Site Of Protease Research Articles

A lable-free SPR biosensor based on one peptide sequence with three recognition sites for O-GlcNAc transferase detection

Abnormal O-linked N-acetylglucosamine (O-GlcNAc) concentrations have been associated with many diseases, but the lack of accurate detection method limited O-GlcNAc to be used as a biomarker in clinical diagnosis. Then O-GlcNAc transferase (OGT) has drawn researchers’ attention as it closed related to the level of O-GlcNAc and be considered to be a promising new target for diseases diagnosis. Nevertheless, the existing OGT detection methods are either need labeling or the sensitity can not meet the needs of clinic testing. Herein, a label-free and sensitive SPR biosensor was developed for accurate detection of OGT based on a multi-functional peptide. The designed peptide contains three recognition sites, one is the cleavage site of protease K, one is the O-GlcNAcylated site by OGT, and another is six histidine which be used as the signal report probe to recognize Ni2+. The immobilized peptide would be cleavaged by proteinase K, then the His-tag residue part will leave the surface of Au film, resulting less His-tag could bind to Ni2+ and a small SPR signal would be record. If the peptide is O-GlcNAcylated by OGT, the cleaving reaction would be limited due to the adjacent site of O-GlcNAcylation. Then more His-tag can be left on the Au film and a bigger SPR signal could be record, this signal is associated with the concentration of OGT. Utilizing the change of the peptide configuration as a signal report probe for OGT detection not only avoids labeling of peptide, but also makes the method more sensitive. The determination linear range of OGT is from 2.00 × 10−13 to 5.00 × 10−8 M with a detection limit of 1.19 × 10−13 M, and the separation of two enzyme reactions ensured the high selectivity of the method. Finally, the sensing system was successfully used for OGT detection in blood samples with satisfied recovery. In summary, the label-free SPR platform for accurate detection of OGT in real samples is helpful to promote OGT serve as a biomarker for early clinical diagnosis of O-GlcNAc related diseases.

Identification of novel human USP2 inhibitor and its putative role in treatment of COVID-19 by inhibiting SARS-CoV-2 papain-like (PLpro) protease

Human ubiquitin carboxyl-terminal hydrolase-2 (USP2) inhibitors, such as thiopurine analogs, have been reported to inhibit 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 in-silico efforts. After an extensive 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.

Understanding Selenium and Glutathione as Antiviral Factors in COVID-19: Does the Viral Mpro Protease Target Host Selenoproteins and Glutathione Synthesis?

Glutathione peroxidases (GPX), a family of antioxidant selenoenzymes, functionally link selenium and glutathione, which both show correlations with clinical outcomes in COVID-19. Thus, it is highly significant that cytosolic GPX1 has been shown to interact with an inactive C145A mutant of Mpro, the main cysteine protease of SARS-CoV-2, but not with catalytically active wild-type Mpro. This seemingly anomalous result is what might be expected if GPX1 is a substrate for the active protease, leading to its fragmentation. We show that the GPX1 active site sequence is substantially similar to a known Mpro cleavage site, and is identified as a potential cysteine protease site by the Procleave algorithm. Proteolytic knockdown of GPX1 is highly consistent with previously documented effects of recombinant SARS-CoV Mpro in transfected cells, including increased reactive oxygen species and NF-κB activation. Because NF-κB in turn activates many pro-inflammatory cytokines, this mechanism could contribute to increased inflammation and cytokine storms observed in COVID-19. Using web-based protease cleavage site prediction tools, we show that Mpro may be targeting not only GPX1, but several other selenoproteins including SELENOF and thioredoxin reductase 1, as well as glutamate-cysteine ligase, the rate-limiting enzyme for glutathione synthesis. This hypothesized proteolytic knockdown of components of both the thioredoxin and glutaredoxin systems is consistent with a viral strategy to inhibit DNA synthesis, to increase the pool of ribonucleotides for RNA synthesis, thereby enhancing virion production. The resulting “collateral damage” of increased oxidative stress and inflammation would be exacerbated by dietary deficiencies of selenium and glutathione precursors.

