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 FtsZ-Dependent Elongation of a Coccoid Bacterium

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Ana R. Pereira ◽  
Jen Hsin ◽  
Ewa Król ◽  
Andreia C. Tavares ◽  
Pierre Flores ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Shape ◽  
Single Point ◽  
Spherical Shape ◽  
Single Point Mutation ◽  
Wild Type ◽  
Dead End ◽  
Dynamics Simulations

ABSTRACT A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ G193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ G193D filaments are more twisted and shorter than wild-type filaments. In vivo , M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.

scholarly journals Sulfated non-anticoagulant heparin derivative modifies intracellular hemoglobin, inhibits cell sickling in vitro, and prolongs survival of sickle cell mice under hypoxia

Haematologica ◽  
2021 ◽  
Author(s):  
Osheiza Abdulmalik ◽  
Noureldien H. E. Darwish ◽  
Vandhana Muralidharan-Chari ◽  
Maii Abu Taleb ◽  
Shaker A. Mousa
Keyword(s):  
Sickle Cell ◽  
Inflammatory Responses ◽  
Anticoagulant Activity ◽  
Single Point ◽  
Bleeding Complications ◽  
Single Point Mutation ◽  
Repeated Dosing ◽  
Heparin Derivative

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by a single point mutation, resulting in abnormal sickle hemoglobin (HbS). During hypoxia or dehydration, HbS polymerizes to form insoluble aggregates and induces sickling of red blood cells (RBCs). RBC sickling increases adhesiveness of RBCs to alter the rheological properties of the blood and triggers inflammatory responses, leading to hemolysis and vaso-occlusive crisis sequelae. Unfractionated heparin (UFH) and low-molecular weight heparins (LMWH) have been suggested as treatments to relieve coagulation complications in SCD. However, they are associated with bleeding complications after repeated dosing. An alternative sulfated nonanticoagulant heparin derivative (S-NACH) was previously reported to have none to low systemic anticoagulant activity and no bleeding side effects, and it interfered with P-selectindependent binding of sickle cells to endothelial cells, with concomitant decrease in the levels of adhesion biomarkers in SCD mice. S-NACH has been further engineered and structurally enhanced to bind with and modify HbS to directly inhibit sickling, thus employing a multimodal approach. Here, we show that S-NACH can (i) directly engage in Schiff-base reactions with HbS to decrease RBC sickling under both normoxia and hypoxia in vitro, ii) prolong the survival of SCD mice under hypoxia, and (iii) regulate the altered steady state levels of pro- and antiinflammatory cytokines. Thus, our proof of concept in vitro and in vivo preclinical studies demonstrate that the multimodal S-NACH is a highly promising candidate for development into an improved and optimized alternative to LMWHs for the treatment of patients with SCD.

scholarly journals The Carboxy-Terminal Fragment of Nucleolin Interacts with the Nucleocapsid Domain of Retroviral Gag Proteins and Inhibits Virion Assembly

2000 ◽  
Vol 74 (23) ◽  
pp. 11027-11039 ◽  
Author(s):  
Eran Bacharach ◽  
Jason Gonsky ◽  
Kimona Alin ◽  
Marianna Orlova ◽  
Stephen P. Goff
Keyword(s):  
Leukemia Virus ◽  
Single Point ◽  
Host Protein ◽  
Single Point Mutation ◽  
Gag Protein ◽  
Virion Assembly ◽  
Gag Proteins ◽  
Carboxy Terminal

ABSTRACT A yeast two-hybrid screen for cellular proteins that interact with the murine leukemia virus (MuLV) Gag protein resulted in the identification of nucleolin, a host protein known to function in ribosome assembly. The interacting fusions contained the carboxy-terminal 212 amino acids of nucleolin [Nuc(212)]. The nucleocapsid (NC) portion of Gag was necessary and sufficient to mediate the binding to Nuc(212). The interaction of Gag with Nuc(212) could be demonstrated in vitro and was manifested in vivo by the NC-dependent incorporation of Nuc(212) inside MuLV virions. Overexpression of Nuc(212), but not full-length nucleolin, potently and specifically blocked MuLV virion assembly and/or release. A mutant of MuLV, selected to specifically disrupt the binding to Nuc(212), was found to be severely defective for virion assembly. This mutant harbors a single point mutation in capsid (CA) adjacent to the CA-NC junction, suggesting a role for this region in Moloney MuLV assembly. These experiments demonstrate that selection for proteins that bind assembly domain(s) can yield potent inhibitors of virion assembly. These experiments also raise the possibility that a nucleolin-Gag interaction may be involved in virion assembly.

