scholarly journals Quantitating drug-target engagement in single cells in vitro and in vivo

2016 ◽  
Vol 13 (2) ◽  
pp. 168-173 ◽  
Author(s):  
J Matthew Dubach ◽  
Eunha Kim ◽  
Katherine Yang ◽  
Michael Cuccarese ◽  
Randy J Giedt ◽  
...  
Keyword(s):  
Drug Target ◽  
Single Cells ◽  
Target Engagement

Monitoring Drug-Target Interactions through Target Engagement-Mediated Amplification on Arrays and in situ

2021 ◽  
Author(s):  
Rasel Al-Amin ◽  
Lars Johansson ◽  
Eldar Abdurakhmanov ◽  
Nils Landegren ◽  
Liza Löf ◽  
...  
Keyword(s):  
Drug Target ◽  
Kinase Inhibitors ◽  
Expression Profiles ◽  
Clinical Samples ◽  
Target Engagement ◽  
Rolling Circle Replication ◽  
Target Proteins ◽  
Dna Conjugates

Abstract Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to a panel of fixed adherent cells agreed with expectations from expression profiles of the cells. These findings were corroborated by competition experiments using kinase inhibitors with overlapping and non-overlapping target specificities, and translated to pathology tissue sections. We also introduce a proximity ligation variant of TEMA in which these drug-DNA conjugates are combined with antibody-DNA conjugates to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug-target interactions at spatial resolution in protein arrays, cells and tissues.

scholarly journals Clonal analysis of basophil differentiation in bone marrow cultures from a Down's syndrome patient with megakaryoblastic leukemia

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1278-1283
Author(s):  
T Suda ◽  
J Suda ◽  
Y Miura ◽  
Y Hayashi ◽  
M Eguchi ◽  
...  
Keyword(s):  
Single Cells ◽  
Down's Syndrome ◽  
Calcium Ionophore ◽  
Down’S Syndrome ◽  
Colony Growth ◽  
Leukemic Cells ◽  
Cytologic Examination ◽  
Megakaryoblastic Leukemia

We present the in vitro differentiation of marrow cells from a patient with Down's syndrome accompanied by megakaryoblastic leukemia into basophils in the presence of phytohemagglutinin-stimulated leukocyte conditioned medium, using a liquid culture and methylcellulose culture system. Identification of basophils was established by metachromatic staining with toluidine blue, transmission electron microscopy, and the presence of histamine. However, these basophils did not release histamine in response to calcium ionophore or chemotactic peptide. Samples from suspension cultures that contained 90% basophils showed chromosomal markers characteristic of leukemic cells (48, XY, +11, +21, t(1;15)) in all examined mitoses. The cellular composition of leukemic colonies grown in methylcellulose culture from single cells was studied using the micromanipulation technique. High plating efficiency and extreme predominance of basophil colonies were observed. In a total 137 cultures, 79 revealed colony growth. Of 59 colonies that were analyzed by cytologic examination, 46 were pure basophil colonies. These basophil colonies showed disperse morphology, similar to that of a normal basophil colony. The clonality of the basophil colonies and skewing of lineage expression were documented from leukemic single-cell cultures. These data showed that leukemic cells have the capacity for differentiation into some lineages that are not expressed in vivo.

scholarly journals Self-organized cell migration across scales – from single cell movement to tissue formation

Development ◽  
2021 ◽  
Vol 148 (7) ◽  
pp. dev191767
Author(s):  
Jessica Stock ◽  
Andrea Pauli
Keyword(s):  
Cell Migration ◽  
Multiple Scales ◽  
Single Cells ◽  
Cell Movement ◽  
Biological Response ◽  
Collective Cell Migration ◽  
Theoretical Concepts ◽  
Self Organizing

ABSTRACTSelf-organization is a key feature of many biological and developmental processes, including cell migration. Although cell migration has traditionally been viewed as a biological response to extrinsic signals, advances within the past two decades have highlighted the importance of intrinsic self-organizing properties to direct cell migration on multiple scales. In this Review, we will explore self-organizing mechanisms that lay the foundation for both single and collective cell migration. Based on in vitro and in vivo examples, we will discuss theoretical concepts that underlie the persistent migration of single cells in the absence of directional guidance cues, and the formation of an autonomous cell collective that drives coordinated migration. Finally, we highlight the general implications of self-organizing principles guiding cell migration for biological and medical research.

scholarly journals A causal role for TRESK loss of function in migraine mechanisms

Brain ◽  
2019 ◽  
Vol 142 (12) ◽  
pp. 3852-3867 ◽  
Author(s):  
Philippa Pettingill ◽  
Greg A Weir ◽  
Tina Wei ◽  
Yukyee Wu ◽  
Grace Flower ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Drug Target ◽  
Causal Role ◽  
Potential Drug Target ◽  
Potential Drug ◽  
Loss Of Function

The two-pore potassium channel TRESK is a potential drug target in pain and migraine. Pettingill et al. show that the F139WfsX2 mutation causes TRESK loss of function and hyperexcitability in nociceptors derived from iPSCs of patients with migraine. Cloxyquin, a TRESK activator, reverses migraine-relevant phenotypes in vitro and in vivo.