Abstract P002: Soluble (pro)renin Receptor Contributes To Angiotensin II-Induced Hypertension In Mice

Activation of (pro)renin receptor (PRR) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during angiotensin II (AngII) infusion. Soluble PRR (sPRR), the extracellular domain of PRR, is generated by multiple proteases including furin or ADAM19, and recently site-1 protease (S1P). The goal of the present study was to test the role of S1P-derived sPRR mediated AngII-induced hypertension. F1 B6129SF1/J mice were infused for 6 days with control (CTR) or AngII at 300 ng/kg/day alone or in combination with S1P inhibitor PF-429242 (PF) and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated AngII-induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histadine-tagged sPRR termed as sPRR-His. Mutagenesis of overlapping recognition site for S1P and furin in PRR for was generated in mice by CRISPR strategy (termed as mutant mice). Mutant mice were fertile and developed normally with a 50% reduction plasma sPRR. These mice exhibited blunted hypertensive response to AngII infusion accompanied with suppressed intrarenal renin levels. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mpkCCD cells exposed for 24 h to AngII, AngII + PF, or AngII + PF + sPRR-His. AngII-induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that sPRR derived from S1P or in combination with furin mediates AngII-induced hypertension through enhancement of intrarenal renin level and activation of ENaC.

Abstract MP18: Mutagenesis Of Protease-mediated Cleavage Site In (pro)renin Receptor Induces Obesity And Type 2 Diabetes In Mice

Soluble (pro)renin receptor (sPRR), the extracellular domain of PRR, is generated by multiple proteases, including furin or ADAM19, and recently site-1 protease (S1P). We have previously reported that the therapeutic potential of histidine-tagged recombinant soluble (pro)renin receptor (sPRR), termed as sPRR-His, in a mouse obesity model induced by high-fat diet (DIO) for management of obesity and hyperglycemia. Conversely, inhibition of endogenous sPRR production by PF429242, an inhibitor of S1P in DIO mice aggravated diabetes and insulin resistance (Wang F et al. JCI Insight In press). However, the existence of the multiple substrates for S1P may impose confounding influence on the function of sPRR. The goal of the present study was to employ CRISPR strategy to mutagenize the overlapping recognition site for S1P and furin in PRR (termed as mutant mice) to examine its impact on metabolism. Mutant mice were fertile and developed normally with a 50% reduction plasma and tissue sPRR. 12-wk-old male mutant mice fed chow diet exhibited increased body weight (45.6 ± 4.8 g vs. 34.8 ± 2.7 g, n = 9, p < 0.05) and fat mass percentage ( fat mass/ body weight: 26% ± 4% vs. 12% ± 3%, n =9, p < 0.01) accompanied by reduced energy expenditure (0.35 ± 0.02 Kcal/kg/h vs. 0.54 ± 0.04 Kcal/kg/h, n = 4, p < 0.05) as assessed by calorimetry. The mutant mice developed hyperglycemia (fasting blood glucose: 172 ± 6.8 in mutant vs. 118 ± 5.4 mg/dL in WT, n = 15, p < 0.01), hyperinsulinemia (serum insulin: 48.7 ± 12.9 ng/ml vs. 3.7 ± 0.4 ng/ml, n = 6, p < 0.01), and impaired GTT and ITT. Additionally, sPRR-His supplement in the mutant mice nearly normalized blood glucose (132.4 ± 12.2 mg/dL) and insulin level (14.1 ± 5.9 ng/ml). Overall, these results support sPRR as an essential regulator of energy metabolism.