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.

In vitro Selection of a Peptide with High Selectivity for Cardiomyocytes In vivo

2004 ◽  
Vol 342 (1) ◽  
pp. 171-182 ◽  
Author(s):  
Michael J. McGuire ◽  
Kausar N. Samli ◽  
Stephen Albert Johnston ◽  
Kathlynn C. Brown
Keyword(s):  
In Vitro Selection ◽  
High Selectivity ◽  
Selection Of

scholarly journals In-Vitro Selection of Ceftazidime/Avibactam Resistance in OXA-48-Like-Expressing Klebsiella pneumoniae: In-Vitro and In-Vivo Fitness, Genetic Basis and Activities of β-Lactam Plus Novel β-Lactamase Inhibitor or β-Lactam Enhancer Combinations

Antibiotics ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1318
Author(s):  
Snehal Palwe ◽  
Yamuna Devi Bakthavatchalam ◽  
Kshama Khobragadea ◽  
Arun S. Kharat ◽  
Kamini Walia ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Whole Genome Sequence ◽  
Serial Transfer ◽  
Resistance Selection ◽  
Clinical Resistance ◽  
Growth Properties ◽  
Outer Membrane Permeability ◽  
Selection Of

Ceftazidime/avibactam uniquely demonstrates activity against both KPC and OXA-48-like carbapenemase-expressing Enterobacterales. Clinical resistance to ceftazidime/avibactam in KPC-producers was foreseen in in-vitro resistance studies. Herein, we assessed the resistance selection propensity of ceftazidime/avibactam in K. pneumoniae expressing OXA-48-like β-lactamases (n = 10), employing serial transfer approach. Ceftazidime/avibactam MICs (0.25–4 mg/L) increased to 16–256 mg/L after 15 daily-sequential transfers. The whole genome sequence analysis of terminal mutants showed modifications in proteins linked to efflux (AcrB/AcrD/EmrA/Mdt), outer membrane permeability (OmpK36) and/or stress response pathways (CpxA/EnvZ/RpoE). In-vitro growth properties of all the ceftazidime/avibactam-selected mutants were comparable to their respective parents and they retained the ability to cause pulmonary infection in neutropenic mice. Against these mutants, we explored the activities of various combinations of β-lactams (ceftazidime or cefepime) with structurally diverse β-lactamase inhibitors or a β-lactam enhancer, zidebactam. Zidebactam, in combination with either cefepime or ceftazidime, overcame ceftazidime/avibactam resistance (MIC range 0.5–8 mg/L), while cefepime/avibactam was the second best (MIC: 0.5–16 mg/L) in yielding lower MICs. The present work revealed the possibility of ceftazidime/avibactam resistance in OXA-48-like K. pneumoniae through mutations in proteins involved in efflux and/or porins without concomitant fitness cost mandating astute monitoring of ceftazidime/avibactam resistance among OXA-48 genotypes.

scholarly journals A Simple Model for Assessment of Anti-Toxin Antibodies

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Alex Skvortsov ◽  
Peter Gray
Keyword(s):  
In Vitro Selection ◽  
Receptor Complex ◽  
Simple Mathematical Model ◽  
Antibody Affinity ◽  
In Vivo Evaluation ◽  
Degree Of Protection ◽  
Protective Antibodies ◽  
Selection Of

The toxins associated with infectious diseases are potential targets for inhibitors which have the potential for prophylactic or therapeutic use. Many antibodies have been generated for this purpose, and the objective of this study was to develop a simple mathematical model that may be used to evaluate the potential protective effect of antibodies. This model was used to evaluate the contributions of antibody affinity and concentration to reducing antibody-receptor complex formation and internalization. The model also enables prediction of the antibody kinetic constants and concentration required to provide a specified degree of protection. We hope that this model, once validated experimentally, will be a useful tool for in vitro selection of potentially protective antibodies for progression to in vivo evaluation.

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