scholarly journals An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants

2021 ◽  
Vol 7 (8) ◽  
pp. eabf1738 ◽  
Author(s):  
Kui K. Chan ◽  
Timothy J. C. Tan ◽  
Krishna K. Narayanan ◽  
Erik Procko
Keyword(s):  
In Vitro Selection ◽  
Protein S ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
Host Cell Membrane ◽  
Decoy Receptor ◽  
Decoy Receptors ◽  
Binding Surface ◽  
Type Receptor

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, because of close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find that an engineered decoy receptor, sACE22.v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain followed by in vitro selection, with wild-type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild-type receptor. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.

scholarly journals An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants

2020 ◽  
Author(s):  
Kui K. Chan ◽  
Timothy J.C. Tan ◽  
Krishna K. Narayanan ◽  
Erik Procko
Keyword(s):  
In Vitro Selection ◽  
Protein S ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
Host Cell Membrane ◽  
Decoy Receptor ◽  
Decoy Receptors ◽  
Binding Surface ◽  
Type Receptor

ABSTRACTThe spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, due to close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find an engineered decoy receptor, sACE22.v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain (RBD) followed by in vitro selection, with wild type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild type receptor. Variant N501Y in the RBD, which has emerged in a rapidly spreading lineage (B.1.1.7) in England, enhances affinity for wild type ACE2 20-fold but remains tightly bound to engineered sACE22.v2.4. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.

scholarly journals Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

2009 ◽  
Vol 54 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Hassan Safi ◽  
Robert D. Fleischmann ◽  
Scott N. Peterson ◽  
Marcus B. Jones ◽  
Behnam Jarrahi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Vitro Selection ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutant Selection ◽  
Preferential Growth ◽  
Allelic Exchange ◽  
High Level ◽  
Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

scholarly journals Multiple Novel Inhibitors of the NorA Multidrug Transporter of Staphylococcus aureus

1999 ◽  
Vol 43 (10) ◽  
pp. 2404-2408 ◽  
Author(s):  
Penelope N. Markham ◽  
Eric Westhaus ◽  
Katya Klyachko ◽  
Michael E. Johnson ◽  
Alex A. Neyfakh
Keyword(s):  
Staphylococcus Aureus ◽  
Clinical Setting ◽  
In Vitro Selection ◽  
Antibacterial Activities ◽  
Multidrug Transporter ◽  
Wild Type ◽  
Chemical Library ◽  
Fluoroquinolone Antibiotics

ABSTRACT The multidrug transporter NorA contributes to the resistance ofStaphylococcus aureus to fluoroquinolone antibiotics by promoting their active extrusion from the cell. Previous studies with the alkaloid reserpine, the first identified inhibitor of NorA, indicate that the combination of a chemical NorA inhibitor with a fluoroquinolone could improve the efficacy of this class of antibiotics. Since reserpine is toxic to humans at the concentrations required to inhibit NorA, we sought to identify new inhibitors of NorA that may be used in a clinical setting. Screening of a chemical library yielded a number of structurally diverse inhibitors of NorA that were more potent than reserpine. The new inhibitors act in a synergistic manner with the most widely used fluoroquinolone, ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. Furthermore, the inhibitors dramatically suppress the emergence of ciprofloxacin-resistant S. aureus upon in vitro selection with this drug. Some of these new inhibitors, or their derivatives, may prove useful for augmentation of the antibacterial activities of fluoroquinolones in the clinical setting.

In vitro selection of Phytomonas serpens cells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps

Parasitology ◽  
2017 ◽  
Vol 145 (3) ◽  
pp. 355-370 ◽  
Author(s):  
SIMONE S. C. OLIVEIRA ◽  
INÊS C. GONÇALVES ◽  
VITOR ENNES-VIDAL ◽  
ANGELA H. C. S. LOPES ◽  
RUBEM F. S. MENNA-BARRETO ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Oncopeltus Fasciatus ◽  
Calpain Inhibitor ◽  
Insect Vector ◽  
Peptidase Activity ◽  
Wild Type ◽  
Calcium Dependent ◽  
Calpain Inhibitors ◽  
Selection Of