Abstract P229: Site-1 Protease-derived Soluble (pro) Renin Receptor Mediates Aldosterone-induced Enac Activation In The Collecting Duct

We have previously shown that activation of (pro)renin receptor (PRR) induces epithelial Na + channel (ENaC) activity in cultured collecting duct cells. Here, we examined the role of soluble PRR (sPRR), generated by site-1 protease (S1P), a newly identified PRR cleavage protease, in ENaC regulation, and further tested its relevance to Aldo signaling. In cultured mpkCCD cells, administration of recombinant histidine-tagged sPRR (sPRR-His) at 10 nM for 24 h induced a significant increase in the amiloride-sensitive short-circuit current as assessed using the Ussing chamber technique ( I eq : 7.5 ± 0.7 μA/cm 2 in sPRR group vs. 3.5 ± 0.5 μA/cm 2 in vehicle group, n = 6, p < 0.01) . In primary cultured rat IMCD cells, the same sPRR-His treatment induced a 1.7 fold increase in protein expression of the α-subunit but not β- or γ-subunit of ENaC, in parallel with upregulation of mRNA expression as well as promoter activity of the α-subunit. The upregulation of α-ENaC transcription depended on β-catenin signaling. Consistent results obtained by epithelial volt ohmmeter measurement of equivalent current and Using chamber determination of short-circuit current showed that Aldo-induced ENaC activity was almost completely abolished by PF-429242 (PF), a S1P inhibitor, and the response was restored by supplement of sPRR-His ( I eq : 7.2 ± 0.7 μA/cm 2 in Aldo group vs. 5.0 ± 0.3 μA/cm 2 in Aldo/PF group vs. 6.8 ± 0.3 μA/cm 2 in Aldo/PF/sPRR-His group, n = 5, p < 0.05). Medium sPRR was elevated by Aldo and inhibited by PF. Male C57BL/6 mice were pretreated with PF (30 mg/kg/day) or vehicle via minipump, followed by 3 days of aldosterone (0.2 mg/kg/day via a second minipump). Amiloride-sensitive Na+ current in freshly isolated CCD as measured by using patch clamp lower in Aldo + PF group than in Aldo group. Together, these results support an essential role of S1P-derived sPRR in mediating Aldo-induced ENaC activation.

Abstract P030: Dct (pro)renin Receptor Via Its Soluble Product Inhibits Ncc Activity During Regulation Of Blood Pressure And Potassium Homeostasis In Mice

Sodium-chloride cotransporter (NCC), uniquely located at the distal convoluted tubule (DCT), is the target of thiazide diuretics and critically involved in renal handling of both Na + and K + . However, the mechanism of how NCC activity is regulated is incompletely understood. Here we report a novel role of (pro)renin receptor (PRR) and its cleavage product soluble PRR (sPRR) via site-1 protease (S1P) as negative regulators of NCC during high-salt or high-K + loading. Mice with DCT-specific deletion of PRR (termed as DCT PRR KO) was generated by crossing PRR floxed mice with a transgenic mouse line expressing Cre under the control of the parvalbumin (PV) promoter. Radiotelemrty demonstrated that under basic condition, DCT PRR KO mice exhibited modest hypertension (~5 mmHg) associated with reduced urinary Na + (Floxed: 0.20±0.02, KO: 0.12±0.01 mmol/24h, p<0.001), K + (Floxed: 0.29±0.02, KO: 0.18±0.03 mmol/24h, p<0.05), and Cl - (Floxed: 0.30±0.03, KO: 0.21±0.02 mmol/24h, p<0.01) excretion and increased in vivo NCC activity as assessed by hydrochlorothiazide (HCTN)-induced natriuresis ( by 61%). Renal protein abundance of pNCC-T53 (by 51%) but not total NCC was elevated in the null mice. Following a high salt diet, DCT PRR KO mice were salt sensitive as evidenced by a ~30 mm Hg increase of mean arterial pressure (MAP) which was sensitive to HCTN (ΔMAP, Floxed+HS: -2.05±1.50, KO+HS: -12.77±2.6 mmHg, p<0.05), contrasting to salt resistance in the floxed control. The null mice also exhibited impaired kaliuresis (Floxed+HK: 3.24±0.13, KO+HK: 2.32±0.23 mmol/24h, p<0.001) and hyperkalemia (Floxed+HK: 3.75±0.05, KO+HK: 4.40±0.08 mM, p<0.001) after high K + intake. This phenotype was recapitulated by treatment of C57/BL6 mice with S1P inhibitor PF429242. In cultured Flp-In T-REx 293 NCC cells, S1P-derived sPRR directly dephosphorylated NCC (by 40%) via activation of angiotensin II receptor type 1 (AT1R) independently of aldosterone. 1 / 2 Taken together, the present study has demonstrated that S1P-derived sPRR via AT1R negatively regulates NCC activity in the DCT to render salt resistance and to promote K + excretion. 2 / 2

Soluble (pro)renin receptor promotes the fibrotic response in renal proximal tubule epithelial cells in vitro via the Akt/β-catenin/Snail signaling pathway.

