Rational Design of Fluorophores for In Vivo Applications

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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Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

Communications Chemistry ◽

10.1038/s42004-020-00437-3 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Yusaku Hontani ◽

Mikhail Baloban ◽

Francisco Velazquez Escobar ◽

Swetta A. Jansen ◽

Daria M. Shcherbakova ◽

...

Keyword(s):

Real Time ◽

Rational Design ◽

Near Infrared ◽

Fluorescent Proteins ◽

Covalent Bond ◽

Binding Pocket ◽

Tissue Imaging ◽

Covalent Attachment ◽

Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

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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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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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TRPM7 and CaV3.2 channels mediate Ca2+ influx required for egg activation at fertilization

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1810422115 ◽

2018 ◽

Vol 115 (44) ◽

pp. E10370-E10378 ◽

Cited By ~ 21

Author(s):

Miranda L. Bernhardt ◽

Paula Stein ◽

Ingrid Carvacho ◽

Christopher Krapp ◽

Goli Ardestani ◽

...

Keyword(s):

Rational Design ◽

Culture Media ◽

Mammalian Development ◽

Developmental Potential ◽

Store Depletion ◽

Offspring Growth ◽

Mechanistic Basis ◽

Genetic Mouse Models

The success of mammalian development following fertilization depends on a series of transient increases in egg cytoplasmic Ca2+, referred to as Ca2+ oscillations. Maintenance of these oscillations requires Ca2+ influx across the plasma membrane, which is mediated in part by T-type, CaV3.2 channels. Here we show using genetic mouse models that TRPM7 channels are required to support this Ca2+ influx. Eggs lacking both TRPM7 and CaV3.2 stop oscillating prematurely, indicating that together they are responsible for the majority of Ca2+ influx immediately following fertilization. Fertilized eggs lacking both channels also frequently display delayed resumption of Ca2+ oscillations, which appears to require sperm–egg fusion. TRPM7 and CaV3.2 channels almost completely account for Ca2+ influx observed following store depletion, a process previously attributed to canonical store-operated Ca2+ entry mediated by STIM/ORAI interactions. TRPM7 serves as a membrane sensor of extracellular Mg2+ and Ca2+ concentrations and mediates the effects of these ions on Ca2+ oscillation frequency. When bred to wild-type males, female mice carrying eggs lacking TRPM7 and CaV3.2 are subfertile, and their offspring have increased variance in postnatal weight. These in vivo findings confirm previous observations linking in vitro experimental alterations in Ca2+ oscillatory patterns with developmental potential and offspring growth. The identification of TRPM7 and CaV3.2 as key mediators of Ca2+ influx following fertilization provides a mechanistic basis for the rational design of culture media that optimize developmental potential in research animals, domestic animals, and humans.

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Lipophilicity and Click Reactivity Determine the Performance of Bioorthogonal Tetrazine Tools in Pretargeted in Vivo Chemistry

10.26434/chemrxiv.13482594 ◽

2020 ◽

Author(s):

Johanna Stéen ◽

Jesper Tranekjær Jørgensen ◽

Denk Christoph ◽

Umberto Maria Battisti ◽

Kamilla Nørregaard ◽

...

Keyword(s):

Targeted Delivery ◽

Rational Design ◽

Structural Parameters ◽

Early Time ◽

High Rate ◽

Drug Conjugates ◽

Compound Libraries ◽

Drug Concentrations ◽

The Impact

The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of in vivo chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody-drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile in vivo chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the in vivo ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the in vivo fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted trans-cyclooctene (TCO)-tagged antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their in vivo performance. In particular, high rate constants (>50,000 M-1s-1) for the reaction with TCO and low calculated logD7.4 values (below -3) of the tetrazine were identified as strong indicators for successful pretargeted in vivo click chemistry. Click-radiolabeling gave access to a set of selected 18F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for in vivo application and will thereby assist the clinical translation of bioorthogonal pretargeting.

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Rational design of protein-specific folding modifiers

10.1101/2020.04.28.064113 ◽

2020 ◽

Author(s):

Anirban Das ◽

Anju Yadav ◽

Mona Gupta ◽

R Purushotham ◽

Vishram L. Terse ◽

...

Keyword(s):

Single Molecule ◽

Rational Design ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Force Measurements ◽

Folding Nucleus ◽

Dynamics Simulations ◽

General Method

AbstractProtein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

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Rational design of peptides for identification of linear epitopes and generation of neutralizing monoclonal antibodies against DKK2 for cancer therapy

Antibody Therapeutics ◽

10.1093/abt/tbaa004 ◽

2020 ◽

Vol 3 (2) ◽

pp. 63-70 ◽

Cited By ~ 1

Author(s):

Rongqing Zhao ◽

Qian Xiao ◽

Maohua Li ◽

Wenlin Ren ◽

Chenxi Xia ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Tumor Growth ◽

Wnt Signaling ◽

Rational Design ◽

Polyclonal Antibodies ◽

Wnt Signaling Pathway ◽

Keyhole Limpet Hemocyanin ◽

Linear Epitopes

Abstract Dickkopf-related protein 2 (DKK2)is a member of the Dickkopf family in Wnt signaling pathway. Recently, we found that antibodies against DKK2 could activate natural killer (NK) and CD8+ T cells in tumors and inhibit tumor growth. In this paper, we report the rational design of peptides for identification of linear epitopes and generation of neutralizing monoclonal anti-DKK2 antibodies. To break the immune tolerance, we designed and chemically synthesized six peptides corresponding to different regions of DKK2 as immunogens and found five of them could generate mouse polyclonal antibodies that can bind to the active recombinant human DKK2 protein. Neutralizing mouse monoclonal antibodies (5F8 and 1A10) against human DKK2 were successfully developed by immunizing the mice with two different peptides (34KLNSIKSSL42 and 240KVWKDATYS248) conjugated to Keyhole limpet hemocyanin (KLH). The monoclonal antibodies not only abolish DKK2’s suppression of Wnt signaling in vitro but also inhibits tumor growth in vivo. Currently, those two mAbs are undergoing humanization as immunotherapy candidates and may offer a new drug for treatment of human cancers.

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