fertilized soils and the unfertilized control under field conditions, but the reduction in activity was substantially greater in the fertilized soils during short-term laboratory incubation. The results show that the interruption of the natural, continuous supply of organic matter or non-soil microbial-derived enzymes, which typically occurs under field conditions, leads to a more significant reduction in apparent enzyme activities in fertilized soils compared to unfertilized control. This may be attributed to the higher abundance of protease in fertilized soils, resulting in faster enzyme degradation. Interestingly, P fertilization alone did not have a similar effect, indicating that N fertilization is likely the main cause of the larger decreases in enzyme activity during incubation in fertilized soils compared to unfertilized control soils, despite our study site being poor in P and rich in N. These findings highlight the importance of considering enzyme degradation when investigating material dynamics in forest ecosystems, including the impact of nutrient addition on enzyme activity, as enzyme production alone may not fully explain changes in apparent enzyme activity.
ARTICLE | doi:10.20944/preprints202302.0304.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: Urban garden; protease; lipase; DNAse; lipase; Spatial statistics.
Online: 17 February 2023 (07:44:32 CET)
The enzymatic activities of bacterial isolates have been widely studied, but a spatial distribution of this activity is rarely focused. New antibiotic-producing microorganisms can be discovered in a more efficient manner if spatial statistical techniques are applied to the distribution of the activity of bacterial isolates in different terrains. This study is focused to generate a series of maps illustrating the spatial distribution of different soil parameters and the inhibition against relative-safe pathogens (like Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella, Pseudomonas fluorencens and Enterobacter cloacae) in an urban park in València (Spain). This spatial data has demonstrated a higher proportion of isolates was specifically allocated. This routine of data presentation is valuable for understanding bacterial population dynamics at a local level.
Subject: Medicine And Pharmacology, Epidemiology And Infectious Diseases Keywords: SARS-Cov-2; main protease; Spike protein; RIN
Online: 15 June 2020 (06:27:35 CEST)
Motivation: The SARS-Cov-2 pandemic has gripped the entire world and a race to find either a cure or a vaccine for this pandemic is on. The public databases have a deluge of information in terms of genomic sequences and protein structures making it possible to study the minute details in terms of conserved motifs and super-motifs in its proteins. In this study we have identified the hidden features of the Spike protein and the Main protease (Mpro) of SARS-Cov2. These domains have been identified using the standard bioinformatics tools and the pfam database. We found four domains reported in the pfam database are present in the spike protein and the Mpro of SARS-Cov2 but have not been reported earlier. These domains are specific to human strains of SARS-Cov2 and are not present in SARS-Cov or the coronaviruses of other animals. Using RIN we also identified the motifs and super-motifs in these two proteins that are important in understanding species wise changes as well as evolution driven variation in amino acids. Our results highlight several interesting features of the spike protein and Mpro of SARS-Cov2 that can be exploited for the development of various drug and vaccine therapies.
ARTICLE | doi:10.20944/preprints202002.0438.v1
Subject: Chemistry And Materials Science, Medicinal Chemistry Keywords: COVID-19; Simeprevir; Protease inhibitor; Virtual screening; Docking
Online: 28 February 2020 (13:14:38 CET)
Coronavirus disease 2019 (COVID-19) has been first appeared in Wuhan, China but its fast transmission, led to its widespread prevalence in various countries and make it a global concern. In addition, lack of a definitive treatment is another concern that needs to be attention. Researchers have come up with several options, which are not certain, but protease inhibitor and some antiviral agent are in the forefront. In this study a virtual screening procedure employing docking of different databases including 1615 FDA approved drugs was used to identify new potential small molecule inhibitors for protease protein of COVID-19. The docking result indicates that among all, simeprevir (Hepatitis C virus (HCV) NS3/4A protease inhibitor) could fit well to the binding pocket of protease and because of some other positive features including ADME profile, might be a helpful treatment option for COVID-19.
ARTICLE | doi:10.20944/preprints201910.0026.v1
Subject: Biology And Life Sciences, Insect Science Keywords: ticks; Ixodes scapularis; serine protease; blood digestion; trypsin
Online: 2 October 2019 (08:56:27 CEST)
Ixodes scapularis is the major vector of Lyme disease in the eastern United States. This species undergoes a life cycle consisting of eggs and three active stages: larva, nymph, and adult. Each active life stage takes a blood meal either for developing and molting to the next stage (larvae and nymphs) or for oviposition (adult females). This protein rich blood meal is the only food taken by Ixodes ticks. Most studies on blood digestion in ticks have shown that the initial stages of blood digestion are carried out by cathepsin proteases within endosomes of acidic digestive cells. However, in other hematophagous arthropods, the serine protease trypsin plays an important role in early protein degradation. In this study, we determined transcript expression of I. scapularis cathepsins and serine proteases, some with previously characterized roles in blood digestion. Gut pH was also determined and a trypsin-benzoyl-D, L-arginine 4-nitoanilide assay was used to measure active trypsin levels during blood digestion. Our data suggest that trypsin levels increase significantly after blood feeding and peaked in larvae, nymphs, and adults at 3, 1, and 1 days post host detachment, respectively. In addition, alkaline gut pH (8.0) conditions after I. scapularis blood feeding were similar to those required for trypsin activity in other arthropods suggesting these enzymes have an important and previously overlooked role in I. scapularis blood digestion.
REVIEW | doi:10.20944/preprints202305.1317.v1
Subject: Chemistry And Materials Science, Food Chemistry Keywords: Starchy food; potato protease inhibitors; phytochemicals; antioxidants; cancer prevention
Online: 18 May 2023 (09:48:15 CEST)
Potato (Solanum tuberosum L) gradually becomes a stable food worldwide since it can be treated as practical nutritional supplement and antioxidant, as well as energy provider for human beings. Financially and nutritionally, cultivation and utility of potatoes is worthy enough attention of the world. To further explore functionality and maximize utilization of component parts, also to develop new products based on potato still be an ongoing issue. To maximize the benefits of potato and induce new high-value products while avoiding unfavorable properties of the crop has been a growing trend in food and medical areas. This review intends to summarize the factors that influence the changes in the key functional components of potatoes and discuss referred research focuses, then help further researchers to make efforts into. Afterwards, it summarizes the application of the latest commercial products and potential value of components existing in potato. Particularly, there are several main tasks for the next potato research: preparing starchy foods for special group of people and developing fiber-rich products to supply the dietary fiber intake as well as manufacturing bio-friendly and specific design films/coating in packaging industry; extracting bioactive proteins and potato protease inhibitors with high biological activity, and continue to build new commercial products based on potato protein and examine their health benefits. Noteworthy, the preservation methods play a key role in phytochemicals contents left in foods and potato performs superior to many common vegetables when meeting demand of mineral daily intake and alleviating mineral deficiencies.
ARTICLE | doi:10.20944/preprints202102.0159.v1
Subject: Biology And Life Sciences, Anatomy And Physiology Keywords: Enterovirus; Coxsackievirus; 2A protease; polyclonal antibody; type 1 diabetes
Online: 5 February 2021 (11:34:57 CET)
The need for antiserum for immunohistochemical (IHC) detection of enterovirus (EV) in formaldehyde fixed and paraffin-embedded (FFPE) specimens is increasing. The standard monoclonal antibody against EV-envelope protein (VP1), clone 5D8/1, was proven to cross-react with other proteins. Another candidate marker of EV proteins is 2A protease (2Apro), which is coded by the EV gene and translated by host cells during EV replication. We raised polyclonal antiserum by immunizing rabbits with an 18-mer peptide of Coxsackievirus B1 (CVB1)-2A protease (2Apro) and examined the specificity and sensitivity for EV on FFPE tissue samples. ELISA study showed a high titer of antibody for CVB1-2Apro. IHC demonstrated that antiserum against 2Apro reacted with CVB1-infected Vero-cells. Confocal microscopy demonstrated that 2Apro labelled by the antibody located in the same cell with VP1 stained with 5D8/1. IHC demonstrated dense positive reactions pancreatic islets of EV-associated fulminant type 1 diabetes (FT1DM), and located in the same cell stained positive with 5D8/1. Specificity of IHC staining FT1DM pancreas was confirmed by absorption with an excessive concentration of immunized peptide. In conclusion, our study provides a new polyclonal antiserum against CVB1 2Apro which may be useful for detection of EV-infected human tissues stored as archive of FFPE tissue samples.
