Rational design of a new mutant of tobacco etch virus protease in order to increase the in vitro solubility

Technetium-99m has a rich coordination chemistry that offers many possibilities in terms of oxidation states and donor atom sets. Modifications in the structure of the technetium complexes could be very useful for fine tuning the physicochemical and biological properties of potential 99mTc radiopharmaceuticals. However, systematic study of the influence of the labelling strategy on the “in vitro” and “in vivo” behaviour is necessary for a rational design of radiopharmaceuticals. Herein we present a review of the influence of the Tc complexes’ molecular structure on the biodistribution and the interaction with the biological target of potential nitroimidazolic hypoxia imaging radiopharmaceuticals presented in the literature from 2010 to the present. Comparison with the gold standard [18F]Fluoromisonidazole (FMISO) is also presented.

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High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

Nature Communications ◽

10.1038/s41467-021-23954-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhou Fang ◽

Junjian Chen ◽

Ye Zhu ◽

Guansong Hu ◽

Haoqian Xin ◽

...

Keyword(s):

Antimicrobial Activity ◽

High Throughput ◽

High Throughput Screening ◽

Rational Design ◽

Click Reaction ◽

Reaction Times ◽

Great Promise ◽

Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

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Experimental and theoretical explorations of nanocarriers’ multistep delivery performance for rational design and anticancer prediction

Science Advances ◽

10.1126/sciadv.aba2458 ◽

2021 ◽

Vol 7 (6) ◽

pp. eaba2458

Author(s):

Weier Bao ◽

Falin Tian ◽

Chengliang Lyu ◽

Bin Liu ◽

Bin Li ◽

...

Keyword(s):

Physical Properties ◽

Rational Design ◽

Theoretical Models ◽

Cell Uptake ◽

Anticancer Efficacy ◽

Delivery Performance ◽

Simulation Based ◽

Multistep Process

The poor understanding of the complex multistep process taken by nanocarriers during the delivery process limits the delivery efficiencies and further hinders the translation of these systems into medicine. Here, we describe a series of six self-assembled nanocarrier types with systematically altered physical properties including size, shape, and rigidity, as well as both in vitro and in vivo analyses of their performance in blood circulation, tumor penetration, cancer cell uptake, and anticancer efficacy. We also developed both data and simulation-based models for understanding the influence of physical properties, both individually and considered together, on each delivery step and overall delivery process. Thus, beyond finding that nanocarriers that are simultaneously endowed with tubular shape, short length, and low rigidity outperformed the other types, we now have a suit of theoretical models that can predict how nanocarrier properties will individually and collectively perform in the multistep delivery of anticancer therapies.

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Tick Importin-α Is Implicated in the Interactome and Regulome of the Cofactor Subolesin

Pathogens ◽

10.3390/pathogens10040457 ◽

2021 ◽

Vol 10 (4) ◽

pp. 457

Author(s):

Sara Artigas-Jerónimo ◽

Margarita Villar ◽

Alejandro Cabezas-Cruz ◽

Grégory Caignard ◽

Damien Vitour ◽

...

Keyword(s):

Rational Design ◽

Fat Body ◽

Animal Health ◽

Direct Interaction ◽

Vaccine Antigen ◽

Tick Feeding ◽

Interacting Protein ◽

Expression Levels ◽

Importin Α

Ticks and tick-borne diseases (TBDs) represent a burden for human and animal health worldwide. Currently, vaccines constitute the safest and most effective approach to control ticks and TBDs. Subolesin (SUB) has been identified as a vaccine antigen for the control of tick infestations and pathogen infection and transmission. The characterization of the molecular function of SUB and the identification of tick proteins interacting with SUB may provide the basis for the discovery of novel antigens and for the rational design of novel anti-tick vaccines. In the present study, we used the yeast two-hybrid system (Y2H) as an unbiased approach to identify tick SUB-interacting proteins in an Ixodes ricinus cDNA library, and studied the possible role of SUB as a chromatin remodeler through direct interaction with histones. The Y2H screening identified Importin-α as a potential SUB-interacting protein, which was confirmed in vitro in a protein pull-down assay. The sub gene expression levels in tick midgut and fat body were significantly higher in unfed than fed female ticks, however, the importin-α expression levels did not vary between unfed and fed ticks but tended to be higher in the ovary when compared to those in other organs. The effect of importin-α RNAi was characterized in I. ricinus under artificial feeding conditions. Both sub and importin-α gene knockdown was observed in all tick tissues and, while tick weight was significantly lower in sub RNAi-treated ticks than in controls, importin-α RNAi did not affect tick feeding or oviposition, suggesting that SUB is able to exert its function in the absence of Importin-α. Furthermore, SUB was shown to physically interact with histone 4, which was corroborated by protein pull-down and western blot analysis. These results confirm that by interacting with numerous tick proteins, SUB is a key cofactor of the tick interactome and regulome. Further studies are needed to elucidate the nature of the SUB-Importin-α interaction and the biological processes and functional implications that this interaction may have.

