Target Proteases Research Articles

Quinoline analogue as a potential inhibitor of SARS-CoV-2 main protease: ADMET prediction, molecular docking and dynamics simulation analysis

The novel coronavirus (COVID-19) has triggered a major human turmoil worldwide by posing challenges regarding infection prevention, disease diagnosis, and treatment. Several drugs including remdesivir (RDV), hydroxychloroquine (HCQ), and others are being used to treat COVID-19, although these are not specifically proven drugs. Thus, it is very critical to understand COVID-19 drug targets and their interactions with candidate drugs. Here, we attempted in silico screening of ten quinoline analogs (Q1-Q10) against the five main proteases of SARS-CoV-2 by docking and dynamics analysis. The prediction of the ADMET profile showed that the best docked quinolines are safe and possess drug-like properties. The molecular interaction and binding affinity of these small molecules were determined with respect to the five protease (Mpro) targets of SARS-CoV-2 (PDB ID: 6LU7, 6W63, 6M03, 6Y84 and 6YB7). The study indicated that the quinoline ligands Q4, Q5, Q6, Q7, Q8, Q9, and Q10as probable inhibitors against SARS-CoV-2 Mpro and showed favorable binding interaction with the amino acid Glu166 of 6Y84, 6LU7and 6M03. Furthermore, Q9 has a highly significant docking score and binding affinity with all fiveCOVID-19 receptors having a minimum of two H-bonds, which is remarkable compared to HCQ, RDV, and other quinolines. The dynamics simulation analysis of this potent drug candidate Q9 with 6LU7 indicated high stability of the complex. In conclusion, our findings indicate that all of these quinolines in general possess good binding affinity and Q9 can serve as a good quinoline scaffold for the design of new antiviral agents to target the active site of SARS-CoV-2 MPro.

Systematic computer analysis of the pharmacology of bioflavonoids in the context of increasing the body's antiviral defenses

Background. The rapidly developing resistance of viruses to synthetic antiviral drugs indicates the need to use substances with multitarget action (to avoid polypharmacy and to improve the safety of treatment).Objective: systematic analysis of the scientific literature on the pharmacology of bioflavonoids with an emphasis on their antiviral action.Material and methods. More than 150,000 references of primary sources were found in the PubMed/MEDLINE database of biomedical publications, including 3282 references on the antiviral effects of bioflavonoids. A systematic computerized analysis of this array of publications was carried out in order to identify the main directions in the pharmacology of bioflavonoids with an emphasis on their antiviral, antibacterial and immunomodulatory effects. The literature analysis was carried out using modern methods of topological and metric analysis of big data.Results. The molecular mechanisms of action of baicalin, hesperidin, rutin, quercetin, leukodelphinidin bioflavonoids and epigallocatechin-3-gallate, curcumin polyphenols, their anti-inflammatory, antioxidant, antiviral, bactericidal, angioprotective, regenerative effects, and their prospects in therapy, prevention and rehabilitation of patients with COVID-19 and other respiratory viral infections were described in detail.Conclusion. Bioflavonoids and synergistic polyphenols exhibit not only multitarget antiviral effects by inhibiting the main protease, spike proteins, and other target proteins, but also pronounced anti-inflammatory, hepatoprotective, and immunomodulatory effects.

In Silico Analysis of Bioactive Compounds from Sea Urchin (Echinometra mathaei) against SARS-COV-2

SARS-CoV-2 is a kind of coronavirus that produces Covid-19 illness, which is still a public health concern in Indonesia. Meanwhile, an effective drug has not yet been found and although vaccination has been carried out, in several regions and neighboring countries there is still an increase in Covid-19 cases. This study aimed to obtain bioactive compounds from sea urchins (Echinometra mathaei) that have greater antiviral potential and lower toxicity than remdesivir. This research was started by predicting druglikeness with SwissADME, followed ADMET predicition with pkCSM online, and docking of molecule using the Molegro Virtual Docker (MVD) 5.5 software against the main protease (Mpro) target (PDB ID: 6W63). The results showed that six compounds from sea urchins (Echinometra mathaei) had antiviral activity, where the bioactive compound from sea urchins (Echinometra mathaei) with the highest affinity was shown by Spinochrome C a smaller rerank score compared with Remdesivir and native ligand (X77). So that Spinochrome C compounds are candidates as SARS-CoV-2 inhibitors potential developed drug.