ARTICLE | doi:10.20944/preprints202004.0329.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: SARS-CoV-2; COVID-19; protease; docking; pharmacophore; Zinc15
Online: 19 April 2020 (05:33:03 CEST)
A big race for the search for novel lead has begun due to the emergence of COVID-19 across the globe. More than 6,00,000 cases of afflicted patients worldwide has been reported till date with high mortality and morbidity. At present no approved drugs are known for COVID-19. Phylogenetic analysis present strong nucleotide sequence similarity of around 80% with SARS-CoV. Therefore, the drugs used for treating SARS-CoV and MERS are being used for SARS-CoV-2 also. Recently, the crystal structure of COVID-19 is reported and hence, we have used this tom predict the binding affinity with SARS-CoV-2-main protease and prepared a pharmacophore that may be used for future design of novel inhibitors.
BRIEF REPORT | doi:10.20944/preprints202002.0242.v2
Subject: Medicine And Pharmacology, Pharmacology And Toxicology Keywords: COVID-19; molecular docking; HIV protease inhibitor; nucleotide analogues
Online: 29 February 2020 (12:43:40 CET)
The outbreak of novel coronavirus (COVID-19) infections in 2019 is in dire need of finding potential therapeutic agents. In this study, we used molecular docking to repurpose HIV protease inhibitors and nucleoside analogues for COVID-19, with evaluations based on docking scores calculated by AutoDock Vina and RosettaCommons. Our results suggest that Indinavir and Remdesivir possess the best docking scores, and comparison of the docking sites of the two drugs reveal a near perfect dock in the overlapping region of the protein pockets. After further investigation of the functional regions inferred from the proteins of SARS coronavirus, we discovered that Indinavir does not dock on any active sites of the protease, which may give rise to concern in regards to the efficacy of Indinavir. On the other hand, the docking site of Remdesivir is not compatible with any known functional regions, including template binding motifs, polymerization motifs and nucleoside triphosphate (NTP) binding motifs. However, when we tested the active form (CHEMBL2016761) of Remdesivir, the docking site revealed a perfect dock in the overlapping region of the NTP binding motif. This result suggests that Remdesivir could be a potential therapeutic agent. Clinical trials still must be done in order to confirm the curative effect of these drugs.
ARTICLE | doi:10.20944/preprints202002.0061.v1
Subject: Medicine And Pharmacology, Medicine And Pharmacology Keywords: Coronavirus; Deep learning; Drug screening; homology modeling; main protease
Online: 5 February 2020 (10:59:09 CET)
A novel coronavirus called 2019-nCoV was recently found in Wuhan, Hubei Province of China, and now is spreading across China and other parts of the world. 2019-nCoV spreads more rapidly than SARS-CoV. Unfortunately, there is no drug to combat the virus. It is of high significance to develop a drug that can combat the virus effectively before the situation gets worse. It usually takes a much longer time to develop a drug using traditional methods. For 2019-nCoV, it is now better to rely on some alternative methods to develop drugs that can combat such a disease effectively since 2019-nCoV is highly homologous to SARS-CoV. In this paper, we first collected virus RNA sequences from the GISAID database, translated the RNA sequences into protein sequences, and built a protein 3D model using homology modeling. Coronavirus main protease is considered to be a major therapeutic target, thus this paper focused on drug screening based on the modeled 2019-nCov_main_protease structure. The deep learning based method DFCNN, developed by our group, can identify/rank the protein-ligand interactions with relatively high accuracy. DFCNN is capable of performing virtual screening quickly since no docking or molecular dynamic simulation is needed. DFCNN identifies potential drugs for 2019-nCoV protease by performing drug screening against 4 chemical compound databases. Also, we performed drug screening for all tripeptides against the binding site of 2019-nCov_main_protease since peptides often show better stability, more bio-availability and negligible immune responses. In the end, we provided the list of possible chemical ligands and peptide drugs for experimental validation.
ARTICLE | doi:10.20944/preprints201905.0319.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: cyanobacteria; protease inhibitors; digestive enzyme; daphnia; HPLC; UV/Vis
Online: 27 May 2019 (12:56:49 CEST)
Cyanobacterial mass developments in eutrophic ponds and lakes are a major concern for lake management, as many cyanobacteria produce a huge variety of toxic secondary metabolites, e.g. microcystins. The aim of this research was to culture a strain of the cyanobacterium Microcystis sp strain BM25, to observe its biomass production and to isolate and purify protease inhibitors from this cyanobacterial biomass. Different secondary metabolites were isolated following a standard bioassay-guideline. Isolation was performed, with an enzymatic protease assay as bioassay. High performance liquid chromatography was used to identify different fractions of secondary metabolite from the strain BM25. Moreover, protease homogenates were isolated from Daphnia magna in order to test the inhibitors against naturally occurring major digestive proteases trypsin and chymotrypsin. It was measured that 60% MeOH and the 80% MeOH C18-SPE fraction inhibits chymotrypsin activity 98% (6 nmol pNA min-1 mg-1) and 99 % (4 nmol pNA min-1 mg-1), respectively. In contrast, trypsin activity was not inhibited by methanolic extracts of this cyanobacterium strain.
ARTICLE | doi:10.20944/preprints202305.1103.v1
Subject: Engineering, Bioengineering Keywords: mutate HIV protease inhibitors; carboxamides; heteroarenes; mammalian cells essay; ADME
Online: 16 May 2023 (05:32:28 CEST)
The Virus HIV-1 infection still represents a serious disease even if actually it is transformed in chronic pathology. Considering the crucial role of the enzyme Protease in life cycle of HIV many efforts have been made in the research of new organic compounds showing inhibitory activity. After development of several series of non peptidic inhibitors we report here the synthesis of novel simple HIV-Protease inhibitors containing heteroaryl carboxamides and their antiviral activity in vitro and in HEK293 cells. Benzofuryl- benzothienyl- and indolyl rings as well as aryl sulfonamides with different electronic properties have been introduced by efficient synthetic procedures. All compounds showed inhibitory activity similar to the commercial drug Darunavir, effective against both wild-type HIV-1 protease and that containing the V32I or V82A mutations. ADME properties were also evaluated, showing the potential of such compounds to be developed as drugs.
ARTICLE | doi:10.20944/preprints202004.0003.v2
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; protease; virtual screening; pharmacophore; inhibitors; natural compounds
Online: 17 April 2020 (11:00:55 CEST)
Background: SARS-CoV-2 that are the causal agent of a current pandemic are enveloped, positive-sense, single-stranded RNA viruses of the Coronaviridae family. Proteases of SARS-CoV-2 are necessary for viral replication, structural assembly and pathogenicity. The ~33.8KDa Mpro protease of SARS-CoV-2 is a non-human homologue and highly conserved among several coronaviruses indicating Mpro could be a potential drug target for Coronaviruses.Methods: Here we performed computational ligand screening of four pharmacophores (OEW, Remdesivir, Hydroxycholoquine and N3) that are presumed to have positive effects against SARS-CoV-2 Mpro protease (6LU7) and also screened 50,000 molecules from the ZINC Database dataset against this protease target.Results: We found 40 pharmacophore-like structures of natural compounds from diverse chemical classes that exhibited better affinity of docking as compared to the known ligands. The 10 best selected ligands namely, ZINC1845382, ZINC1875405, ZINC2092396, ZINC2104424, ZINC44018332, ZINC2101723, ZINC2094526, ZINC2094304, ZINC2104482, ZINC3984030, and ZINC1531664, are mainly classified as β-carboline, Alkaloids and Polyflavonoids, and all of them displayed interactions with dyad CYS145 and HIS41 from the protease pocket in a similar way as with other known ligands.Conclusion: Our results suggest that these 10 molecules could be effective against SARS-CoV-2 protease and may be tested in vitro and in vivo to develop novel drugs against this virus.
