High-throughput Identification of FLT3 Wild-type and Mutant Kinase Substrate Preferences and Application to Design of SensitiveIn VitroKinase Assay Substrates

SummaryAcute myeloid leukemia (AML) is an aggressive disease that is characterized by abnormal increase of immature myeloblasts in blood and bone marrow. The FLT3 receptor tyrosine kinase plays an integral role in haematopoiesis, and one third of AML diagnoses exhibit gain-of-function mutations in FLT3, with the juxtamembrane domain internal tandem duplication (ITD) and the kinase domain D835Y variants observed most frequently. Few FLT3 substrates or phosphorylation sites are known, which limits insight into FLT3’s substrate preferences and makes assay design particularly challenging. We applied in vitro phosphorylation of a cell lysate digest (adaptation of the Kinase Assay Linked with Phosphoproteomics (KALIP) technique and similar methods) for high-throughput identification of substrates for three FLT3 variants (wild-type, ITD mutant, and D835Y mutant). Incorporation of identified substrate sequences as input into the KINATEST-ID substrate preference analysis and assay development pipeline facilitated the design of several peptide substrates that are phosphorylated efficiently by all three FLT3 kinase variants. These substrates could be used in assays to identify new FLT3 inhibitors that overcome resistant mutations to improve FLT3-positive AML treatment.

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High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells

Blood Cancer Journal ◽

10.1038/s41408-021-00531-2 ◽

2021 ◽

Vol 11 (7) ◽

Author(s):

Ruochen Jia ◽

Leon Kutzner ◽

Anna Koren ◽

Kathrin Runggatscher ◽

Peter Májek ◽

...

Keyword(s):

High Throughput ◽

Drug Screening ◽

Synthetic Lethality ◽

Myeloproliferative Neoplasms ◽

Hematopoietic Cells ◽

Replication Stress ◽

Phase Cell ◽

Nuclear Staining ◽

Wild Type ◽

High Throughput Drug Screening

AbstractMutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia and primary myelofibrosis. To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we searched for small molecules that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89 172 compounds using isogenic cell lines carrying CALR mutations and identified synthetic lethality with compounds targeting the ATR-CHK1 pathway. The selective inhibitory effect of these compounds was validated in a co-culture assay of CALR mutated and wild-type cells. Of the tested compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing wild-type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to ATR-CHK1 inhibition, thus suggesting specificity for the oncogenic activation by the mutant CALR. CHK1 inhibitors induced replication stress in CALR mutated cells revealed by elevated pan-nuclear staining for γH2AX and hyperphosphorylation of RPA2. This was accompanied by S-phase cell cycle arrest due to incomplete DNA replication. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by oncogenic CALR, suggesting an intrinsic vulnerability to CHK1 perturbation. This study reveals the ATR-CHK1 pathway as a potential therapeutic target in CALR mutated hematopoietic cells.

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Local adaptation mediated niche expansion in correlation with genetic richness

10.1101/2021.11.22.469607 ◽

2021 ◽

Author(s):

Masaomi Kurokawa ◽

Issei Nishimura ◽

Bei-Wen YING

Keyword(s):

Escherichia Coli ◽

Local Adaptation ◽

High Throughput ◽

Experimental Evolution ◽

Environmental Gradient ◽

Growth Curves ◽

Quantitative Relationship ◽

Central Issue ◽

Wild Type ◽

Niche Expansion

As a central issue in evolution and ecology, the quantitative relationship among the genome, adaptation and the niche was investigated. Local adaptation of five Escherichia coli strains carrying either the wild-type genome or reduced genomes was achieved by experimental evolution. A high-throughput fitness assay of the ancestor and evolved populations across an environmental gradient of eight niches resulted in a total of 80 fitness curves generated from 2,220 growth curves. Further analyses showed that the increases in both local adaptiveness and niche broadness were negatively correlated with genetic richness. Local adaptation caused common niche expansion, whereas niche expansion for generality or speciality was decided by genetic richness. The order of the mutations accumulated stepwise was correlated with the magnitude of the fitness increase attributed to mutation accumulation. Pre-adaptation probably participated in coordination among genetic richness, local adaptation and niche expansion.

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Chemical Mutagenesis and Fluorescence-Based High-Throughput Screening for Enhanced Accumulation of Carotenoids in a Model Marine Diatom Phaeodactylum tricornutum

Marine Drugs ◽

10.3390/md16080272 ◽

2018 ◽

Vol 16 (8) ◽

pp. 272 ◽

Cited By ~ 8

Author(s):

Zhiqian Yi ◽

Yixi Su ◽

Maonian Xu ◽

Andreas Bergmann ◽

Saevar Ingthorsson ◽

...