Tubulointerstitial fibrosis has been regarded as a critical event in the pathogenesis of chronic kidney disease. The soluble form of (pro)renin receptor (sPRR), generated by site-1 protease (S1P) cleavage of full-length PRR, can be detected in biological fluid and elevated under certain pathological conditions. The present study was designed to evaluate the potential role of sPRR in the regulation of the fibrotic response in a cultured human renal proximal tubular cell line (HK-2 cells) in the setting of transforming growth factor (TGF)-β or sPRR-His treatment. The TGF-β-induced fibrotic response of HK-2 cells was indicated by upregulation of fibronectin (FN) expression; meanwhile, TGF-β could also induce the generation of sPRR, due to enhanced cleavage of full-length PRR. To explore the role of sPRR in the fibrotic response of HK-2 cells, we blocked the production of sPRR with a the S1P inhibitor PF429242 and found that PF429242 remarkably suppressed TGF-β-induced sPRR generation and FN expression in HK-2 cells. Administration of sPRR-His restored the PF429242-attenuated FN expression in HK-2 cells, indicating that sPRR could promote the TGF-β-induced fibrotic response. Furthermore, sPRR-His alone also increased the abundance of FN in HK-2 cells. These data suggested that sPRR was sufficient and necessary for the TGF-β-induced fibrotic response of HK-2 cells. Mechanistically, sPRR activated the AKT and β-catenin pathway in HK-2 cells, and blockade of the AKT or β-catenin pathway significantly abrogated sPRR-induced FN and Snail expression. Taking together, sPRR promoted the fibrotic response of HK-2 cells by activating Akt/β-catenin/Snail signaling, and it may serve as a potential therapeutic target in renal fibrosis.

Discovery of Potent SARS-CoV-2 Inhibitors from Approved Antiviral Drugs via Docking and Virtual Screening.

Coronavirus Disease 2019 (COVID-19) pandemic continues to threaten patients, societies and healthcare systems around the world. There is an urgent need to search for possible medications. This article intends to use virtual screening and molecular docking methods to find potential inhibitors from existing drugs that can respond to COVID-19. To take part in the current research investigation and to define a potential target drug that may protect the world from the pandemic of corona disease, a virtual screening study of 129 approved drugs was carried out which showed that their metabolic characteristics, dosages used, potential efficacy and side effects are clear as they have been approved for treating existing infections. Especially 12 drugs against chronic hepatitis B virus, 37 against chronic hepatitis C virus, 37 against human immunodeficiency virus, 14 anti-herpesvirus, 11 anti-influenza, and 18 other drugs currently on the market were considered for this study. These drugs were then evaluated using virtual screening and molecular docking studies on the active site of the (SARS-CoV-2) main protease (6lu7). Once the efficacy of the drug is determined, it can be approved for its in vitro and in vivo activity against the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which can be beneficial for the rapid clinical treatment of patients. These drugs were considered potentially effective against SARS-CoV-2 and those with high molecular docking scores were proposed as novel candidates for repurposing. The N3 inhibitor cocrystallized with protease (6lu7) and the anti-HIV protease inhibitor Lopinavir were used as standards for comparison. The results suggest the effectiveness of Beclabuvir, Nilotinib, Tirilazad, Trametinib and Glecaprevir as potent drugs against SARS-CoV-2 since they tightly bind to its main protease. These promising drugs can inhibit the replication of the virus; hence, the repurposing of these compounds is suggested for the treatment of COVID-19. No toxicity measurements are required for these drugs since they were previously tested prior to their approval by the FDA. However, the assessment of these potential inhibitors as clinical drugs requires further in vivo tests of these drugs.