ARTICLE | doi:10.20944/preprints202003.0277.v1
Subject: Medicine And Pharmacology, Medicine And Pharmacology Keywords: COVID-19; hydrolysable tannins; protease; molecular docking; structural-relationship activity
Online: 17 March 2020 (15:43:48 CET)
Coronavirus disease 2019 (COVID-19) was recently appeared all over the world. The viral main protease (3-chymotrypsin-like cysteine enzyme) controls COVID-19 duplication and manages its life cycle, making it a drug discovery target. Therefore, herein, we analyzed the theoretical approaches of 10 structurally different hydrolysable tannins as natural anti-COVID-19 through binding with the main protease of 2019-nCoV using molecular docking modelling via Molecular Operating Environment (MOE v2009) software. Our results revealed that there are top three hits may serve as potential anti-COVID-19 lead molecules for further optimization and drug development to control COVID-19. Pedunculagin, tercatain, and punicalin were found to faithfully interact with the receptor binding site and catalytic dyad (Cys145 and His41) of COVID-19 main protease, showing their successfully inhibit the protease enzyme of 2019-nCoV. We anticipated that this study would pave way for tannins based novel small molecules as more efficacious and selective anti-COVID-19 therapeutic compounds.
HYPOTHESIS | doi:10.20944/preprints202002.0254.v2
Subject: Medicine And Pharmacology, Pharmacology And Toxicology Keywords: 2019-nCoV; novel coronavirus pneumonia; docking; ACE2; viral main protease
Online: 23 February 2020 (02:09:52 CET)
The 2019 novel coronavirus (2019-nCoV) causes novel coronavirus pneumonia (NCP). Given that approved drug repurposing becomes a common strategy to quickly find antiviral treatments, a collection of FDA-approved drugs can be powerful resources for new anti-NCP indication discoveries. In addition to synthetic compounds, Chinese Patent Drugs (CPD), also play a key role in the treatment of virus related infections diseases in China. Here we compiled major components from 38 CPDs that are commonly used in the respiratory diseases and docked them against two drug targets, ACE2 receptor and viral main protease. According to our docking screening, 10 antiviral components, including hesperidin, saikosaponin A, rutin, corosolic acid, verbascoside, baicalin, glycyrrhizin, mulberroside A, cynaroside, and bilirubin, can directly bind to both host cell target ACE2 receptor and viral target main protease. In combination of the docking results, the natural abundance of the substances, and botanical knowledge, we proposed that artemisinin, rutin, glycyrrhizin, cholic acid, hyodeoxycholic acid, puerarin, oleanic acid, andrographolide, matrine, codeine, morphine, chlorogenic acid, and baicalin (or Yinhuang Injection containing chlorogenic acid and baicalin) might be of value for clinical trials during a 2019-nCov outbreak.
ARTICLE | doi:10.20944/preprints201904.0166.v1
Subject: Biology And Life Sciences, Insect Science Keywords: antimicrobial peptides; cellular defense; insect pathology; phenoloxidae; phospholipase A2; protease
Online: 15 April 2019 (11:45:11 CEST)
Xenorhabdus nematophila and Photorhabdus luminescens are entomopathogenic symbionts that produce several toxic proteins that can interfere with the immune system of insects. Here, we showed that outer membrane proteins (OMPs) could be involved as virulence factors during bacterial symbiont pathogenesis. Purified OMPs from bacterial culture were injected fifth instar larvae of Spodoptera exigua Hübner. Larvae were surveyed for fluctuations in total haemocyte counts (THC), granulocyte percentage (cellular defence), protease, phospholipase A2 (PLA2), and phenoloxidase (PO) activities (humoral defence) at specific time intervals. Changes in the expression of the three antimicrobial peptides (AMPs), cecropin, attacin, and spodoptericin, were also measured. Larvae treated with both types of OMPs had more haemocytes than did the negative controls. OMPs of X. nematophila caused more haemocyte destruction than did the OMPs of P. luminescens. The OMPs of both bacterial species initially activated insect defensive enzymes post-injection, their activating fluctuated in different ways. Attacin, cecropin and spodoptericin were up-regulated by OMP injections more than in normal larvae. The expression of these three AMPs was maximal at four hpi with P. luminescens OMPs treatment. Expression of the three AMPs in X. nematophila treatment was irregular and lower than in the P. luminescens OMPs treatment. These findings provide insights into the role of OMPs of entomopathogenic nematode bacterial symbionts in countering the physiological defenses of insects.
ARTICLE | doi:10.20944/preprints201811.0550.v1
Subject: Biology And Life Sciences, Virology Keywords: poliovirus 2A protease (PV2Apro); expression; purification; MALDI-TOF; 3D structure
Online: 22 November 2018 (14:41:00 CET)
Poliovirus 2A protease (PV2Apro) plays a vital role in viral replication and down-regulation of host cell protein synthesis. In order to understand more concerning PV2Apro, the protein was over-expressed in bacteria following amplification using sense and antisense primers and cloning in pET15b. Several expression hosts were tested and BL21 (DE3) pLysS cells gave the best expression of PV2Apro with minimal unwanted protein expression following IPTG induction. The 2Apro protein was purified to homogeneity using column chromatography, its solubility determined and its molecular weight and composition determined by MALDI-TOF mass spectrometry. The protease was found in the insoluble fraction and the purified protein had a slightly lower molecular weight than predicted. Moreover, three dimensional structure was modelled using template 1z8r with 58% identity and validated using ramachandran plot. Results revealed that most of the residues lie in favoured and allowed regions. These findings could help in a better understanding of PV2Apro structure and inhibition thus, highlighting potential targets for antiviral drug development.
ARTICLE | doi:10.20944/preprints201811.0150.v2
Subject: Biology And Life Sciences, Anatomy And Physiology Keywords: meagre; Argyrosomus regius; protease activity; inhibitions; live foods; in vitro
Online: 9 November 2018 (10:43:38 CET)
The aim of this study was to gather preliminary data about the potential inhibitory effects and contributions of live foods used from 3 to 32 days after hatching (DAH) in routine feeding protocols on protease activities of meagre, Argyrosomus regius (Asso 1801) larvae, using in vitro techniques. Enriched rotifer, Artemia nauplii and Artemia metanauplii were tested in the present study. The highest values of protease activities of meagre larvae at 7 DAH in 2013 and 2014 years were established. The lowest values at 15 DAH in 2013 and at 20 DAH in 2014 were observed. Protease activities of enriched rotifer, Artemia nauplii, and Artemia metanauplii were 21.76±0.31, 36.00±1.48–29.33±0.93, and 416.44±19.7–403.53±11.85 U/mg protein, respectively (p< 0.05). The highest inhibitions of live foods were observed at 7 DAH. The positive contributions of live food Artemia metanauplii’s on protease activities of meagre larvae were significant (p< 0.05). The inhibitory effects and positive contributions of live foods on survival and growth rates of meagre larvae, should be taken into account in meagre and other marine fish larvae’s future studies. Cysteine protease activities of Artemia sp. should be investigated to provide the higher growth and survival ratio from the feeding protocols used in marine fish larvae.
REVIEW | doi:10.20944/preprints202204.0097.v1
Subject: Biology And Life Sciences, Plant Sciences Keywords: Prolyl endoprotease; prolyl oligopeptidase; protease; protein engineering; proteolytic enzyme; proteomics; therapeutics
Online: 11 April 2022 (11:21:44 CEST)
Proteases or peptidases are hydrolases that catalyze the breakdown of polypeptide chains into smaller peptide subunits. Proteases exist in all life forms, including archaea, bacteria, protozoa, insects, animals, and plants, due to their vital functions in cellular processing and regulation. There are several classes of proteases in the MEROPS database based on their catalytic mecha-nisms. This review focuses on the post-proline cleaving enzymes (PPCEs), especially the prolyl endoprotease/oligopeptidase (PEP/POP). To date, most PPCEs studied are of microbial and ani-mal origins. Recently, there are reports of new plant PPCEs. The most common PEP/POP are members of the S9 family that comprise two conserved domains. The substrate-limiting β-propeller domain prevents unwanted digestion, while the α/β hydrolase catalyzes reaction at the carboxyl-terminal of proline residues. PPCEs have diverse applications, are widely used in the beer brewing industry, and have potential as therapeutic agents for Alzheimer’s disease and celiac disease by targeting proline-rich substrates. Protein engineering via mutagenesis has been performed to improve heat resistance, pepsin-resistant capability, specificity, and protein turno-ver of PPCEs for pharmacological applications. This is the first comprehensive review to cover the biotechnological applications of PPCEs and discuss the unique prolyl cleaving activity of dif-ferent enzymes based on the recent structure-function studies from diverse taxa.