SARS-CoV-2 Main Protease Targets Host Selenoproteins and Glutathione Biosynthesis for Knockdown via Proteolysis, Potentially Disrupting the Thioredoxin and Glutaredoxin Redox Cycles

Associations between dietary selenium status and the clinical outcome of many viral infections, including SARS-CoV-2, are well established. Multiple independent studies have documented a significant inverse correlation between selenium status and the incidence and mortality of COVID-19. At the molecular level, SARS-CoV-2 infection has been shown to decrease the expression of certain selenoproteins, both in vitro and in COVID-19 patients. Using computational methods, our group previously identified a set of six host proteins that contain potential SARS-CoV-2 main protease (Mpro) cleavage sites. Here we show experimentally that Mpro can cleave four of the six predicted target sites, including those from three selenoproteins: thioredoxin reductase 1 (TXNRD1), selenoprotein F, and selenoprotein P, as well as the rate-limiting enzyme in glutathione synthesis, glutamate-cysteine ligase catalytic subunit (GCLC). Cleavage was assessed by incubating recombinant SARS-CoV-2 Mpro with synthetic peptides spanning the proposed cleavage sites, and analyzing the products via UPLC-MS. Furthermore, upon incubation of a recombinant Sec498Ser mutant of the full TXNRD1 protein with SARS-CoV-2 Mpro, the predicted cleavage was observed, destroying the TXNRD1 C-terminal redox center. Mechanistically, proteolytic knockdown of both TXNRD1 and GCLC is consistent with a viral strategy to inhibit DNA synthesis, conserving the pool of ribonucleotides for increased virion production. Viral infectivity could also be enhanced by GCLC knockdown, given the ability of glutathione to disrupt the structure of the viral spike protein via disulfide bond reduction. These findings shed new light on the importance of dietary factors like selenium and glutathione in COVID-19 prevention and treatment.

In-Silico Lead Druggable Compounds Identification against SARS COVID-19 Main Protease Target from In-House, Chembridge and Zinc Databases by Structure-Based Virtual Screening, Molecular Docking and Molecular Dynamics Simulations.

Pharmacological strategies to lower the viral load among patients suffering from severe diseases were researched in great detail during the SARS-CoV-2 outbreak. The viral protease Mpro (3CLpro) is necessary for viral replication and is among the main therapeutic targets proposed, thus far. To stop the pandemic from spreading, researchers are working to find more effective Mpro inhibitors against SARS-CoV-2. The 33.8 kDa Mpro protease of SARS-CoV-2, being a nonhuman homologue, has the possibility of being utilized as a therapeutic target against coronaviruses. To develop drug-like compounds capable of preventing the replication of SARS-main CoV-2's protease (Mpro), a computer-aided drug design (CADD) approach is extremely viable. Using MOE, structure-based virtual screening (SBVS) of in-house and commercial databases was carried out using SARS-CoV-2 proteins. The most promising hits obtained during virtual screening (VS) were put through molecular docking with the help of MOE. The virtual screening yielded 3/5 hits (in-house database) and 56/66 hits (commercial databases). Finally, 3/5 hits (in-house database), 3/5 hits (ZINC database), and 2/7 hits (ChemBridge database) were chosen as potent lead compounds using various scaffolds due to their considerable binding affinity with Mpro protein. The outcomes of SBVS were then validated using an analysis based on molecular dynamics simulation (MDS). The complexes' stability was tested using MDS and post-MDS. The most promising candidates were found to exhibit a high capacity for fitting into the protein-binding pocket and interacting with the catalytic dyad. At least one of the scaffolds selected will possibly prove useful for future research. However, further scientific confirmation in the form of preclinical and clinical research is required before implementation.

Safety of Intravenous Administration of an AAV8 Vector Coding for an Oxidation-Resistant Human α1-Antitrypsin for the Treatment of α1-Antitrypsin Deficiency.