Keyword(s):

High Throughput ◽

Metabolic Pathways ◽

Phaeodactylum Tricornutum ◽

Mass Spectrometry Data ◽

Marine Diatom ◽

Chemical Mutagenesis ◽

Carotenoid Content ◽

Enzymatic Reactions ◽

Wild Type ◽

A Genome

Diatoms are a major group of unicellular algae that are rich in lipids and carotenoids. However, sustained research efforts are needed to improve the strain performance for high product yields towards commercialization. In this study, we generated a number of mutants of the model diatom Phaeodactylum tricornutum, a cosmopolitan species that has also been found in Nordic region, using the chemical mutagens ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). We found that both chlorophyll a and neutral lipids had a significant correlation with carotenoid content and these correlations were better during exponential growth than in the stationary growth phase. Then, we studied P. tricornutum common metabolic pathways and analyzed correlated enzymatic reactions between fucoxanthin synthesis and pigmentation or lipid metabolism through a genome-scale metabolic model. The integration of the computational results with liquid chromatography-mass spectrometry data revealed key compounds underlying the correlative metabolic pathways. Approximately 1000 strains were screened using fluorescence-based high-throughput method and five mutants selected had 33% or higher total carotenoids than the wild type, in which four strains remained stable in the long term and the top mutant exhibited an increase of 69.3% in fucoxanthin content compared to the wild type. The platform described in this study may be applied to the screening of other high performing diatom strains for industrial applications.

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Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays

Viruses ◽

10.3390/v10110655 ◽

2018 ◽

Vol 10 (11) ◽

pp. 655 ◽

Cited By ~ 6

Author(s):

Yíngyún Caì ◽

Masaharu Iwasaki ◽

Brett Beitzel ◽

Shuīqìng Yú ◽

Elena Postnikova ◽

...

Keyword(s):

Green Fluorescent Protein ◽

High Throughput ◽

Reverse Genetics ◽

Fluorescent Protein ◽

Cultured Cells ◽

Neutralizing Antibody ◽

Quantitative Detection ◽

Lassa Virus ◽

Wild Type ◽

Green Fluorescent

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000–300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

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High-Throughput Production and Biophysical Characterization of Wild Type and Variant Titin Domains

Biophysical Journal ◽

10.1016/j.bpj.2019.11.228 ◽

2020 ◽

Vol 118 (3) ◽

pp. 6a

Author(s):

Martin Rees ◽

Alexander Alexandrovich ◽

Roksana Nikoopour ◽

Sarah Grover ◽

Anna Laddach ◽

...

Keyword(s):

High Throughput ◽

Wild Type ◽

Biophysical Characterization

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APOL1 polymorphism modulates sphingolipid profile of human podocytes

Glycoconjugate Journal ◽

10.1007/s10719-020-09944-w ◽

2020 ◽

Vol 37 (6) ◽

pp. 729-744 ◽

Cited By ~ 1

Author(s):

Manuela Valsecchi ◽

Valentina Cazzetta ◽

Ferdinando Oriolo ◽

Xiqian Lan ◽

Rocco Piazza ◽

...

Keyword(s):

Mass Spectrometry ◽

Plasma Membrane ◽

High Throughput ◽

Living Cells ◽

Bioactive Components ◽

Wild Type ◽

Drastic Reduction ◽

Metabolic Labelling ◽

Layer Chromatography ◽

Apolipoprotein L1

AbstractApolipoprotein L1 (APOL1) wild type (G0) plays a role in the metabolism of sphingolipids, glycosphingolipids, sphingomyelin and ceramide, which constitute bioactive components of the lipid rafts (DRM). We asked whether APOL1 variants (APOL1-Vs) G1 and G2 carry the potential to alter the metabolism of sphingolipids in human podocytes. The sphingolipid pattern in HPs overexpressing either APOL1G0 or APOL1-Vs was analysed by using a thin mono- and bi-dimensional layer chromatography, mass-spectrometry and metabolic labelling with [1-3H]sphingosine. HP G0 and G1/G2-Vs exhibit a comparable decrease in lactosylceramide and an increase in the globotriaosylceramide content. An analysis of the main glycohydrolases activity involved in glycosphingolipid catabolism showed an overall decrease in the activeness of the tested enzymes, irrespective of the type of APOL1-Vs expression. Similarly, the high throughput cell live-based assay showed a comparable increased action of the plasma membrane glycosphingolipid-glycohydrolases in living cells independent of the genetic APOL1 expression profile. Importantly, the most significative modification of the sphingolipid pattern induced by APOL1-Vs occurred in DRM resulted with a drastic reduction of radioactivity associated with sphingolipids. G1/G2-Vs present a decrease amount of globotriaosylceramide and globopentaosylceramide compared to G0. Additionally, ceramide at the DRM site and lactosylceramide in general, showed a greatest fall in G1/G2 in comparison with G0. Additionally, the levels of glucosylceramide decreased only in the DRM of human podocytes overexpressing G1/G2-Vs. These findings suggest that altered sphingolipidsprofiles may contribute to the deranged functionality of the plasma membrane in APOL1 risk milieu.