HYPOTHESIS | doi:10.20944/preprints202012.0813.v1
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: Alzheimer’s disease; beta-amyloid; protease; membrane; membrane channel; lysosome; autophagy; mitochondrion
Online: 31 December 2020 (13:32:26 CET)
Alzheimer's disease (AD) is the most common cause of dementia and affects millions of people around the world. Neuronal death in AD is initiated by the toxic action of oligomeric amyloid-β (Aβ) peptides. The formation of membrane channels by Aβ is a primary molecular action and does not require any other proteins. Channels are formed by short amyloid fragments faster and more frequently than by full-length peptides. The channel formation is dependent on an electrostatic interaction between a positively charged peptide and a negatively charged membrane. Negative membranes can be found in several locations of a cell – the inner leaflet of plasma membrane, mitochondria, and lysosomes, which all are well-known cellular targets in AD. Considering that the amyloid enters a cell by endocytosis and is exposed to lysosomal enzymes, we propose the amyloid degradation toxicity hypothesis. Endopeptidases degrade the endocytosed peptide. Produced fragments form membrane channels, which can transfer various ions (including protons) and even relatively large compounds. The neutralization of lysosomal content inactivates enzymes, which fails the whole system of recycling cellular content, including autophagy. The permeabilization of lysosomes could also lead to cell death through necrotic and apoptotic mechanisms. We discuss several mechanisms that describe how amyloid degradation products reach plasma and mitochondrial membranes, and form membrane channels. The pathogenesis of AD is discussed at various levels in a context of how the primary molecular mechanism of membrane channel formation could progress into the disease state. The discussion starts at the molecular level and extends to why the development of a disease takes years and is closely associated with aging. The proposed hypothesis offers an interpretation to several clinical observations such as the involvement of iron metabolism and an inverse association between developing Alzheimer's disease and cancer. Predictions about potential biomarkers and effectiveness of future treatments are discussed.
CONCEPT PAPER | doi:10.20944/preprints202006.0206.v1
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: serine protease; interferon; ribonucleoproteins; RNA binding proteins; SERPIN; A1AT; hemostasis rebalancing
Online: 17 June 2020 (03:20:54 CEST)
Emerging paradigms in interferon (IFN) biology suggest a dynamic INF induced interactome that extends through broader Interferon Stimulated Gene (ISG)- induction, which implicates interferon- ISG coordinated cross-talk with mRNA processing, post-translational modification and metabolic processes that underlie pathological (viral, autoimmune and tumor biology) and physiological (stem cell regenerative pathways) processes. INF immune responses can also be triggered by endogenous host-derived molecules that are generated in response to cellular stress or hemostasis imbalance to establish tissue repair and regeneration in first place, however, overactivation or lack of countermeasures can result in host tissue damage. The proteases are integral to viral and tumor pathology, and importantly serine proteases TMPRSS2 and trypsin have been identified as important molecular determinants underlying COVID-19 pathology, and emergence of coronaviruses cultured in vitro, respectively. We propose that pathogen associated proteases can act as novel stress-inducers to facilitate viral- competent immunomodulation. We term it as Protease Induced Transcriptomic/ epi-Transcriptomic Reshaping (PITTR) of host cells to counter cellular stress. We present a novel experimental model and our preliminary findings of trypsin- primed Caco-2 cells (CPT) that result in translational halt comparable to cells grown under serum-starvation conditions (CSS). CPT at escalating trypsin concentration (CPT- EC) induce upregulation of selective proteins that majorly map to ribosomal, RNA transport, and spliceosome ribonucleoproteins (RNPs). The inclusion of proinflammatory IL1-b to CPT (CPT- IL) resulted in global overexpression of proteins comparable to Caco-2 cells cultured in growth-factor rich serum conditions (CFBS), indicating a likely de-repression of trypsin- induced translational halt. Caco-2 cells display abortive interferon proteome under differential trypsin conditions (CPT, CPT-EC and CPT-IL), which is marked by complete lack of INF generation despite induction of intermediate ISGs, suggestive of protease (trypsin)- dependent regulation of INF response. Viruses regulate the proteome of stress granules (SGs) that are induced to cope transient translational halt as a central adaptive response to pathogen induced cellular stress. The integral components of SGs include non-translating mRNA, ribonucleoproteins (RNPs) and RNA binding proteins (RBPs), which together form biological condensates through a biophysical process involving weak electrostatic interactions through intrinsically disordered regions in RBPs resulting in liquid- liquid phase separation. We compared the CPT- EC proteome to the Mammalian Stress Granules Proteome (MSGP) database to explore potential RBPs that could possibly regulate INF response (and could act as potential anti-viral targets). Notably, differentially upregulated RNPs and potential RBPs from ISG family including ADAR and PRKRA, and RNA helicases implicated in viral pathogenesis were found to be upregulated in the CPT- EC proteome further strengthening the role of proteases (trypsin) in regulating INF pathways independent of the pathogen. We propose that the supplementation of viable SARS-CoV-2 viral loads to trypsin- primed host cells could recapitulate an infectious disease model, which may closely phenocopy pathogen- driven inflammation and signaling events. Based on the global downregulation of seven SERPINS (serine protease inhibitors) linked to thromboinflammation in our LCMS profiling data, we support the candidature of serine protease inhibitors for protease mediated viral pathologies. COVID-19 is increasingly linked to coagulopathy and resemblance to Neutrophil Extracellular Trap (NET) related thromboinflammatory features; SERPIN A1AT (alpha 1 antitrypsin) being a potent neutrophil- elastase inhibitor and a negative regulator of coagulation complement pathway may be a promising candidate for establishing hemostasis rebalancing in COVID-19 pathology.
REVIEW | doi:10.20944/preprints201802.0029.v1
Subject: Biology And Life Sciences, Plant Sciences Keywords: protease; plant; pathogen; defence; substrate; immunity; hypersensitive response; recognition; signalling; priming
Online: 5 February 2018 (04:11:42 CET)
Proteases are integral enzymes of the plant immune system. Multiple aspects of defence are regulated by proteases, including the hypersensitive response, pathogen recognition, priming and peptide hormone release. These processes are regulated by unrelated proteases residing at different subcellular locations. In this review we discuss ten prominent plant proteases contributing to the plant immune system, highlighting the diversity of roles they perform in plant defence.
REVIEW | doi:10.20944/preprints202305.1788.v1
Subject: Biology And Life Sciences, Endocrinology And Metabolism Keywords: adipose tissue; corin; hepsin; matriptase-2; type II transmembrane serine protease; thermogenesis
Online: 25 May 2023 (09:51:58 CEST)
Adipose tissue is a crucial organ in energy metabolism and thermoregulation. Adipose tissue phenotype is controlled by various signaling mechanisms under pathophysiological conditions. Type II transmembrane serine proteases (TTSPs) are a group of trypsin-like enzymes anchoring on the cell surface. These proteases act in diverse tissues to regulate physiological processes, such as food digestion, salt-water balance, iron metabolism, epithelial integrity, and auditory nerve development. More recently, several members of the TTSP family, namely, hepsin, matriptase-2, and corin, have been shown to play a role in regulating lipid metabolism, adipose tissue phenotype, and thermogenesis, via direct growth factor activation or indirect hormonal mechanisms. In mice, hepsin deficiency increases adipose browning and protects from high-fat diet-induced hyperglycemia, hyperlipidemia, and obesity. Similarly, matriptase-2 deficiency increases fat lipolysis and reduces obesity and hepatic steatosis in high-fat diet-fed mice. In contrast, corin deficiency increases white adipose weights and cell sizes, suppresses adipocyte browning and thermogenic responses, and causes cold intolerance in mice. These findings highlight an important role of TTSPs in modifying cellular phenotype and function in adipose tissue. In this review, we provide a brief description about TTSPs and discuss recent findings regarding the role of hepsin, matriptase-2, and corin in regulating adipose tissue phenotype, energy metabolism, and thermogenic responses.