Cell Mechanics Drives Migration Modes

2020 ◽  
Vol 15 (01) ◽  
pp. 1-34 ◽  
Author(s):  
Claudia Tanja Mierke
Keyword(s):  
Extracellular Matrix ◽  
Cell Mechanics ◽  
Single Cells ◽  
Biochemical Properties ◽  
Migration And Invasion ◽  
Environmental Cues ◽  
Connective Tissues ◽  
Migration Of Cells

The classical migration modes, such as mesenchymal or amoeboid migration modes, are essentially determined by molecular, morphological or biochemical properties of the cells. These specific properties facilitate the cell migration and invasion through artificial extracellular matrices mimicking the environmental conditions of connective tissues. However, during the migration of cells through narrow extracellular matrix constrictions, the specific extracellular matrix environments can either support or impair the invasion of cells. Beyond the classical molecular or biochemical properties, the migration and invasion of cells depends on intracellular cell mechanical characteristics and extracellular matrix mechanical features. The switch between cell states, such as epithelial, mesenchymal or amoeboid states, seems to be mainly based on epigenetic changes and environmental cues that induce the reversible transition of cells toward another state and thereby promote a specific migration mode. However, the exact number of migration modes is not yet clear. Moreover, it is also unclear whether every individual cell, independent of the type, can undergo a transition between all different migration modes in general. A newer theory states that the transition from the jamming to unjamming phase of clustered cells enables cells to migrate as single cells through extracellular matrix confinements. This review will highlight the mechanical features of cells and their matrix environment that regulate and subsequently determine individual migration modes. It is discussed whether each migration mode in each cell type is detectable or whether some migration modes are limited to artificially engineered matrices in vitro and can therefore not or only rarely be detected in vivo. It is specifically pointed out how the intracellular architecture and its contribution to cellular stiffness or contractility favors the employment of a distinct migration mode. Finally, this review envisions a connection between mechanical properties of cells and matrices and the choice of a distinct migration mode in confined 3D microenvironments.

scholarly journals Trifluoromethionine, a Prodrug Designed against Methionine γ-Lyase-Containing Pathogens, Has Efficacy In Vitro and In Vivo against Trichomonas vaginalis

2001 ◽  
Vol 45 (6) ◽  
pp. 1743-1745 ◽  
Author(s):  
Graham H. Coombs ◽  
Jeremy C. Mottram
Keyword(s):  
Drug Target ◽  
Trichomonas Vaginalis ◽  
Anaerobic Bacteria ◽  
Protozoan Parasite ◽  
Chemotherapeutic Agents ◽  
Single Step ◽  
Lesion Formation ◽  
Highly Active

ABSTRACT Methionine γ-lyase, the enzyme which catalyzes the single-step conversion of methionine to α-ketobutyrate, ammonia, and methanethiol, is highly active in many anaerobic pathogenic microorganisms but has no counterpart in mammals. This study tested the hypothesis that this pathogen-specific enzyme can be exploited as a drug target by prodrugs that are exclusively activated by it. Trifluoromethionine was confirmed as such a prodrug and shown to be highly toxic in vitro to the anaerobic protozoan parasiteTrichomonas vaginalis, to anaerobic bacteria containing methionine γ-lyase, and to Escherichia coli expressing the trichomonad gene. The compound also has exceptional activity against the parasite growing in vivo, with a single dose preventing lesion formation in five of the six mice challenged. These findings suggest that trifluoromethionine represents a lead compound for a novel class of anti-infective drugs with potential as chemotherapeutic agents against a range of prokaryotic and eukaryotic anaerobic pathogens.

scholarly journals The Opportunities and Use of Imaging to Measure Target Engagement

2019 ◽  
Vol 25 (2) ◽  
pp. 127-136
Author(s):  
Juliana Maynard ◽  
Philippa Hart
Keyword(s):  
Ex Vivo ◽  
Clinical Success ◽  
Receptor Occupancy ◽  
Accurate Information ◽  
Target Engagement ◽  
Drug Molecule ◽  
Drug Discovery And Development ◽  
Ex Vivo Imaging