Drug repurposing studies targeting SARS-CoV-2: an ensemble docking approach on drug target 3C-like protease (3CLpro)

The COVID-19 pandemic has been responsible for several deaths worldwide. The causative agent behind this disease is the Severe Acute Respiratory Syndrome – novel Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 belongs to the category of RNA viruses. The main protease, responsible for the cleavage of the viral polyprotein is considered as one of the hot targets for treating COVID-19. Earlier reports suggest the use of HIV anti-viral drugs for targeting the main protease of SARS-CoV, which caused SARS in the year 2002–2003. Hence, drug repurposing approach may prove to be useful in targeting the main protease of SARS-CoV-2. The high-resolution crystal structure of the main protease of SARS-CoV-2 (PDB ID: 6LU7) was used as the target. The Food and Drug Administration approved and SWEETLEAD database of drug molecules were screened. The apo form of the main protease was simulated for a cumulative of 150 ns and 10 μs open-source simulation data was used, to obtain conformations for ensemble docking. The representative structures for docking were selected using RMSD-based clustering and Markov State Modeling analysis. This ensemble docking approach for the main protease helped in exploring the conformational variation in the drug-binding site of the main protease leading to the efficient binding of more relevant drug molecules. The drugs obtained as top hits from the ensemble docking possessed anti-bacterial and anti-viral properties. This in silico ensemble docking approach would support the identification of potential candidates for repurposing against COVID-19. Communicated by Ramaswamy H. Sarma

Computational investigation of potential inhibitors of novel coronavirus 2019 through structure-based virtual screening, molecular dynamics and density functional theory studies

Despite the intensive research efforts towards antiviral drug against COVID-19, no potential drug or vaccines has not yet discovered. Initially, the binding site of COVID-19 main protease was predicted which located between regions 2 and 3. Structure-based virtual screening was performed through a hierarchal mode of elimination technique after generating a grid box. This led to the identification of five top hit molecules that were selected on the basis of docking score and visualization of non-bonding interactions. The docking results revealed that the hydrogen bonding and hydrophobic interactions are the major contributing factors in the stabilization of complexes. The docking scores were found between −7.524 and −6.711 kcal/mol indicating strong ligand-protein interactions. Amino acid residues Phe140, Leu141, Gly143, Asn142, Thr26, Glu166 and Thr190 (hydrogen bonding interactions) and Phe140, Cys145, Cys44, Met49, Leu167, Pro168, Met165, Val42, Leu27 and Ala191 (hydrophobic interactions) formed the binding pocket of COVID-19 main protease. From identified hits, ZINC13144609 and ZINC01581128 were selected for atomistic MD simulation and density functional theory calculations. MD simulation results confirm that the protein interacting with both hit molecules is stabilized in the chosen POPC lipid bilayer membrane. The presence of lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) in the hydrophobic region of the hit molecules leads to favorable ligand-protein contacts. The calculated pharmacokinetic descriptors were found to be in their acceptable range and therefore confirming their drug-like properties. Hence, the present investigation can serve as the basis for designing and developing COVID-19 inhibitors. Communicated by Ramaswamy H. Sarma

POST1/C12ORF49 regulates the SREBP pathway by promoting site-1 protease maturation