COMMUNICATION | doi:10.20944/preprints202003.0423.v1
Subject: Medicine And Pharmacology, Pulmonary And Respiratory Medicine Keywords: COVID-19; SARS-CoV2; SARS-CoV; variable residues; main protease; structural analysis
Online: 29 March 2020 (06:22:06 CEST)
The novel coronavirus SARS-CoV2 (CoV2) emerged in December 2019. This virus has 88% genomic similarity with SARS-CoV (CoV), and both viruses largely depend on their main protease (Mpro) to regulate infection. Mpro thus represents an attractive target for anti-SARS drug design. The CoV and CoV2 Mpro are 97% identical at the sequence level, with 12 variable residues, and their X-ray structures appear similar. We thus structurally analysed how these variable residues affect the intra-molecular interactions between key residues in the CoV2 Mpro active-site. Compared to CoV Mpro, the 12 divergent residues in CoV2 Mpro exhibit modified intra-molecular interaction networks that ultimately restructure the molecular micro-environment. These altered networks also indirectly affect the networks of other active-site residues at the entrance (T26, M49 and Q192) and near the catalytic region (F140, H163, H164, M165 and H172) of the Mpro. This suggest CoV2 indirectly (via neighbours) reshape key molecular networks around the Mpro active-site. It seems that the CoV2 Mpro deceives us with its apparent structurally identical to the CoV Mpro while this viral system accumulates mass mutations (12 variable residues) at key positions. Some of these identified CoV2 Mpro networks at the active-site might guide design of efficient CoV2 Mpro inhibitors.
ARTICLE | doi:10.20944/preprints202003.0349.v1
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: novel coronavirus; COVID-19; protease; molecular docking; drug designing; ADME; drug repurposing
Online: 23 March 2020 (09:47:49 CET)
The Novel Coronavirus (COVID-19) is a positive-sense single-stranded RNA ((+)ssRNA) virus. The COVID-19 Main Proteases play very important role in the propagation of the Novel Coronavirus (COVID-19). It has already killed more than 8000 people around the world and thousands of people are getting infected every day. Therefore, it is very important to identify a potential inhibitor against COVID-19 Main Proteases to inhibit the propagation of the Novel Coronavirus (COVID-19). We have applied a drug repurposing approach of computational methodology, depending on the synergy of molecular docking and virtual screening techniques, aimed to identify possible potent inhibitors against Novel Coronavirus (COVID-19) from FDA approved antiviral compounds and from the library of active phytochemicals. On the basis of recently resolved COVID-19 Main Protease crystal structure (PDB:6LU7), the library of 100 FDA approved antiviral compounds and 1000 active components of Indian Medicinal Plants extracted for screening against COVID-19 Main Protease. The compounds were further screened using Pyrex virtual screening tool and then best inhibitors, top 19 compounds optimally docked to the COVID-19 Main Protease structure to understand the participation of specific amino acids with inhibitors at active sites. Total 19 best compounds were identified after screening based on their highest binding affinity with respect to the other screened compounds. Out of 19, 6 best compounds were further screened based on their binding affinity and best ADME properties. Nelfinavir exhibited highest binding energy -8.4 kcal/mol and strong stability with the TRP207, ILE281, LEU282, PHE3, PHE291, GLN127, ARG4, GLY283, GLU288, LYS5, LYS137, TYR126, GLY138, TYR126, SER139 and VAL135 amino acid residues of COVID-19 Main Protease participating in the interaction at the binding pocket. In addition to Nelfinavir (-8.4), Rhein (-8.1), Withanolide D (-7.8), Withaferin A (-7.7), Enoxacin (-7.4), and Aloe-emodin (-7.4) also showed good binding affinity and best ADME properties. Our findings suggest that these compounds can be used as potential inhibitors against COVID-19 Main Protease, which could be helpful in inhibiting the propagation of the Novel Coronavirus (COVID-19). Moreover, further in vitro and in vivo validation of these findings would be very helpful to bring these inhibitors to next level study.
ARTICLE | doi:10.20944/preprints201806.0244.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Clostridium botulinum; botulinum neurotoxin; serotype F; subtype; VAMP-2; synaptobrevin; Zn2+ protease
Online: 15 June 2018 (05:32:49 CEST)
In the recent past about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons, but are also recognized pharmaceuticals for efficient counteraction of hyperactive nerve terminals in a variety of human diseases. The detailed biochemical characterization of subtypes as the basis for development of suitable countermeasures and possible novel therapeutic applications is lagging behind the increase in new subtypes. Here we report the primary structure of a ninth subtype of BoNT/F. Its amino acid sequence diverges by at least 8.4% at the holotoxin and 13.4% on the enzymatic domain level from all other known BoNT/F subtypes. We found that BoNT/F9 shares the scissile Q58/K59 bond in its substrate vesicle associated membrane protein 2 with the prototype BoNT/F1. Comparative biochemical analyses of four BoNT/F enzymatic domains showed that the catalytic efficiencies decrease in the order F1 > F7 > F9 > F6 and vary by up to factor eight. KM values increase in the order F1 > F9 > F6 ≈ F7, whereas kcat decreases in the order F7 > F1 > F9 > F6. Comparative substrate scanning mutagenesis studies revealed a unique pattern of crucial substrate residues for each subtype. Based upon structural coordinates of F1 bound to an inhibitor polypeptide the mutational analyses suggest different substrate interactions in the substrate binding channel of each subtype.
ARTICLE | doi:10.20944/preprints202109.0375.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: HIV-protease inhibitors; pseudo-symmetric core; heteroaryl carboxyamides; synthesis; biological screening; molecular modeling
Online: 22 September 2021 (10:44:16 CEST)
New series of compounds containing both heterocycle moieties and pseudo-symmetric hydroxyethylamine core were obtained using a simple synthetic path that can provide a library of compounds in few steps and high yields. Furthermore, diversity-oriented synthesis was studied to change different functionalities according to needs. The in vitro inhibition activity against recombinant HIV-1 protease was evaluated. A beneficial effect of this class of compounds can be obtained either for the presence of a bis-benzyl group into the core and for the heterocyclic moiety in P1, specifically the indole ring. Docking analysis was also reported.
ARTICLE | doi:10.20944/preprints202010.0346.v1
Subject: Biology And Life Sciences, Anatomy And Physiology Keywords: Trypanosoma cruzi; Presenilin; aspartic protease; SPOT-synthesis; anti-peptide antibodies; immunolocalization; transmembrane domains
Online: 16 October 2020 (12:01:25 CEST)
The increasing detection of infections of Trypanosoma cruzi, the etiological agent of Chagas disease, in non-endemic regions beyond Latin America has risen to be a major public health issue. With an impact in the millions of people, current treatments rely on antiquated drugs that produce severe side effects and are considered nearly ineffective for the chronic phase. The minimal progress in the development of new drugs highlights the need for advances in basic research on crucial biochemical pathways in T. cruzi to identify new targets. Here, we report on the T. cruzi presenilin-like transmembrane aspartyl enzyme, a protease of the aspartic class in a unique phylogenetic subgroup with T. vivax separate from protozoans. Computational analyses suggests it contains 9 transmembrane domains and an active site with the characteristic PALP motif of the A22 family. Multiple linear B-cell epitopes were identified by SPOT synthesis analysis with Chagasic patient sera. Two were chosen to generate rabbit antisera, whose signal was primarily localized to the flagellar pocket, intracellular vesicles and endoplasmic reticulum in parasites by whole cell immunofluorescence. The results suggest that the parasitic presenilin-like enzyme could have a role in the secretory pathway and serve as a biomarker for infections.
ARTICLE | doi:10.20944/preprints202004.0161.v1
Subject: Medicine And Pharmacology, Pharmacology And Toxicology Keywords: COVID-19; SARS-CoV-2; main protease inhibition; virtual screening; drugs; natural products
Online: 9 April 2020 (14:59:37 CEST)
We use state-of-the-art computer-aided drug design (CADD) techniques to identify prospective inhibitors of the main protease enzyme, Mpro of the COVID-19 virus. With the high-resolution X-ray crystallography structure of this viral enzyme recently being solved, CADD provides a veritable tool for rapidly screening diverse sets of compounds with the aim of identifying ligands capable of forming energetically favorable complexes with Mpro . From our screening of 1,082,653 compounds derived from the ZINC, the DrugBank, and our in-house African natural product libraries, and a rescreening protocol incorporating enzyme dynamics via ensemble docking, we have been able to identify a range of prospective Mpro inhibitors, which include FDA-approved drugs, drug candidates in clinical trials, as well as natural products. The top-ranking compounds are characterized by the presence of an extended ring system combined with functional groups that allow the ligands to adapt flexibly to the Mpro active site as, for example, present in the biflavonoid amentoflavone, one of the most promising compounds identified here. This particular chemical architecture leads to considerable stronger binding than found for reference compounds with in vitro demonstrated M pro inhibition and anticoronavirus activity. The compounds determined in this work thus represent a good starting point for the design of inhibitors of SARS-CoV-2 replication.