α1-antitrypsin (AAT) deficiency is a common autosomal recessive hereditary disorder, with a high risk for the development of early-onset panacinar emphysema. AAT, produced primarily in the liver, functions to protect the lung from neutrophil protease; with AAT deficiency, unimpeded neutrophil proteases destroy the lung parenchyma. AAT is susceptible to oxidative damage resulting in an inability to inhibit its target proteases, neutrophil elastase, and cathepsin G. The major sites of oxidative modification on the AAT molecule are methionine residues 351 and 358. We have previously demonstrated that an engineered variant of AAT that resists oxidation by modifying both protein surface methionines (M351V and M358L) provides antiprotease protection, despite oxidative stress. In mice, intravenous delivery of the modified AAT(AVL) variant by AAV serotype 8, AAV8hAAT(AVL), primarily to the liver resulted in long-term expression of an AAT that resists oxidative inactivation. In this study, we evaluated the safety of intravenous administration of AAV8hAAT(AVL) in a dose-escalating, blinded, placebo-controlled toxicology study in wild-type mice. The study assessed organ histology and clinical pathology findings of mice, intravenously administered AAV8hAAT(AVL) at three doses (5.0 × 1011, 5.0 × 1012, and 5.0 × 1013 genome copies [gc]/kg), compared to control mice injected intravenously with phosphate-buffered saline. As previously demonstrated, administration of AAV8hAAT(AVL) resulted in dose-dependent expression of high, potentially therapeutic, levels of serum human AAT protein that persist for at least 6 months. Antibodies against the AAV8 capsid were elicited as expected, but there was no antibody detected against the AAT(AVL) protein generated by the AAV8hAAT(AVL) vector. There was no morbidity or mortality observed in the study. The data demonstrate that intravenous administration of AAV8hAAT(AVL) is safe with no significant adverse effect attributed to AAV8hAAT(AVL) vector at any dose. This study demonstrates that AAV8hAAT(AVL) has a safety profile consistent with the requirements for proceeding to a clinical study.

Virtual screening of novel pyridine derivatives as effective inhibitors of DNA gyrase (GyrA) of salmonella typhi

In a bid to discovering novel antibiotics to combat growing trend of multi-drug resistance strains of Salmonella typhi, 48 new pyridine derivatives with significant inhibitory activities against the aforementioned bacterium were subjected to molecular docking against DNA gyrase protease of the bacterium, drug likeness evaluation and pharmacokinetics profiling. All the 48 leads displayed better binding affinity values when compared with Amoxicillin, Ciprofloxacin, Ceftriaxone, Ampicillin, and chloramphenicol, the standard antibiotics used herein for quality assurance. Furthermore, the majority of the compounds were, however, screened out due to their poor pharmacokinetics profiles and drug-likeness. Only five compounds emerged as the most promising leads and they include C4 with binding affinity of -8.0 kcal/mol, C8 (-8.6 kcal/mol), C9 (-8.1 kcal/mol), C26 (-8.3 kcal/mol), and C27 (-8.0 kcal/mol). These compounds not only displayed better binding affinity when compared with the reference antibiotics but also exhibit different modes of interactions with the target protease of the bacterium making them more potent and drug like. Toxicity evaluation of the leads also revealed that the compounds are neither tumorigenic nor mutagenic. In view of the excellent binding affinity, high pharmacokinetics profile and positive drug-likeness of the novel ligands, we recommend these promising compounds for in vitro and in vivo studies in order to discover novel antibiotics that could curb the dangerous trend of multiple drug resistance by Salmonella typhi.

Embracing enzyme promiscuity with activity-based compressed biosensing

The development of protease-activatable drugs and diagnostics requires identifying substrates specific to individual proteases. However, this process becomes increasingly difficult as the number of target proteases increases because most substrates are promiscuously cleaved by multiple proteases. We introduce a method-substrate libraries for compressed sensing of enzymes (SLICE)-for selecting libraries of promiscuous substrates that classify protease mixtures (1) without deconvolution of compressed signals and (2) without highly specific substrates. SLICE ranks substrate libraries using a compression score (C), which quantifies substrate orthogonality and protease coverage. This metric is predictive of classification accuracy across 140 in silico (Pearson r= 0.71) and 55 invitro libraries (r= 0.55). Using SLICE, we select a two-substrate library to classify 28 samples containing 11 enzymes in plasma (area under the receiver operating characteristic curve [AUROC]= 0.93). We envision that SLICE will enable the selection of libraries that capture information from hundreds of enzymes using fewer substrates for applications like activity-based sensors for imaging and diagnostics.