Sterol-regulatory element binding proteins (SREBPs) are the key transcriptional regulators of lipid metabolism. The activation of SREBP requires translocation of the SREBP precursor from the endoplasmic reticulum to the Golgi, where it is sequentially cleaved by site-1 protease (S1P) and site-2 protease and releases a nuclear form to modulate gene expression. To search for new genes regulating cholesterol metabolism, we perform a genome-wide CRISPR/Cas9 knockout screen and find that partner of site-1 protease (POST1), encoded by C12ORF49, is critically involved in the SREBP signaling. Ablation of POST1 decreases the generation of nuclear SREBP and reduces the expression of SREBP target genes. POST1 binds S1P, which is synthesized as an inactive protease (form A) and becomes fully mature via a two-step autocatalytic process involving forms B’/B and C’/C. POST1 promotes the generation of the functional S1P-C’/C from S1P-B’/B (canonical cleavage) and, notably, from S1P-A directly (non-canonical cleavage) as well. This POST1-mediated S1P activation is also essential for the cleavages of other S1P substrates including ATF6, CREB3 family members and the α/β-subunit precursor of N-acetylglucosamine-1-phosphotransferase. Together, we demonstrate that POST1 is a cofactor controlling S1P maturation and plays important roles in lipid homeostasis, unfolded protein response, lipoprotein metabolism and lysosome biogenesis.

Probing the Interaction between HIV-1 Protease and the Homodimeric p66/p66' Reverse Transcriptase Precursor by Double Electron-Electron Resonance EPR Spectroscopy.

Following excision from the Gag-Pol polyprotein, HIV-1 reverse transcriptase is released as an asymmetric homodimer comprising two p66 subunits that are structurally dissimilar but identical in amino acid sequence. Subsequent cleavage of the RNase H domain from only one of the subunits, denoted p66', results in the formation of the mature p66/p51 enzyme in which catalytic activity resides in the p66 subunit, and the p51 subunit (derived from p66') provides a supporting structural scaffold. Here, we probe the interaction of the p66/p66' asymmetric reverse transcriptase precursor with HIV-1 protease by pulsed Q-band double electron-electron resonance EPR spectroscopy to measure distances between nitroxide labels introduced at surface-engineered cysteine residues. The data suggest that the flexible, exposed linker between the RNaseH and connection domains in the open state of the p66' subunit binds to the active site of protease in a configuration that is similar to that of extended peptide substrates.

Identification of a novel dual-target scaffold for 3CLpro and RdRp proteins of SARS-CoV-2 using 3D-similarity search, molecular docking, molecular dynamics and ADMET evaluation

The new SARS-CoV-2 coronavirus is the causative agent of the COVID-19 pandemic outbreak that affected whole the world with more than 6 million confirmed cases and over 370,000 deaths. At present, there are no effective treatments or vaccine for this disease, which constitutes a serious global health crisis. As the pandemic still spreading around the globe, it is of interest to use computational methods to identify potential inhibitors for the virus. The crystallographic structures of 3CLpro (PDB: 6LU7) and RdRp (PDB 6ML7) were used in virtual screening of 50000 chemical compounds obtained from the CAS Antiviral COVID19 database using 3D-similarity search and standard molecular docking followed by ranking and selection of compounds based on their binding affinity, computational techniques for the sake of details on the binding interactions, absorption, distribution, metabolism, excretion, and toxicity prediction; we report three 4-(morpholin-4-yl)-1,3,5-triazin-2-amine derivatives; two compounds (2001083-68-5 and 2001083-69-6) with optimal binding features to the active site of the main protease and one compound (833463-19-7) with optimal binding features to the active site of the polymerase for further consideration to fight COVID-19. The structural stability and dynamics of lead compounds at the active site of 3CLpro and RdRp were examined using molecular dynamics (MD) simulation. Essential dynamics demonstrated that the three complexes remain stable during simulation of 20 ns, which may be suitable candidates for further experimental analysis. As the identified leads share the same scaffold, they may serve as promising leads in the development of dual 3CLpro and RdRp inhibitors against SARS-CoV-2. Communicated by Ramaswamy H. Sarma.