SUMMARYThe species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µm (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

scholarly journals Robotic QM/MM-driven maturation of antibody combining sites

2016 ◽  
Vol 2 (10) ◽  
pp. e1501695 ◽  
Author(s):  
Ivan V. Smirnov ◽  
Andrey V. Golovin ◽  
Spyros D. Chatziefthimiou ◽  
Anastasiya V. Stepanova ◽  
Yingjie Peng ◽  
...  
Keyword(s):  
Immune Response ◽  
In Vitro Selection ◽  
Single Point ◽  
Combinatorial Libraries ◽  
Single Point Mutation ◽  
Wild Type ◽  
Selection Of ◽  
Maturation Function

In vitro selection of antibodies from large repertoires of immunoglobulin (Ig) combining sites using combinatorial libraries is a powerful tool, with great potential for generating in vivo scavengers for toxins. However, addition of a maturation function is necessary to enable these selected antibodies to more closely mimic the full mammalian immune response. We approached this goal using quantum mechanics/molecular mechanics (QM/MM) calculations to achieve maturation in silico. We preselected A17, an Ig template, from a naïve library for its ability to disarm a toxic pesticide related to organophosphorus nerve agents. Virtual screening of 167,538 robotically generated mutants identified an optimum single point mutation, which experimentally boosted wild-type Ig scavenger performance by 170-fold. We validated the QM/MM predictions via kinetic analysis and crystal structures of mutant apo-A17 and covalently modified Ig, thereby identifying the displacement of one water molecule by an arginine as delivering this catalysis.

scholarly journals Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine

2021 ◽  
Vol 1 ◽  
Author(s):  
Eun Young Hong ◽  
Sun-Gu Lee ◽  
Hyungdon Yun ◽  
Byung-Gee Kim
Keyword(s):  
Disulfide Bond ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Arginine Decarboxylase ◽  
Saturation Mutagenesis ◽  
Alkaline Ph ◽  
Wild Type ◽  
Active Site Residues ◽  
Cellular Mechanisms

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (>89.0% yield based on 100 mM of Arg within 5  h).

scholarly journals Protein scaffold-based multimerization of soluble ACE2 efficiently blocks SARS-CoV-2 infection in vitro

2021 ◽  
Author(s):  
Alisan Kayabolen ◽  
Ugur Akcan ◽  
Dogancan Ozturan ◽  
Ehsan Sarayloo ◽  
Elif Nurtop ◽  
...  
Keyword(s):  
Treatment Approach ◽  
Viral Proteins ◽  
Spike Protein ◽  
Decoy Receptor ◽  
Highly Active ◽  
Decoy Receptors ◽  
Novel Variants ◽  
Promising Treatment ◽  
Nanomolar Range

Soluble ACE2 (sACE2) decoy receptors are promising agents to inhibit SARS-CoV-2 as they are not affected by common escape mutations in viral proteins. However, their success may be limited by their relatively poor potency. To address these challenges, we developed a highly active multimeric sACE2 decoy receptor via a SunTag system that could neutralize both pseudoviruses bearing SARS-CoV-2 spike protein and SARS-CoV-2 clinical isolates. This fusion protein demonstrated a neutralization efficiency nearly 250-fold greater than monomeric sACE2. SunTag in combination with a more potent version of sACE2 achieved near complete neutralization at a sub-nanomolar range, which is comparable with clinical monoclonal antibodies. We demonstrate that this activity is due to greater occupancy of the multimeric decoy receptors on Spike protein as compared to monomeric sACE2. Overall, these highly potent multimeric sACE2 decoy receptors offer a promising treatment approach against SARS-CoV-2 infections including its novel variants.

scholarly journals Characterization of a Novel Human Immunodeficiency Virus Type 1 Protease Inhibitor, A-790742

2008 ◽  
Vol 52 (4) ◽  
pp. 1337-1344 ◽  
Author(s):  
Tatyana Dekhtyar ◽  
Teresa I. Ng ◽  
Liangjun Lu ◽  
Sherie Masse ◽  
David A. DeGoey ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protease Inhibitor ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
In Vitro Selection ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A-790742 is a potent human immunodeficiency virus type 1 (HIV-1) protease inhibitor, with 50% effective concentrations ranging from 2 to 7 nM against wild-type HIV-1. The activity of this compound is lowered by approximately sevenfold in the presence of 50% human serum. A-790742 maintained potent antiviral activity against lopinavir-resistant variants generated in vitro as well as against a panel of molecular clones containing proteases derived from HIV-1 patient isolates with multiple protease mutations. During in vitro selection, A-790742 selected two primary mutations (V82L and I84V) along with L23I, L33F, K45I, A71V/A, and V77I in the pNL4-3 background and two other mutations (A71V and V82G) accompanied by M46I and L63P in the HIV-1 RF background. HIV-1 pNL4-3 clones with a single V82L or I84V mutation were phenotypically resistant to A-790742 and ritonavir. Taking these results together, A-790742 displays a favorable anti-HIV-1 profile against both the wild type and a large number of mutants resistant to other protease inhibitors. The selection of the uncommon V82L and V82G mutations in protease by A-790742 suggests the potential for an advantageous resistance profile with this protease inhibitor.