COMMUNICATION | doi:10.20944/preprints202004.0146.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Coronavirus Disease 2019; SARS-CoV-2; main protease; molecular docking-based virtual screening
Online: 9 April 2020 (09:34:45 CEST)
Coronavirus Disease 2019 (Covid-19) was first described in December 2019 in Wuhan, Hubei Province, China; and produced by a novel coronavirus designed as the acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Covid-19 has become a pandemic reaching over 1.3 million confirmed cases and 73,000 deaths. Several efforts have been done to identify pharmacological agents that can be used to treat patients and protect healthcare professionals. The sequencing of the virus genome not only has offered the possibility to develop a vaccine, but also to identified and characterize the virus proteins. Among these proteins, main protease (Mpro) has been identified as a potential therapeutic target, since it is essential for the processing other viral proteins. Crystal structures of SARS-CoV-2 Mpro and inhibitors has been described during the last months. To describe additional compounds that can inhibit SARS-CoV-2 Mpro, in this study we performed a molecular docking-based virtual screening against a library of experimental and approved drugs. Top 10 hits included Pictilisib, Nimorazole, Ergoloid mesylates, Lumacaftor, Cefuroxime, Cepharanhine, and Nilotinib. These compounds were predicted to have higher binding affinity for SARS-CoV-2 Mpro than previously reported inhibitors for this protein, suggesting a higher potential to inhibit virus replication. Since the identified drugs have both pre-clinical and clinical information, we consider that these results may contribute to the identification of treatment alternative for Covid-19. Nevertheless, in vitro and in vivo confirmation should be performed before these compounds could be translated to the clinic.
ARTICLE | doi:10.20944/preprints202002.0308.v1
Subject: Medicine And Pharmacology, Pharmacology And Toxicology Keywords: SARS-CoV-2; COVID-19; 3CL protease; molecular docking; molecular dynamics and simulations
Online: 23 February 2020 (02:17:46 CET)
The SARS-CoV-2 was confirmed to cause the regional outbreak of coronavirus disease 2019 (COVID-19) in Wuhan, China. The 3C-like protease (3CLpro), an essential enzyme for viral replication, is a valid target to compacts SARS-CoV and MERS-CoV. In this research, an integrated library consisting of 1000 compounds from Asinex Focused Covalent (AFCL) library and 16 FDA-approved protease inhibitors were screened against SARS-CoV-2 3CLpro. Top compounds with significant docking scores and making stable interactions with catalytic dyad residues were obtained. The screening results in identification of compound 621 from AFCL library as well as Paritaprevir and Simeprevir from FDA-approved protease inhibitors as potential inhibitors of SARS-CoV-2 3CLpro. The mechanism and dynamic stability of binding between the identified compounds and SARS-CoV-2 3CLpro were characterized using 50 nanoseconds (ns) molecular dynamic (MD) simulation approach. The identified compounds are potential inhibitors worthy of further development as SARS-CoV-2 3CLpro inhibitors/drugs. Importantly, the identified FDA-approved therapeutics could be ready for clinical trials to treat infected patients and help to curb the COVID-19.
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: mast cell; tryptase; chymase; serine protease; human chymase; cleavage specificity; cytokine; chemokine; th2
Online: 26 September 2019 (12:00:55 CEST)
Mast cells (MC) are resident tissue cells found primarily at the interphase between tissues and environment. These evolutionary old cells store large amounts of proteases within cytoplasmic granules, and one of the most abundant of these proteases is the tryptase. To look deeper into the question their in vivo targets, we have analyzed the activity of the human MC tryptase on 69 different human cytokines and chemokines, and the activity of the mouse tryptase (mMCP-6) on 56 mouse cytokines and chemokines. These enzymes were found to be remarkably restrictive in their cleavage of these potential targets. Only five were efficiently cleaved by the human tryptase: TSLP, IL-21, MCP3, MIP-3b and eotaxin. This strict specificity indicates a regulatory function of these proteases and not primarily as unspecific degrading enzymes. We recently showed that the human MC chymase also had a relatively strict specificity, indicating that both of these proteases have regulatory functions. One of the most interesting such regulatory functions may involve controlling excessive TH2 mediated inflammation by cleaving several of the most important TH2-promoting inflammatory cytokines, including IL-18, IL-33, TSLP, IL-15 and IL-21 indicating a potent negative feedback loop on TH2 immunity.
ARTICLE | doi:10.20944/preprints202211.0516.v1
Subject: Chemistry And Materials Science, Medicinal Chemistry Keywords: Antivirals; coronavirus; COVID-19; covalent drugs; dynamic light scattering; inhibitors; main protease; natural products
Online: 28 November 2022 (13:40:01 CET)
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has forced the development of direct-acting antiviral drugs due to the coronavirus disease 2019 (COVID-19) pandemic. The main protease of SARS-CoV-2 is a crucial enzyme that breaks down polyproteins synthesized from the viral RNA, making it a validated target for the development of SARS-CoV-2 therapeutics. New chemical phenotypes are frequently discovered in natural goods. In the current study, we used a fluorogenic assay to test a variety of natural products for their ability to inhibit SARS-CoV-2 Mpro. Several compounds were discovered to inhibit the Mpro at low micromolar concentrations. It was possible to crystallize robinetin together with SARS-CoV-2 Mpro, and the X-ray structure revealed covalent interaction with the protease's catalytic Cys145 site. Selected potent molecules also exhibited antiviral properties without cytotoxicity. Some of these powerful inhibitors might be utilized as lead compounds for COVID-19 research.
ARTICLE | doi:10.20944/preprints202304.0323.v1
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: ImmunoDefender; Essential oils (EO),; Bioactive molecules; antiviral, SARS-CoV-2; Main-Protease; active & allosteric sites.
Online: 13 April 2023 (12:41:47 CEST)
Essential oils have demonstrated antiviral activity, but their toxicity can hinder their use as therapeutic agents. Recently, some essential oil components have been used within safe levels of acceptable daily intake limits without causing toxicity. The "ImmunoDefender," a novel antiviral compound made from a well-known mixture of essential oils, is considered highly effective in treating SARS-CoV-2 infections. The components and doses were chosen based on existing information about their structure and toxicity. Blocking the Main Protease (Mpro) of SARS-CoV-2 with high affinity and capacity is critical for inhibiting the virus's pathogenesis and transmission. In-silico studies were conducted to examine the molecular interactions between the main essential oil components in "ImmunoDefender" and SARS-CoV-2 Mpro. The screening results showed that six key components of ImmunoDefender formed stable complexes with Mpro via its active catalytic site with binding energies ranging from -8.75 to -10.30 kcal/mol, respectively for Cinnamtannin B1, Cinnamtannin B2, Pavetannin C1, Syzyginin B, Procyanidin C1, and Tenuifolin. Furthermore, three essential oil bioactive inhibitors, Cinnamtannin B1, Cinnamtannin B2, and Pavetannin C, have a significant ability to bind to the allosteric site of the main protease with binding energies of -11.12, -10.74, and -10.79 kcal/mol, These results suggest that these essential oil bioactive compounds may play a role in preventing the attachment of the translated polyprotein to Mpro, inhibiting the virus's pathogenesis and transmission. These components also had drug-like characteristics similar to approved and effective drugs, suggesting further pre-clinical and clinical studies are needed to confirm the generated in-silico outcomes.
REVIEW | doi:10.20944/preprints202302.0058.v1
Subject: Biology And Life Sciences, Virology Keywords: HIV-1 PR; host factors; host cell shut-off; protease; antiviral therapy; cell death; apoptosis
Online: 3 February 2023 (04:42:32 CET)
The human immunodeficiency virus 1 (HIV-1) viral protease (PR) is one of the most studied viral enzymes, and approval of drugs targeting its catalytic activity opened the door to the develop-ment of highly active antiretroviral therapy (HAART). Despite the fact that its crucial role in viri-on maturation is well characterized, an increasing body of research is starting to focus on its abil-ity to cleave host cell proteins, based on recent advances in proteomics and genomics technologies. Such findings are apparently in contrast with the dogma of HIV-1 PR activity being restricted to the interior of nascent virions, and suggest catalytic activity within the host cell environment. Given the limited amount of PR present in the virion at the time of infection, it is tempting to specu-late that such events mainly occur during viral late gene expression, mediated by newly synthe-sized Gag-Pol polyprotein precursors, rather than at a very early stage of infection, before pro-viral integration. Among cellular targets of HIV-1 PR, three major clusters can be identified: pro-teins involved in viral and cellular translation, those controlling cell survival, and restriction fac-tors responsible for innate/intrinsic antiviral responses. Indeed, by cleaving host cell translation initiation factors HIV-1 PR can impair cap-dependent translation, thus promoting IRES-mediated translation of late viral transcripts and viral production, while by targeting several apoptotic fac-tors it modulates cell survival, thus promoting immune evasion and viral dissemination. Addi-tionally, HIV-1 PR counteracts restriction factors incorporated in the virion that would otherwise interfere with nascent virus vitality. Thus, HIV-1 PR appears to modulate host cell function at dif-ferent times and locations during its life cycle, to ensure efficient viral persistency and propaga-tion. This kind of PR-mediated host cell modulation is found in a plethora of different viruses and HIV-1 is no exception, and although we are far from having a complete picture, it is clear that the PR has a multifaceted role in interfering with host machineries to better suit viral replication, and is a field that needs to be explored further.