An in silico approach for identification of novel inhibitors as potential therapeutics targeting COVID-19 main protease

Respiratory disease caused by a novel coronavirus, COVID-19, has been labeled a pandemic by the World Health Organization. Very little is known about the infection mechanism for this virus. More importantly, there are no drugs or vaccines that can cure or prevent a person from getting COVID-19. In this study, the binding affinity of 2692 protease inhibitor compounds that are known in the protein data bank, are calculated against the main protease of the novel coronavirus with docking and molecular dynamics (MD). Both the docking and MD methods predict the macrocyclic tissue factor–factor VIIa (PubChem ID: 118098670) inhibitor to bind strongly with the main protease with a binding affinity of −10.6 and −10.0 kcal/mol, respectively. The TF-FVIIa inhibitors are known to prevent the coagulation of blood and have antiviral activity as shown in the case of SARS coronavirus. Two more inhibitors, phenyltriazolinones (PubChem ID: 104161460) and allosteric HCV NS5B polymerase thumb pocket 2 (PubChem ID: 163632044) have shown antiviral activity and also have high affinity towards the main protease of COVID-19. Furthermore, these inhibitors interact with the catalytic dyad in the active site of the COVID-19 main protease that is especially important in viral replication. The calculated theoretical dissociation constants of the proposed COVID-19 inhibitors are found to be very similar to the experimental dissociation constant values of similar protease-inhibitor systems. Communicated by Ramaswamy H. Sarma

Molecular Docking Study on Some Isonicotinoyl Hydrazide Derivatives as Potential Inhibitors of COVID-19

Coronavirus (COVID-19) is more than a health disaster;it is the greatest challenge that the world confrontsnowadays. There is a race to slow the spread of this disease. Searching for an antiviral agent to stop COVID-19 is an essential demand since there is no approved drug for COVID-19 till now. Molecular docking is a powerful tool in predicting new drugs. In this study, Favpiravir (Avigan), Hydroxychloroquine, and a series of biologically active compounds derived from iso-nicotinoyl hydrazide have been chosen for molecular docking study. Molecular docking was carried out by theMolegro virtual docker program on proteaseenzyme of COVID-19.The results showed that all the studied molecules are located in the active sites of protease after molecular docking. The tested nicotinoyl hydrazide derivatives showed a higher ranking docking score than Favpiravir (Avigan). According to the docking score ranking rearrangement, Hydroxychloroquine comes the third, and Favpiravir comes the last among the tested compounds. N(2-iso-nicotinoyl hydrazine-carbonthioyl)benzamide(2) and the enol form of (E)-N-(1-phenylethylidene)-nicotinohydrazide(7) have shown the highest docking score (123.23 and -123.12 kcal/mol respectively) among the tested compounds. Ligands (2) and (7) are expected to be potential inhibitors of the main protease enzyme of coronavirus.

Site-1 protease ablation in the osterix-lineage in mice results in bone marrow neutrophilia and hematopoietic stem cell alterations.

ABSTRACTSite-1 protease (S1P) ablation in the osterix-lineage in mice drastically reduces bone development and downregulates bone marrow-derived skeletal stem cells. Here we show that these mice also suffer from spina bifida occulta with a characteristic lack of bone fusion in the posterior neural arches. Molecular analysis of bone marrow-derived non-red blood cell cells, via single-cell RNA-Seq and protein mass spectrometry, demonstrate that these mice have a much-altered bone marrow with a significant increase in neutrophils and Ly6C-expressing leukocytes. The molecular composition of bone marrow neutrophils is also different as they express more and additional members of the stefin A (Stfa) family of proteins. In vitro, recombinant Stfa1 and Stfa2 proteins have the ability to drastically inhibit osteogenic differentiation of bone marrow stromal cells, with no effect on adipogenic differentiation. FACS analysis of hematopoietic stem cells show that despite a decrease in hematopoietic stem cells, S1P ablation results in an increased production of granulocyte-macrophage progenitors, the precursors to neutrophils. These observations indicate that S1P has a role in the lineage specification of hematopoietic stem cells and/or their progenitors for development of a normal hematopoietic niche. Our study designates a fundamental requirement of S1P for maintaining a balanced regenerative capacity of the bone marrow niche.

Potential of Flavonoid-Inspired Phytomedicines against COVID-19.