Deficient APC-cofactor activity of protein S Heerlen in degradation of factor Va Leiden: a possible mechanism of synergism between thrombophilic risk factors

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 523-531 ◽  
Author(s):  
Tusar Kanti Giri ◽  
Tomio Yamazaki ◽  
Núria Sala ◽  
Björn Dahlbäck ◽  
Pablo Garcı́a de Frutos
Keyword(s):  
Recombinant Protein ◽  
Factor V Leiden ◽  
Protein S ◽  
Factor V ◽  
Wild Type ◽  
Genetic Traits ◽  
Factor Va ◽  
Type Protein ◽  
Wild Type Protein

In protein S Heerlen, an S-to-P (single-letter amino acid codes) mutation at position 460 results in the loss of glycosylation of N458. This polymorphism has been found to be slightly more prevalent in thrombophilic populations than in normal controls, particularly in cohorts of patients having free protein S deficiency. This suggests that carriers of the Heerlen allele may have an increased risk of thrombosis. We have now characterized the expression in cell cultures of recombinant protein S Heerlen and investigated the anticoagulant functions of the purified recombinant protein in vitro. Protein S Heerlen was synthesized and secreted equally well as wild-type protein S by transiently transfected COS-1 cells. The recombinant protein S Heerlen interacted with conformation-dependent monoclonal antibodies and bound C4b-binding protein to the same extent as wild-type protein S. Protein S Heerlen displayed reduced anticoagulant activity as cofactor to activated protein C (APC) in plasma-based assays, as well as in a factor VIIIa–degradation system. In contrast, protein S Heerlen functioned equally well as an APC cofactor in the degradation of factor Va as wild-type protein S did. However, when recombinant activated factor V Leiden (FVa:Q506) was used as APC substrate, protein S Heerlen was found to be a poor APC cofactor as compared with wild-type protein S. These in vitro results suggest a possible mechanism of synergy between protein S Heerlen and factor V Leiden that might be involved in the pathogenesis of thrombosis in individuals carrying both genetic traits.

Deficient APC-cofactor activity of protein S Heerlen in degradation of factor Va Leiden: a possible mechanism of synergism between thrombophilic risk factors

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 523-531 ◽  
Author(s):  
Tusar Kanti Giri ◽  
Tomio Yamazaki ◽  
Núria Sala ◽  
Björn Dahlbäck ◽  
Pablo Garcı́a de Frutos
Keyword(s):  
Recombinant Protein ◽  
Factor V Leiden ◽  
Protein S ◽  
Factor V ◽  
Wild Type ◽  
Genetic Traits ◽  
Factor Va ◽  
Type Protein ◽  
Wild Type Protein

Abstract In protein S Heerlen, an S-to-P (single-letter amino acid codes) mutation at position 460 results in the loss of glycosylation of N458. This polymorphism has been found to be slightly more prevalent in thrombophilic populations than in normal controls, particularly in cohorts of patients having free protein S deficiency. This suggests that carriers of the Heerlen allele may have an increased risk of thrombosis. We have now characterized the expression in cell cultures of recombinant protein S Heerlen and investigated the anticoagulant functions of the purified recombinant protein in vitro. Protein S Heerlen was synthesized and secreted equally well as wild-type protein S by transiently transfected COS-1 cells. The recombinant protein S Heerlen interacted with conformation-dependent monoclonal antibodies and bound C4b-binding protein to the same extent as wild-type protein S. Protein S Heerlen displayed reduced anticoagulant activity as cofactor to activated protein C (APC) in plasma-based assays, as well as in a factor VIIIa–degradation system. In contrast, protein S Heerlen functioned equally well as an APC cofactor in the degradation of factor Va as wild-type protein S did. However, when recombinant activated factor V Leiden (FVa:Q506) was used as APC substrate, protein S Heerlen was found to be a poor APC cofactor as compared with wild-type protein S. These in vitro results suggest a possible mechanism of synergy between protein S Heerlen and factor V Leiden that might be involved in the pathogenesis of thrombosis in individuals carrying both genetic traits.

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