SARS-CoV-2 proteases Mpro and PLpro: Design of inhibitors with predicted high potency and low mammalian toxicity using artificial neural networks, ligand-protein docking, molecular dynamics simulations, and ADMET calculations

The main (Mpro) and papain-like (PLpro) proteases are highly conserved viral proteins essential for replication of the COVID-19 virus, SARS-COV-2. Therefore, a logical plan for producing new drugs against this pathogen is to discover inhibitors of these enzymes. Accordingly, the goal of the present work was to devise a computational approach to design, characterize, and select compounds predicted to be potent dual inhibitors – effective against both Mpro and PLpro. The first step employed LigDream, an artificial neural network, to create a virtual ligand library. Ligands with computed ADMET profiles indicating drug-like properties and low mammalian toxicity were selected for further study. Initial docking of these ligands into the active sites of Mpro and PLpro was done with GOLD, and the highest-scoring ligands were redocked with AutoDock Vina to determine binding free energies (ΔG). Compounds 89–00, 89–07, 89–32, and 89–38 exhibited favorable ΔG values for Mpro (−7.6 to −8.7 kcal/mol) and PLpro (−9.1 to −9.7 kcal/mol). Global docking of selected compounds with the Mpro dimer identified prospective allosteric inhibitors 89–00, 89–27, and 89–40 (ΔG -8.2 to −8.9 kcal/mol). Molecular dynamics simulations performed on Mpro and PLpro active site complexes with the four top-scoring ligands from Vina demonstrated that the most stable complexes were formed with compounds 89–32 and 89–38. Overall, the present computational strategy generated new compounds with predicted drug-like characteristics, low mammalian toxicity, and high inhibitory potencies against both target proteases to form stable complexes. Further preclinical studies will be required to validate the in silico findings before the lead compounds could be considered for clinical trials.

Peptide Amphiphile Mediated Co‐assembly for Nanoplasmonic Sensing

AbstractAromatic interactions are commonly involved in the assembly of naturally occurring building blocks, and these interactions can be replicated in an artificial setting to produce functional materials. Here we describe a colorimetric biosensor using co‐assembly experiments with plasmonic gold and surfactant‐like peptides (SLPs) spanning a wide range of aromatic residues, polar stretches, and interfacial affinities. The SLPs programmed in DDD−(ZZ)x−FFPC self‐assemble into higher‐order structures in response to a protease and subsequently modulate the colloidal dispersity of gold leading to a colorimetric readout. Results show the strong aggregation propensity of the FFPC tail without polar DDD head. The SLPs were specific to the target protease, i.e., Mpro, a biomarker for SARS‐CoV‐2. This system is a simple and visual tool that senses Mproin phosphate buffer, exhaled breath condensate, and saliva with detection limits of 15.7, 20.8, and 26.1 nM, respectively. These results may have value in designing other protease testing methods.

Peptide Amphiphile Mediated Co-assembly for Nanoplasmonic Sensing.

Aromatic interactions are commonly involved in the assembly of naturally occurring building blocks, and these interactions can be replicated in an artificial setting to produce functional materials. Here we describe a colorimetric biosensor using co-assembly experiments with plasmonic gold and surfactant-like peptides (SLPs) spanning a wide range of aromatic residues, polar stretches, and interfacial affinities. The SLPs programmed in DDD-(ZZ)x -FFPC self-assemble into higher-order structures in response to a protease and subsequently modulate the colloidal dispersity of gold leading to a colorimetric readout. Results show the strong aggregation propensity of the FFPC tail without polar DDD head. The SLPs were specific to the target protease, i.e., Mpro , a biomarker for SARS-CoV-2. This system is a simple and visual tool that senses Mpro in phosphate buffer, exhaled breath condensate, and saliva with detection limits of 15.7, 20.8, and 26.1 nM, respectively. These results may have value in designing other protease testing methods.