REVIEW | doi:10.20944/preprints202012.0167.v2
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: Matrix metalloproteinase; MMPs; protease; TIMPs; exosite; small molecule inhibitors; monoclonal antibodies; proteomics; N-terminomics; TAILS
Online: 24 March 2021 (16:23:28 CET)
Matrix metalloproteinases (MMPs) have been demonstrated to have both detrimental and protective functions in inflammatory diseases. Several MMP inhibitors, with the exception of Periostat®, have failed in Phase III clinical trials. As an alternative strategy, recent efforts have been focussed on the development of more selective inhibitors or targeting other domains than their active sites (e.g., exosites, ectosites) through specific small molecule inhibitors or monoclonal antibodies. Here, we present some examples that aim to better understand the mechanisms of conformational changes/allosteric control of MMPs functions. In addition to MMP inhibitors, we discuss unbiased global approaches such as proteomics and N-terminomics to identify new MMP substrates and achieve a better understanding of the roles of these proteases in diseases.
REVIEW | doi:10.20944/preprints202301.0420.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: Cryptococcus; extracellular protease; biofilm, secretory vesicles; hypoxia; hydrolytic enzyme; homeostasis; transcription factors; membrane permeases; membrane transporters
Online: 24 January 2023 (03:08:39 CET)
In this review, we present several extracellular proteases, enzymes, membrane permeases, and transporters as essential accessories proteins for nutrient assimilation, conservation, and transportation as determined by nutrient repletion or depletion. As an obligate aerobic pathogen, it is crucial for invading Cryptococcus (C.) neoformans to negotiate its adaptation to human internal organs like the brain and spinal cord, where the oxygen level is low compared to peripheral organs. Besides, essential metals like copper and iron are important cofactors to functional proteins; however, these metals are not usually freely available to invading human pathogens. Again, the phagolysosome low pH with glucose paucity, internal temperature, immune response, and complex extracellular matrixes are challenging environments that must be circumvented by C. neoformans in the systemic tissues for survival, adaptation, and infection in humans. We review extensive works on several extracellular proteases, enzymes, membrane permeases and transporters orchestrated by different transcription factors and present these proteins as weapons needed to outwit systemic resistance to invading pathogens. Lastly, we examine the extracellular secretory vesicles of C. neoformans as “an exosomal virulence bag” that harbours urease, laccase, phosphatase, and capsular components as additional secretory weapons for tissue invasion and persistence.
ARTICLE | doi:10.20944/preprints202204.0008.v1
Subject: Medicine And Pharmacology, Pharmacy Keywords: COVID-19 pandemic; KERRA; SARS-CoV-2 main protease; RNA-dependent RNA polymerase; anti-FIPV activity
Online: 1 April 2022 (14:53:44 CEST)
The COVID-19 pandemic affects all parameters, especially health care professionals, drugs and medical supplies. The KERRA is a mixed medicinal plant capsule that is used for the treatment of patients with high fever with food and drug administration approved by FDA Thailand. Recently, KERRA showed quicker recovery for COVID-19 patients. Therefore, it is possible that some ingredients in KERRA could inhibit SARS-CoV-2. In this study, two important replication-related enzymes in SARS-CoV-2, a main protease and an RNA-dependent RNA polymerase (RdRp), were used to study the effect of KERRA. The results showed that KERRA inhibited the SARS-CoV-2 main protease and SARS-CoV-2 RdRp with IC50 values of 49.91 ± 1.75 ng/mL and 36.23 ± 5.23 µg/mL, respectively. KERRA displayed no cytotoxic activity on macrophage cells at concentrations lower than 1 mg/mL and exhibited anti-inflammatory activity. Additionally, KERRA was against a feline coronavirus (feline infectious peritonitis [FIP]) infection with an EC50 value of 134.3 g/mL. This study supports the potential use of KERRA as a candidate drug for COVID-19.
REVIEW | doi:10.20944/preprints202002.0342.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: cathepsin; chronic obstructive pulmonary disease (COPD); cigarette smoke; cysteine; cysteine protease; emphysema; oxidation; proteolysis; ROS; thiol
Online: 24 February 2020 (02:48:00 CET)
Besides their primary involvement in the recycling and degradation of proteins in endo-lysosomal compartments but also in specialized biological functions, cysteine cathepsins are pivotal proteolytic contributors of various deleterious diseases. While the molecular mechanisms of regulation by their natural inhibitors have been exhaustively studied, less is currently known about how their enzymatic activity is modulated during the redox imbalance associated with an oxidative stress and their exposure resistance to oxidants. More specifically, there is only patchy information on the regulation of lung cysteine cathepsins, while the respiratory system is directly exposed to countless exogenous oxidants contained in dust, tobacco, combustion fumes, and industrial or domestic particles. Papain-like enzymes (clan CA, family C1, subfamily C1A) encompass a conserved catalytic thiolate-imidazolium pair (Cys25-His159) in their active site. Despite the sulfhydryl group (with a low acidic pKa) is a potent nucleophile highly susceptible to chemical modifications, some cysteine cathepsins reveal an unanticipated resistance to oxidative stress. Beside an introductory chapter and a peculiar attention to lung cysteine cathepsins, the purpose of this review is to afford a concise update of the current knowledge on molecular mechanisms associated to the regulation of cysteine cathepsins by redox balance and by oxidants (e.g. Michael acceptors, reactive oxygen and nitrogen species).
ARTICLE | doi:10.20944/preprints202004.0068.v2
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: coronavirus; COVID-19; hACE-2; MPro; multi-target-directed ligand; protease inhibito; RdRp; SARS-CoV-2 virus
Online: 9 April 2020 (05:13:05 CEST)
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in the current COVID-19 pandemic. Worldwide this disease has infected around 1.5 million individuals with a mortality rate ranging from 5 to 10%. It has also imposed extreme challenges on global health, economy, and social behavior. Due to the unavailability of therapeutics, several efforts are going on in the drug discovery to control the SARS-CoV-2 viral infection. The main protease (MPro) plays a critical role in viral replication and maturation, thus can serve as the primary drug target. To understand the structural evolution of MPro, we have performed phylogenetic and SSN analysis, that depicted divergence of Coronaviridae MPro in five clusters specific to viral hosts. This clustering was also corroborated with the comparison of MPro structures. Furthermore, it has been observed that backbone and binding site conformations are conserved despite variation in some of the residues. This conservation can be exploited to repurpose available viral protease inhibitors against SARS-CoV-2 MPro. In agreement with this, we performed screening of the custom-made library of ~7100 molecules including active ingredients present in the Ayurvedic anti-tussive medicines, anti-viral phytochemicals and synthetic anti-virals against SARS-CoV-2 MPro as the primary target. We identified several natural molecules that strongly binds to SARS-CoV-2 MPro among which top seven molecules are d-Viniferin, Myricitrin, Taiwanhomoflavone A, Lactucopicrin 15-oxalate, Nympholide A, Biorobin and Phyllaemblicin B. Most of the predicted lead molecules are from Vitis vinifera, also reported for anti-tussive and/or antiviral activities. These molecules also showed strong binding with other main targets RdRp and hACE-2. We anticipate that our approach for identification of multi-target-directed ligand will provide new avenues for drug discovery against SARS-CoV-2 infection.