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Phagocytic and macropinocytic activity in MARCKS-deficient macrophages and fibroblasts

AJP Cell Physiology ◽

10.1152/ajpcell.1999.277.1.c163 ◽

1999 ◽

Vol 277 (1) ◽

pp. C163-C173 ◽

Cited By ~ 23

Author(s):

Ester Carballo ◽

Diana M. Pitterle ◽

Deborah J. Stumpo ◽

Robert T. Sperling ◽

Perry J. Blackshear

Keyword(s):

Phorbol Esters ◽

Fetal Liver ◽

Fluid Phase ◽

Wild Type ◽

Rhodamine Phalloidin ◽

Kinase Substrate ◽

Latex Beads ◽

Fluid Phase Endocytosis ◽

A Minor ◽

Mannose Receptors

Macrophages express high levels of the myristoylated, alanine-rich, C kinase substrate (MARCKS), an actin cross-linking protein. To investigate a possible role of MARCKS in macrophage function, fetal liver-derived macrophages were generated from wild-type and MARCKS knockout mouse embryos. No differences between the wild-type and MARCKS-deficient macrophages with respect to morphology (Wright’s stain) or actin distribution (staining with rhodamine-phalloidin, under basal conditions or after treatment with phorbol esters, lipopolysaccharide, or both) were observed. We then evaluated phagocytosis mediated by different receptors: Fc receptors tested with IgG-coated sheep red blood cells, complement C3b receptors tested with C3b-coated yeast, mannose receptors tested with unopsonized zymosan, and nonspecific phagocytosis tested with latex beads. We also studied fluid phase endocytosis in macrophages and mouse embryo fibroblasts by using FITC-dextran to quantitate this process. In most cases, there were no differences between the cells derived from wild-type and MARCKS-deficient mice. However, a minor but significant and reproducible difference in rates of zymosan phagocytosis at 45–60 min was observed, with lower rates of phagocytosis in the MARCKS-deficient cells. Our data indicate that MARCKS deficiency may lead to slightly decreased rates of zymosan phagocytosis.

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Molecular dynamics simulations of the interaction of wild type and mutant human CYP2J2 with polyunsaturated fatty acids

BMC Research Notes ◽

10.1186/s13104-019-4797-8 ◽

2019 ◽

Vol 12 (1) ◽

Author(s):

K. K. Abelak ◽

D. Bishop-Bailey ◽

I. Nobeli

Keyword(s):

Molecular Dynamics ◽

Arachidonic Acid ◽

Molecular Dynamics Simulations ◽

Molecular Mechanisms ◽

Homology Model ◽

Cytochrome P450 Enzyme ◽

Wild Type ◽

Substrate Preferences ◽

P450 Enzyme ◽

Dynamics Simulations

Abstract Objectives The data presented here is part of a study that was aimed at characterizing the molecular mechanisms of polyunsaturated fatty acid metabolism by CYP2J2, the main cytochrome P450 enzyme active in the human cardiovasculature. This part comprises the molecular dynamics simulations of the binding of three eicosanoid substrates to wild type and mutant forms of the enzyme. These simulations were carried out with the aim of dissecting the importance of individual residues in the active site and the roles they might play in dictating the binding and catalytic specificity exhibited by CYP2J2. Data description The data comprise: (a) a new homology model of CYP2J2, (b) a number of predicted low-energy complexes of CYP2J2 with arachidonic acid, docosahexaenoic acid and eicosapentaenoic acid, produced with molecular docking and (c) a series of molecular dynamics simulations of the wild type and four mutants interacting with arachidonic acid as well as simulations of the wild type interacting with the two other eicosanoid ligands. The simulations may be helpful in identifying the determinants of substrate specificity of this enzyme and in unraveling the role of individual mutations on its function. They may also help guide the generation of mutants with altered substrate preferences.

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