Absolute binding free energies of the antiviral peptide ATN-161 with protein targets of SARS-CoV-2

The interactions of the antiviral pentapeptide ATN-161 with the closed and open conformations of the α5β1 integrin, the SARS-CoV-2 major protease, and the omicron variant spike protein complexed with hACE2 were studied using molecular docking and molecular dynamics simulation. Molecular docking was performed to obtain ATN-161 binding poses with these studied protein targets. Subsequently, molecular dynamics simulations were performed to verify the ligand stability at the binding site of each protein target. Pulling simulations, umbrella sampling, and weighted histogram analysis method were used to obtain the potential of mean force of each system and calculate the Gibbs free energy of binding for the ATN-161 peptide in each binding site of these protein targets. The results showed that ATN-161 binds to α5β1 integrin in its active and inactive form, binds weakly to the omicron variant spike protein complexed with hACE2, and strongly binds to the main protease target. Communicated by Ramaswamy H. Sarma

A common variant close to the "tripwire" linker region of NLRP1 contributes to severe COVID-19.

The heterogeneity of response to SARS-CoV-2 infection is directly linked to the individual genetic background. Genetic variants of inflammasome-related genes have been pointed as risk factors for several inflammatory sterile and infectious disease. In the group of inflammasome receptors, NLRP1 stands out as a good novel candidate as severity factor for COVID-19 disease. To address this question, we performed an association study of NLRP1, DPP9, CARD8, IL1B, and IL18 single nucleotide variants (SNVs) in a cohort of 945 COVID-19 patients. The NLRP1 p.Leu155His in the linker region, target of viral protease, was significantly associated to COVID-19 severity, which could contribute to the excessive cytokine release reported in severe cases. Inflammasome genetic background contributes to individual response to SARS-CoV-2.

Loss-of-function mutations in CST6 cause dry skin, desquamation and abnormal keratosis without hypotrichosis.

Cystatin M/E (encoded by the CST6 gene) is a cysteine protease inhibitor, that exerts regulatory and protective effects against uncontrolled proteolysis mainly by directly regulating cathepsin V, cathepsin L, and legumain activities. Previous studies have suggested that CST6 may exert a regulatory role in epidermal differentiation and hair follicle formation by inhibiting the activity of respective cognate target proteases. However, until recently, studies have revealed that loss- or gain-of-function of the CST6 gene causes dry skin with hypotrichosis in humans. Here, we reported two siblings of Chinese origin with dry skin, desquamation and abnormal keratosis without hypotrichosis. By applying whole-exome sequencing, we identified homozygous loss-of-function mutation c.251G > A (p.Gly84Asp) in the CST6 gene as the underlying genetic cause. Further fluorimetric enzyme assays demonstrated the mutant cystatin M/E protein lost its inhibitory function on the protease activity of cathepsins. Moreover, the corresponding mutation in mice resulted in excessive cornification, desquamation, impaired skin barrier function, and abnormal proliferation and differentiation of keratinocytes. In conclusion, the homozygous missense mutation c.251G > A in CST6 gene resulted in dry skin, desquamation, as well as abnormal keratosis of the skin, promoting our understanding of the role of protease-antiprotease balance in human skin disorders.

Aptamer-Small Molecule Conjugates As Potent and Reversible Surgical Anticoagulants Targeting Thrombin