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: SARS-CoV-2; COVID-19; acute kidney injury; angiotensin converting enzyme II (ACE2); transmembrane serine protease (TMPRSSs)
Online: 23 February 2020 (15:42:24 CET)
Purpose: Acute kidney injury (AKI) is a severe symptom of the 2019 novel coronavirus disease (COVID-19), especially for patients in a critical condition.This study explored the potential mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on AKI at the single-cell level. Methods: 15 normal human kidney samples were collected and analyzed using single-cell RNA sequencing (scRNA-seq). Subsequently, we analyzed the components and proportions of kidney cells expressing the host cellular receptor ACE2 and the key protease TMPRSSs family, and analyzed the expression differences in Occidental and Asian populations. Results: We drafted the currently available world's largest human kidney cell atlas with 42,589 cells and identified 19 clusters through unsupervised hierarchical clustering analysis. ACE2 and TMPRSSs genes were significantly co-expressed in podocytes and proximal convoluted tubules as potential host cells targeted by SARS-CoV-2. Comparative analysis showed that ACE2 expression in kidney cells was no less than that in the lung, esophagus, small intestine and colon, suggesting that the kidney may be an important target organ for SARS-CoV-2. In addition, given the high expression of ACE2 and kidney disease-related genes in Occidental donors relative to Asian donors, Occidental populations with SARS-CoV-2 infection might be a higher risk of of kidney injury.
ARTICLE | doi:10.20944/preprints201906.0074.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: agrobacterium tumefaciens-mediated transformation; citrus fruit; metal ion chelators; fruit-fungal interaction; transcription factor; virulence; protease inhibitors
Online: 10 June 2019 (09:56:16 CEST)
Penicillium digitatum is the major postharvest pathogen of citrus fruit under Mediterranean climate conditions. In the present work, we have addressed the study of the role of P. digitatum’s proteases in virulence following two complementary approaches. In a first approach, we have undertaken the functional characterization of the P. digitatum prtT gene, which codes for a transcription factor previously shown to regulate extracellular proteases in other filamentous fungi. Deletion of prtT caused a significant loss in secreted protease activity during in vitro growth assays. However, there was no effect on virulence. Gene expression of the two major secreted acid proteases was barely affected in the ΔprtT deletant during infection of citrus fruit. Hence, no conclusion could be drawn on the role of these secreted acidic proteases on the virulence of P. digitatum. In a second approach, we have studied the effect of different protease inhibitors and chelators in virulence. Co-inoculation of citrus fruit with P. digitatum conidia and a cocktail of protease inhibitors resulted in almost a complete absence of disease development. Analysis of individual inhibitors revealed that the metalloprotease inhibitor 1,10-phenanthroline was responsible for the observed effect. The application of metal ions reverted the protective effect caused by the metallopeptidase inhibitor. These results may set the basis for the development of new alternative treatments to combat this important postharvest pathogen.
REVIEW | doi:10.20944/preprints202105.0414.v1
Subject: Medicine And Pharmacology, Epidemiology And Infectious Diseases Keywords: : mineralocorticosteroid receptor antagonist (MRA); angiotensin converting enzyme 2 (ACE2); SARS-CoV-2; transmembrane protease receptor serine 2; furin; plasmin
Online: 18 May 2021 (10:39:03 CEST)
Aims: Spironolactone is a steroidal mineralocoricosteroid receptor antagonist (MRA) used for treatment of resistant hypertension, heart failure and edema. It exerts class specific adverse effects that are shared by other MRAs. Additionally, it exerts unique “off target” steroidal effects that include gynecomastia, impotence and loss of libido in males and menstrual irregularity in females. Together, these have led to a poor tolerability and limited use despite positive results in many randomized, controlled clinical trials. We review the off-target effects of spironolactone that may summate with its MRA action to provide an advantageous profile for prevention or treatment of patients with COVID-19. Methods: Literature review using PubMed Central. Results: The blockade by spironolactone of the androgen receptor should diminish the expression of transmembrane protease serine 2 (TMPRSS2) that has an androgen promoter while its MRA action should enhance the expression of protease nexin1 (PN1) that inhibits furin and plasmin. TMPRSS2, furin and plasmin cooperated to process the SARS-CoV-2 spike protein to increase its high affinity binding to the angiotensin converting enzyme 2 (ACE2) and thereby promote viral cell entry. Its actions as an MRA may reduce inflammation and preserve pulmonary, cardiac and vascular functions. Its anti-plasmin action may combat hemostatic dysfunction. Conclusion: The hypothesis that the off-target effects of spironolactone summate with its MRA actions to provide special benefits for COVID-19 is worthy of direct investigation and clinical trial.
ARTICLE | doi:10.20944/preprints202212.0206.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Cell-to-cell fusion; proprotein convertases; metalloproteases; mutagenesis; PCSK9; protease inhibitors; SARS-CoV-2; shedding; SKI-1/S1P; SREBP-2.
Online: 12 December 2022 (14:20:01 CET)
Proprotein convertases activate various envelope glycoproteins and participate in cellular entry of many viruses. We recently showed that the convertase furin is critical for the infectivity of SARS-CoV-2. This study investigated the implication of the two cholesterol-regulating convertases SKI-1 and PCSK9 in SARS-CoV-2 entry. We used cell-to-cell fusion assays in HeLa cells and pseudoparticle entry into Calu-3 cells. SKI-1 increases cell-to-cell fusion by enhancing the activation of SREBP-2, whereas PCSK9 reduces cell-to-cell fusion by promoting the cellular degradation of ACE2. Metalloprotease activation is sensitive to enhanced cholesterol levels resulting from SKI-1-activated SREBP-2 that leads to enhanced S2’ formation. However, high metalloprotease activity results in S2’ shedding into a new C-terminal fragment (S2”), leading to reduced cell-to-cell fusion. Indeed, S-mutants that increase S2’’ formation, abolish S2’ and cell-to-cell fusion, as well as pseudoparticles entry, indicating that the formation of S2’’ prevents SARS-CoV-2 cell-to-cell fusion and entry. We next demonstrated that PCSK9 enhanced the cellular degradation of ACE2, thereby reducing cell-to-cell fusion. However, different from the LDLR, a canonical target of PCSK9, the C-terminal CHRD domain of PCSK9 is dispensable for the PCSK9-induced degradation of ACE2. Molecular modeling suggested binding of ACE2 to the Pro/Catalytic domains of mature PCSK9. Thus, both cholesterol-regulating convertases SKI-1 and PCSK9 can modulate SARS-CoV-2 entry via two independent mechanisms.
ARTICLE | doi:10.20944/preprints201907.0144.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: longevity; life expectancy; CODAS syndrome; Perrault syndrome; protease target substrates; respiratory complex assembly; oxidative stress; glutathione pathway; lysosomal degradation; fidelity protein synthesis
Online: 10 July 2019 (10:18:22 CEST)
Research on healthy ageing shows that lifespan reductions are often caused by mitochondrial dysfunction. Thus, it is very interesting that the deletion of mitochondrial matrix peptidase LonP1 was observed to abolish embryogenesis, while deletion of the mitochondrial matrix peptidase ClpP prolonged survival. To unveil the targets of each enzyme, we documented the global proteome of LonP1+/- mouse embryonal fibroblasts (MEF), for comparison with ClpP-/- depletion. Proteomic profiles of LonP1+/- MEF generated by label-free mass spectrometry were further processed with the STRING webserver Heidelberg for protein interactions. ClpP was previously reported to degrade Eral1 as a chaperone involved in mitoribosome assembly, so ClpP deficiency triggers accumulation of mitoribosomal subunits and inefficient translation. LonP1+/- MEF also showed Eral1 accumulation, but no systematic effect on mitoribosomal subunits. In contrast to ClpP-/- profiles, several components of the respiratory complex I membrane arm were accumulated, whereas the upregulation of numerous innate immune defense components was similar. Overall, LonP1 as opposed to ClpP appears to have no effect on translational machinery, instead it shows enhanced respiratory dysfunction; this agrees with reports on the human CODAS syndrome caused by LonP1 mutations.
ARTICLE | doi:10.20944/preprints202002.0391.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Highly Pathogenic Avian Influenza Virus, Low Pathogenic Avian Influenza Virus, Evolution, Virulence Determinants, non-H5/H7, Cleavage Site, Chicken-to-Chicken Transmission, Virulence, Protease
Online: 26 February 2020 (11:13:35 CET)
Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in the hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥1.2. Only few non-H5/H7 viruses fulfill the criteria of HPAIVs; nevertheless, it remains unknown why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIVs H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Likewise, replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIVs in poultry.