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pH and Reduction Dual-Responsive Bi-Drugs Conjugated Dextran Assemblies for Combination Chemotherapy and In Vitro Evaluation

Polymers ◽

10.3390/polym13091515 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1515

Author(s):

Xiukun Xue ◽

Yanjuan Wu ◽

Xiao Xu ◽

Ben Xu ◽

Zhaowei Chen ◽

...

Keyword(s):

Combination Chemotherapy ◽

Laser Scanning ◽

Rational Design ◽

A549 Cells ◽

Chemotherapeutic Agents ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Tumor Resistance ◽

Oxidized Dextran

Polymeric prodrugs, synthesized by conjugating chemotherapeutic agents to functional polymers, have been extensively investigated and employed for safer and more efficacious cancer therapy. By rational design, a pH and reduction dual-sensitive dextran-di-drugs conjugate (oDex-g-Pt+DOX) was synthesized by the covalent conjugation of Pt (IV) prodrug and doxorubicin (DOX) to an oxidized dextran (oDex). Pt (IV) prodrug and DOX were linked by the versatile efficient esterification reactions and Schiff base reaction, respectively. oDex-g-Pt+DOX could self-assemble into nanoparticles with an average diameter at around 180 nm. The acidic and reductive (GSH) environment induced degradation and drug release behavior of the resulting nanoparticles (oDex-g-Pt+DOX NPs) were systematically investigated by optical experiment, DLS analysis, TEM measurement, and in vitro drugs release experiment. Effective cellular uptake of the oDex-g-Pt+DOX NPs was identified by the human cervical carcinoma HeLa cells via confocal laser scanning microscopy. Furthermore, oDex-g-Pt+DOX NPs displayed a comparable antiproliferative activity than the simple combination of free cisplatin and DOX (Cis+DOX) as the extension of time. More importantly, oDex-g-Pt+DOX NPs exhibited remarkable reversal ability of tumor resistance compared to the cisplatin in cisplatin-resistant lung carcinoma A549 cells. Take advantage of the acidic and reductive microenvironment of tumors, this smart polymer-dual-drugs conjugate could serve as a promising and effective nanomedicine for combination chemotherapy.

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A high-throughput screen for TMPRSS2 expression identifies FDA-approved compounds that can limit SARS-CoV-2 entry

Nature Communications ◽

10.1038/s41467-021-24156-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yanwen Chen ◽

Travis B. Lear ◽

John W. Evankovich ◽

Mads B. Larsen ◽

Bo Lin ◽

...

Keyword(s):

Rational Design ◽

Surface Expression ◽

In Vitro Models ◽

Lung Epithelial Cells ◽

Cell Surface Receptor ◽

Pharmacokinetic Data ◽

Drug Induced ◽

Global Pandemic ◽

Surface Receptor

AbstractSARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identify small molecules that reduce surface expression of TMPRSS2 using a library of 2,560 FDA-approved or current clinical trial compounds. We identify homoharringtonine and halofuginone as the most attractive agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrate marked resistance to SARS-CoV-2 infection in both live and pseudoviral in vitro models. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat active COVID-19 infection.

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Streptococcal Adhesion and Colonization

Critical Reviews in Oral Biology & Medicine ◽

10.1177/10454411970080020601 ◽

1997 ◽

Vol 8 (2) ◽

pp. 175-200 ◽

Cited By ~ 201

Author(s):

H.F. Jenkinson ◽

RJ Lamont

Keyword(s):