Unfractionated heparin (UFH) is the first-line anticoagulant used in millions of heart surgeries annually. The high potency of UFH is attributed to its effective activation of the antithrombin-mediated anticoagulation pathway. However, the prolonged use of heparin in heart surgeries is often associated with bleeding disorders as it depletes both patient's coagulation and anticoagulation capacity. Moreover, UFH is immunogenic, which can trigger potentially life-threatening heparin-induced thrombocytopenia (HIT) when used repeatedly. Aptamers are short oligonucleotides isolated in vitro that bind to protein or small molecule targets with high affinity and specificity. Several aptamers have been created that act as anticoagulants as they can competitively block substrate binding to anticoagulation protease exosites. Compared to UFH, aptamers have advantages, including not activating antithrombin, reversibility by administration of a well-tolerated antisense oligonucleotide, and low immunogenicity. However, the anticoagulant potency of aptamers is often limited by their inability to effectively inhibit the catalytic sites of their protease targets. Here we provide a strategy to solve this potency problem by conjugating a protease active-site inhibitor to an exosite-binding aptamer via a polynucleotide linker. The resulting bivalent conjugate binds to both the active site and exosite of the target protease to completely inhibit protease function. Meanwhile, the synergy between two binding elements significantly improves the binding affinity compared to the small molecule inhibitor or the aptamer alone. As a proof of concept, we conjugated the thrombin active site inhibitor DAB to the 5’ terminus of anti-thrombin aptamer HD22 via a poly (7) adenosine linker (HD22-7A-DAB) and characterized its thrombin inhibition activity using a fluorogenic substrate cleavage assay. The results reveal that HD22-7A-DAB has a much higher thrombin inhibition activity (IC50 = 0.2 nM) than DAB (IC50 = 80 nM) or the unconjugated aptamer (IC50 > 1000 nM). The significantly higher inhibitory effect of HD22-7A-DAB compared to a 1:1 mixture of unconjugated DAB and HD22 also indicates that conjugation is essential to create an effective synergistic inhibitor. Notably, in the presence of 2 µM complementary DNA to HD22, the effect of HD22-7A-DAB conjugate was drastically diminished (IC50 > 1000 nM), which indicates that both aptamer and small molecule in ADIC can be reversed using an antidote oligonucleotide. HD22-7A-DAB also shows a UFH-rivaling anticoagulation effect and antidote-reversible behavior in a series of in vitro clotting assays, including thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT). Thus the development of a thrombin-targeting small molecule aptamer conjugate inhibitor yields a potent and reversible anticoagulant with clinically relevant anticoagulant activity. In addition, these studies describe a generalizable strategy for rapidly developing aptamer-small molecule active site inhibitor conjugates for generate other potent and reversible inhibitors of the other coagulation proteases. These potent and reversible anticoagulant aptamer-active site drug conjugates have the potential to become UFH substituents in surgeries to reduce the risk of UFH-associated bleeding, UFH-insensitivity and HIT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Structure and inhibition of SARS-CoV-1 and SARS-CoV-2 main proteases by oral antiviral compound AG7404

Severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) pose a threat to global public health. The 3C-like main protease (Mpro), which presents structural similarity with the active site domain of enterovirus 3C protease, is one of the best-characterized drug targets of these viruses. Here we studied the antiviral activity of the orally bioavailable enterovirus protease inhibitor AG7404 against SARS-CoV-1 and SARS-CoV-2 from a structural, biochemical, and cellular perspective, comparing it with the related molecule rupintrivir (AG7800). Crystallographic structures of AG7404 in complex with SARS-CoV-1 Mpro and SARS-CoV-2 Mpro and of rupintrivir in complex with SARS-CoV-2 Mpro were solved, revealing that all protein residues interacting with the inhibitors are conserved between the two proteins. A detailed analysis of protein-inhibitor interactions indicates that AG7404 has a better fit to the active site of the target protease than rupintrivir. This observation was further confirmed by biochemical FRET assays showing IC50 values of 47 μM and 101 μM for AG7404 and rupintrivir, respectively, in the case of SARS-CoV-2 Mpro. Equivalent IC50 values for SARS-CoV-1 also revealed greater inhibitory capacity of AG7404, with a value of 29 μM vs. 66 μM for rupintrivir. Finally, the antiviral activity of the two inhibitors against SARS-CoV-2 was confirmed in a human cell culture model of SARS-CoV-2 infection, although rupintrivir showed a higher potency and selectivity index in this assay.

A novel strategy for sensitive detection of thrombin via subtly integrated polypeptide substrate and aggregation-induced emission fluorophores in carotid artery thrombosis