Lack of efficacy and poor safety outcomes are deemed to be the greatest causes of clinical failure of novel therapeutics. The use of biomarkers that give accurate information on target engagement, providing confidence that pharmacological activity in the target organ is being achieved, is key in optimizing clinical success. Without a measurement of target engagement, it can be very difficult to discern the basis for any lack of efficacy of a drug molecule within the pharmaceutical industry. Target engagement can be measured in both an in vitro and in vivo setting, and in recent years imaging measurements have been used frequently in drug discovery and development to assess target engagement and receptor occupancy in both human and animal models. From this perspective, we assess and look at the advancements in both in vivo and ex vivo imaging to demonstrate the enormous potential that imaging has as an application to provide a greater understanding of target engagement with a correlative therapeutic impact.

scholarly journals Increases in circulating megakaryocyte growth-promoting activity in the plasma of rats following whole body irradiation

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1060-1066 ◽  
Author(s):  
M Miura ◽  
CW Jackson ◽  
SA Lyles
Keyword(s):  
Bone Marrow ◽  
Plasma Concentration ◽  
Bone Marrow Cells ◽  
Single Cells ◽  
Rat Plasma ◽  
Whole Body ◽  
Growth Promoting ◽  
Marrow Cells

Abstract To gain insight into the regulation of megakaryocyte precursors in vivo, we assayed (in vitro) megakaryocyte growth-promoting activity (Meg-GPA) in plasma of rats in which both marrow hypoplasia and thrombocytopenia had been induced by irradiation. Rats received whole body irradiation of 834 rad from a 137Cs source. Plasma was collected at intervals of hours to days, up through day 21 postirradiation, and was tested, at a concentration of 30%, for Meg-GPA on bone marrow cells cultured in 1.1% methylcellulose with 5 X 10(-5) M 2-mercaptoethanol. With normal rat plasma, no megakaryocyte colonies (defined as greater than or equal to 4 megakaryocytes) were seen and only a few single megakaryocytes and clusters (defined as 2 or 3 megakaryocytes) were formed. Two peaks of plasma Meg-GPA were observed after irradiation. The first appeared at 12 hr, before any decrease in marrow megakaryocyte concentration or platelet count. The second occurred on days 10–14 after irradiation, after the nadir in megakaryocyte concentration and while platelet counts were at their lowest levels. A dose-response study of plasma concentration and megakaryocyte growth, using plasma collected 11 days postirradiation, demonstrated that patterns of megakaryocyte growth were related to plasma concentration; formation of single megakaryocytes was optimal over a range of 20%-30% plasma concentration, while cluster and colony formation were optimal at a plasma concentration of 30%. All forms of megakaryocyte growth were decreased with 40% plasma. There was a linear relationship between the number of bone marrow cells plated and growth of single cells, clusters, and colonies using a concentration of 30% plasma collected 11 days after irradiation. We conclude that irradiation causes time- related increases in circulating megakaryocyte growth-promoting activity. We suggest that the irradiated rat is a good model for studying the relationships between Meg-GPA and megakaryocyte and platelet concentration in vivo.

A Comparative Study of Preparation Techniques for Improving the Viability of Striatal Grafts Using Vital Stains, in Vitro Cultures, and in Vivo Grafts

1996 ◽  
Vol 5 (6) ◽  
pp. 599-611 ◽  
Author(s):  
Rosemary A. Fricker ◽  
Roger A. Barker ◽  
James W. Fawcett ◽  
Stephen B. Dunnett
Keyword(s):  
Cell Survival ◽  
Cell Suspension ◽  
Single Cells ◽  
Rat Striatum ◽  
Adult Rat ◽  
Striatal Grafts ◽  
Vital Stains ◽  
And Function

Cell suspension grafts from embryonic striatal primordia placed into the adult rat striatum survive well and are able to alleviate a number of behavioral deficits caused by excitotoxic lesions to this structure. However, neither the anatomical connectivity between the graft and host nor the functional recovery elicited by the grafts is completely restored. One way in which the survival and function of embryonic striatal grafts may be enhanced is by the improvement of techniques for the preparation of the cell suspension prior to implantation, an issue that has been addressed only to a limited extent. We have evaluated a number of parameters during the preparation procedure, looking at the effects on cell survival over the first 24 h from preparation using vital dyes and the numbers of surviving neurons in vitro, after 4 days in culture, in addition to graft survival and function in vivo. Factors influencing cell survival include the type of trypsinization procedure and the age of donor tissues used for suspension preparation. The presence of DNase has no effect on cell viability but aids the dissociation of the tissue to form single cells. These results have important implications for the use of embryonic striatal grafts in animal models of Huntington's disease, and in any future clinical application of this research.

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