Immune Modulation ◽

Rational Design ◽

Repeated Sequence ◽

Host Cells ◽

Mammalian Host ◽

Related Factors ◽

Bacterial Populations ◽

Acellular Vaccines

Streptococci express arrays of adhesins on their cell surfaces that facilitate adherence to substrates present in their natural environment within the mammalian host. A consequence of such promiscuous binding ability is that streptococcal cells may adhere simultaneously to a spectrum of substrates, including salivary glycoproteins, extracellular matrix and serum components, host cells, and other microbial cells. The multiplicity of streptococcal adherence interactions accounts, at least in part, for their success in colonizing the oral and epithelial surfaces of humans. Adhesion facilitates colonization and may be a precursor to tissue invasion and immune modulation, events that presage the development of disease. Many of the streptococcal adhesins and virulence-related factors are cell-wall-associated proteins containing repeated sequence blocks of amino acids. Linear sequences, both within the blocks and within non-repetitive regions of the proteins, have been implicated in substrate binding. Sequences and functions of these proteins among the streptococci have become assorted through gene duplication and horizontal transfer between bacterial populations. Several adhesins identified and characterized through in vitro binding assays have been analyzed for in vivo expression and function by means of animal models used for colonization and virulence. Information on the molecular structure of adhesins as related to their in vivo function will allow for the rational design of novel acellular vaccines, recombinant antibodies, and adhesion agonists for the future control or prevention of streptococcal colonization and streptococcal diseases.

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Exploring the Mutations on Improving Oxidative Stability of Tobacco Etch Virus Protease for Tag-removal in Refolding of Two Disulfide-rich Proteins

10.21203/rs.3.rs-473834/v1 ◽

2021 ◽

Author(s):

Jun Fan ◽

Enkhtuya Bayar ◽

Yuanyuan Ren ◽

Yafang Hu ◽

Yinghua Chen ◽

...

Keyword(s):

Oxidative Stability ◽

Disulfide Bonds ◽

Redox State ◽

Protein Solubility ◽

Tobacco Etch Virus ◽

Tobacco Etch Virus Protease ◽

Amorphous Cellulose ◽

E Coli ◽

Cleavage Activity ◽

Cellulose Binding

Abstract Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tag, but wild type TEVp shows less oxidative stability, which limits its application under the oxidized redox state to facilitate disulfide bonds formation for refolding disulfide-bonded proteins. Previously, we combined six mutations into the TEVp to generate the TEVp5M for obviously increasing the protein solubility and decreasing the auto-cleavage. In this work, we introduced and combined C19S, C110S and C130S mutations into the TEVp5M to generate seven variants, analyzed protein solubility and the cleavage activity of the constructs in each of three E. coli strains including BL21(DE3), BL21(DE3)pLys, and Rossetta(DE3), and those of the optimized soluble variants in the oxidative cytoplasm of Origami(DE3) under the same induction conditions. The results suggested that desirable protein solubility, cleavage activity and oxidative stability are not combined. Unlike that of the C19S, introduction of the C110S and/or C130S less affected protein solubility but increased tolerance to the oxidative redox state. Use of the TEVp5MC110S/C130S variant, the refolded disulfide-rich bovine enteropeptidase or maize peroxidase was released via cleaving the sequence between the target protein and the cellulose-binding module bound to regenerated amorphous cellulose.

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Screening of mucoadhesive vaginal gel formulations

Brazilian Journal of Pharmaceutical Sciences ◽

10.1590/s1984-82502014000400029 ◽

2014 ◽

Vol 50 (4) ◽

pp. 931-941 ◽

Cited By ~ 11

Author(s):

Ana Ochoa Andrade ◽

María Emma Parente ◽

Gastón Ares

Keyword(s):

Xanthan Gum ◽

Rational Design ◽

Point Of View ◽

Vaginal Fluid ◽

Multiple Factor Analysis ◽

Vaginal Drug Delivery ◽

Rheological Measurements ◽

Gel Formulations ◽

Positive Results

Rational design of vaginal drug delivery formulations requires special attention to vehicle properties that optimize vaginal coating and retention. The aim of the present work was to perform a screening of mucoadhesive vaginal gels formulated with carbomer or carrageenan in binary combination with a second polymer (carbomer, guar or xanthan gum). The gels were characterised using in vitroadhesion, spreadability and leakage potential studies, as well as rheological measurements (stress and frequency sweep tests) and the effect of dilution with simulated vaginal fluid (SVF) on spreadability. Results were analysed using analysis of variance and multiple factor analysis. The combination of polymers enhanced adhesion of both primary gelling agents, carbomer and carrageenan. From the rheological point of view all formulations presented a similar behaviour, prevalently elastic and characterised by loss tangent values well below 1. No correlation between rheological and adhesion behaviour was found. Carbomer and carrageenan gels containing the highest percentage of xanthan gum displayed good in vitro mucoadhesion and spreadability, minimal leakage potential and high resistance to dilution. The positive results obtained with carrageenan-xanthan gum-based gels can encourage the use of natural biocompatible adjuvants in the composition of vaginal products, a formulation field that is currently under the synthetic domain.

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