Accurate monitoring thrombin played an important role in the diagnosis of carotid artery thrombosis. In this investigation, a novel strategy was designed by one-step process for sensitive and specific detection of thrombin via integrated 2-(4-bromophenyl)− 3-(4-(4-(diphenylamino)styryl) phenyl) fumaronitrile (TPABDFN) and polypeptide substrate. More concretely, TPABDFN was gathered and wrapped by the peculiar short chain polypeptide substrate (SPS) with the assistance of glutaric dialdehyde in water containing 10 % tetrahydrofuran (THF), possessing an “ON” state owing to the aggregation-induced emission (AIE) characteristics. In the present of thrombin, the TPABDFN-SPS could be specifically degraded, leading the “OFF” state by the specific hydrolysis of thrombin. Indeed, the fluorescence intensity of the TPABDFN-SPS would decline with the increasing concentrations of thrombin. The results demonstrated that this “turn-off” fluorescent complex had a linear response in the range of 1.56–200 ng mL−1 for thrombin with the detection limit of 0.42 ng mL−1. More fascinatingly, this strategy could further applied to monitor thrombin in the plasma sample, which displayed that the thrombin concentration in the plasma of carotid artery thrombosis (CAT) rats was higher than that in the sham group. Conceivably, the proposed strategy showed prospective promise in the detection of target proteases for the diagnose of certain disease.

Deep mutational scanning and massively parallel kinetics of plasminogen activator inhibitor-1 functional stability to probe its latency transition

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48h at 37 °C. Of the 7201 possible single amino acid substitutions in PAI-1, we identified 439 that increased the functional stability of PAI-1 beyond that of the WT protein. We also found 1549 single amino acid substitutions that retained inhibitory activity toward the canonical target protease of PAI-1 (urokinase-like plasminogen activator), whereas exhibiting functional stability less than or equal to that of WT PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying the latency transition of PAI-1 and provide a database for interpreting human PAI-1 genetic variants.

Two Tags in One Probe: Combining Fluorescence- and Biotin-based Detection of the Trypanosomal Cysteine Protease Rhodesain.

Rhodesain is the major cysteine protease of the protozoan parasite Trypanosoma brucei and a therapeutic target for sleeping sickness, a fatal neglected tropical disease. We designed, synthesized and characterized a bimodal activity-based probe that binds to and inactivates rhodesain. This probe exhibited an irreversible mode of action and extraordinary potency for the target protease with a kinac /Ki value of 37,000 M-1 s-1 . Two reporter tags, a fluorescent coumarin moiety and a biotin affinity label, were incorporated into the probe and enabled highly sensitive detection of rhodesain in a complex proteome by in-gel fluorescence and on-blot chemiluminescence. Furthermore, the probe was employed for microseparation and quantification of rhodesain and for inhibitor screening using a competition assay. The developed bimodal rhodesain probe represents a new proteomic tool for studying Trypanosoma pathobiochemistry and antitrypanosomal drug discovery.

Identification and validation of two alternatively spliced novel isoforms of human α-1-antichymotrypsin

α-1-antichymotrypsin (ACT) is a serine proteinase inhibitor that controls the activity of proteases like chymotrypsin, cathepsin G and mast cell chymase. Familial variants of ACT results in liver and lung diseases, but it is also reported to be associated with several other disease conditions. ACT is mainly synthesized in the liver using four coding exons, namely E1, E2, E3 and E4 encoding a 423 amino acid protein that also includes a 23 amino acid signal peptide. It is found to be associated with amyloid plaques and is elevated during inflammatory response and modulates cytokine based signal transduction pathways, independent of its anti-protease activity. Therefore, the multispecificity of ACT and its non-inhibitory roles in diseased conditions warrants an assessment of possible existence of the other isoforms. Consequently, scanning of introns, 5′ and 3′ region of the ACT gene using computational tools like FGENESH and FEX did indicate the presence of coding regions. Using a combined approach of bioinformatics and molecular biology, we have found one novel exon located in the intronic region between exons E1 and E2, that splices with exon E2 and replaces N-terminal exon E1, generating an ACT isoform with a novel 151 base pair N-terminus. This isoform was found to lack the signal sequence and is smaller in size but its reactive centre loop remains intact. A truncated transcript was also confirmed with an extension of the E3 by a 12 nucleotide intronic region including a stop codon. Modelling studies show that due to removal of E4 this isoform lacks the RCL. Novel isoform ACT-N lacks E1 but has a conserved RCL. However, due to loss of strands of β-sheet A, it may also be inactive, but with ability to bind the target proteases. The novel truncated ACT-T isoform lacks the RCL and may have a non-inhibitory role. These hypothesis will need further work for functional validation.