Flavonoids are widely used as phytomedicines. Here, we report on flavonoid phytomedicines with potential for development into prophylactics or therapeutics against coronavirus disease 2019 (COVID-19). These flavonoid-based phytomedicines include: caflanone, Equivir, hesperetin, myricetin, and Linebacker. Our in silico studies show that these flavonoid-based molecules can bind with high affinity to the spike protein, helicase, and protease sites on the ACE2 receptor used by the severe acute respiratory syndrome coronavirus 2 to infect cells and cause COVID-19. Meanwhile, in vitro studies show potential of caflanone to inhibit virus entry factors including, ABL-2, cathepsin L, cytokines (IL-1β, IL-6, IL-8, Mip-1α, TNF-α), and PI4Kiiiβ as well as AXL-2, which facilitates mother-to-fetus transmission of coronavirus. The potential for the use of smart drug delivery technologies like nanoparticle drones loaded with these phytomedicines to overcome bioavailability limitations and improve therapeutic efficacy are discussed.

Mutations in SREBF1, Encoding Sterol Regulatory Element Binding Transcription Factor 1, Cause Autosomal-Dominant IFAP Syndrome

IFAP syndrome is a rare genetic disorder characterized by ichthyosis follicularis, atrichia, and photophobia. Previous research found that mutations in MBTPS2, encoding site-2-protease (S2P), underlie X-linked IFAP syndrome. The present report describes the identification via whole-exome sequencing of three heterozygous mutations in SREBF1 in 11 unrelated, ethnically diverse individuals with autosomal-dominant IFAP syndrome. SREBF1 encodes sterol regulatory element-binding protein 1 (SREBP1), which promotes the transcription of lipogenes involved in the biosynthesis of fatty acids and cholesterols. This process requires cleavage of SREBP1 by site-1-protease (S1P) and S2P and subsequent translocation into the nucleus where it binds to sterol regulatory elements (SRE). The three detected SREBF1 mutations caused substitution or deletion of residues 527, 528, and 530, which are crucial for S1P cleavage. Invitro investigation of SREBP1 variants demonstrated impaired S1P cleavage, which prohibited nuclear translocation of the transcriptionally active form of SREBP1. As a result, SREBP1 variants exhibited significantly lower transcriptional activity compared to the wild-type, as demonstrated via luciferase reporter assay. RNA sequencing of the scalp skin from IFAP-affected individuals revealed a dramatic reduction in transcript levels of low-density lipoprotein receptor (LDLR) and of keratin genes known to be expressed in the outer root sheath of hair follicles. An increased rate of in situ keratinocyte apoptosis, which might contribute to skin hyperkeratosis and hypotrichosis, was also detected in scalp samples from affected individuals. Together with previous research, the present findings suggest that SREBP signaling plays an essential role in epidermal differentiation, skin barrier formation, hair growth, and eye function.

Increased soluble (pro)renin receptor protein by autophagy inhibition in cultured cancer cells.

(Pro)renin receptor ((P)RR) regulates the renin-angiotensin system and functions as an essential accessory subunit of vacuolar H+ -ATPase. There is accumulating evidence that shows close relationship between (P)RR and autophagy. Soluble (P)RR consisting of the extracellular domain of (P)RR is generated from (P)RR by proteolytic enzymes. The aim of the present study was to clarify the influence of autophagy inhibition on soluble (P)RR expression in cancer cells. Autophagy was inhibited by treatment of bafilomycin A1 or chloroquine in MCF-7 and A549 cells for 72hr. Western blot analysis showed that protein levels of soluble (P)RR were markedly elevated by autophagy inhibition, whereas no noticeable increases were observed in full-length (P)RR. Secretion of soluble (P)RR into the medium was increased dose-dependently by bafilomycin A1 or chloroquine. Autophagy inhibition was confirmed by enhanced accumulation of autophagy-related proteins, LC3, p62 and LAMP1 in intracellular vesicles. Increased amount of soluble (P)RR by autophagy inhibition was decreased by site-1 protease inhibitor, whereas no noticeable increase in site-1 protease immunoreactivity was observed in cells with autophagy inhibition by immunocytochemistry. These findings suggest that soluble (P)RR protein accumulates by autophagy inhibition, possibly because of the reduced degradation of soluble (P)RR in the intracellular vesicles during autophagy inhibition.