Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and cell death

ABSTRACTCDC25 phosphatases have a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational approaches we defined a minimal common pharmacophore in established CDC25 inhibitors and performed a virtual screening of a proprietary library. Taking advantage of the availability of crystal structures for CDC25A and CDC25B and using a molecular docking strategy, we carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, studies on 3D organoids derived from intestinal crypt stem cells of Apc/K-Ras mice revealed that the compounds caused arrest of proliferation.

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Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis

Molecular Microbiology ◽

10.1046/j.1365-2958.1998.00808.x ◽

1998 ◽

Vol 28 (5) ◽

pp. 883-892 ◽

Cited By ~ 147

Author(s):

Chris D. Webb ◽

Peter L. Graumann ◽

Jason A. Kahana ◽

Aurelio A. Teleman ◽

Pamela A. Silver ◽

...

Keyword(s):

Cell Cycle ◽

Bacillus Subtilis ◽

Replication Origin ◽

Time Lapse ◽

Time Lapse Microscopy ◽

Rapid Movement ◽

Use Of Time ◽

Origin Region

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Microtubules in Candida albicans Hyphae Drive Nuclear Dynamics and Connect Cell Cycle Progression to Morphogenesis

Eukaryotic Cell ◽

10.1128/ec.4.10.1697-1711.2005 ◽

2005 ◽

Vol 4 (10) ◽

pp. 1697-1711 ◽

Cited By ~ 48

Author(s):

Kenneth R. Finley ◽

Judith Berman

Keyword(s):

Cell Cycle ◽

Candida Albicans ◽

Basal Cell ◽

Time Lapse ◽

Nuclear Migration ◽

Microtubule Dynamics ◽

Growth Defect ◽

Cell Cortex ◽

Long Distance ◽

Time Lapse Microscopy

ABSTRACT Candida albicans is an opportunistic fungal pathogen whose virulence is related to its ability to switch between yeast, pseudohyphal, and true-hyphal morphologies. To ask how long-distance nuclear migration occurs in C. albicans hyphae, we identified the fundamental properties of nuclear movements and microtubule dynamics using time-lapse microscopy. In hyphae, nuclei migrate to, and divide across, the presumptive site of septation, which forms 10 to 15 μm distal to the basal cell. The mother nucleus returns to the basal cell, while the daughter nucleus reiterates the process. We used time-lapse microscopy to identify the mechanisms by which C. albicans nuclei move over long distances and are coordinated with hyphal morphology. We followed nuclear migration and spindle dynamics, as well as the time and position of septum specification, defined it as the presumptum, and established a chronology of nuclear, spindle, and morphological events. Analysis of microtubule dynamics revealed that premitotic forward nuclear migration is due to the repetitive sliding of astral microtubules along the cell cortex but that postmitotic forward and reverse nuclear migrations are due primarily to spindle elongation. Free microtubules exhibit cell cycle regulation; they are present during interphase and disappear at the time of spindle assembly. Finally, a growth defect in strains expressing Tub2-green fluorescent protein revealed a connection between hyphal elongation and the nuclear cell cycle that is coordinated by hyphal length and/or volume.

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Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and tumor regression

Scientific Reports ◽

10.1038/s41598-019-38579-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Zeynep Kabakci ◽

Simon Käppeli ◽

Claudio Cantù ◽

Lasse D. Jensen ◽

Christiane König ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Tumor Regression ◽

Guided Discovery ◽

Cycle Arrest

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Low Doses of Cisplatin Induce Gene Alterations, Cell Cycle Arrest, and Apoptosis in Human Promyelocytic Leukemia Cells

Biomarker Insights ◽

10.4137/bmi.s39445 ◽

2016 ◽

Vol 11 ◽

pp. BMI.S39445 ◽

Cited By ~ 20

Author(s):

Venkatramreddy Velma ◽

Shaloam R. Dasari ◽

Paul B. Tchounwou

Keyword(s):

Cell Cycle ◽

Molecular Level ◽

Promyelocytic Leukemia ◽

Leukemia Cells ◽

Test Model ◽

Low Doses ◽

Cellular Functions ◽

Cycle Arrest ◽

Modulation Of Gene Expression ◽

Gene Alterations

Cisplatin is a known antitumor drug, but its mechanisms of action are not fully elucidated. In this research, we studied the anticancer potential of cisplatin at doses of 1, 2, or 3 μM using HL-60 cells as a test model. We investigated cisplatin effects at the molecular level using RNA sequencing, cell cycle analysis, and apoptotic assay after 24, 48, 72, and 96 hours of treatment. The results show that many genes responsible for molecular and cellular functions were significantly altered. Cisplatin treatment also caused the cells to be arrested at the DNA synthesis phase, and as the time increases, the cells gradually accumulated at the sub-G1 phase. Also, as the dose increases, a significant number of cells entered into the apoptotic and necrotic stages. Altogether, the data show that low doses of cisplatin significantly impact the viability of HL-60 cells, through modulation of gene expression, cell cycle, and apoptosis.

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Initiation of chromosome replication controls both division and replication cycles in E. coli through a double-adder mechanism

eLife ◽

10.7554/elife.48063 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 7

Author(s):

Guillaume Witz ◽

Erik van Nimwegen ◽

Thomas Julou

Keyword(s):

Cell Cycle ◽

Time Lapse ◽

Growth Conditions ◽

Replication Initiation ◽

Stochastic Simulations ◽

E Coli ◽

Time Lapse Microscopy ◽

Statistical Framework ◽

Wide Range ◽

Cell Cycle Models

Living cells proliferate by completing and coordinating two cycles, a division cycle controlling cell size and a DNA replication cycle controlling the number of chromosomal copies. It remains unclear how bacteria such as Escherichia coli tightly coordinate those two cycles across a wide range of growth conditions. Here, we used time-lapse microscopy in combination with microfluidics to measure growth, division and replication in single E. coli cells in both slow and fast growth conditions. To compare different phenomenological cell cycle models, we introduce a statistical framework assessing their ability to capture the correlation structure observed in the data. In combination with stochastic simulations, our data indicate that the cell cycle is driven from one initiation event to the next rather than from birth to division and is controlled by two adder mechanisms: the added volume since the last initiation event determines the timing of both the next division and replication initiation events.

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Optimization of Cell Cycle Measurement by Time-Lapse Microscopy

Methods in Cell Biology - Laboratory Methods in Cell Biology ◽

10.1016/b978-0-12-405914-6.00007-x ◽

2012 ◽

pp. 143-161 ◽

Cited By ~ 3

Author(s):

Gabor Nagy ◽

Gabor Kiraly ◽

Gaspar Banfalvi

Keyword(s):

Cell Cycle ◽

Time Lapse ◽

Time Lapse Microscopy

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Time‐Lapse Microscopy Approaches to Track Cell Cycle and Lineage Progression at the Single‐Cell Level

Current Protocols in Cytometry ◽

10.1002/0471142956.cy1204s64 ◽

2013 ◽

Vol 64 (1) ◽

Cited By ~ 2

Author(s):

Rachel J. Errington ◽

Sally C. Chappell ◽

Imtiaz A. Khan ◽

Nuria Marquez ◽

Marie Wiltshire ◽

...

Keyword(s):

Cell Cycle ◽

Single Cell ◽

Time Lapse ◽

Single Cell Level ◽

Cell Level ◽

Time Lapse Microscopy

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The Gadd45 Family As Mediators of Hematopoietic Stem Cell Differentiation - from Genotoxic Stress to Cytokine-Induced Differentiation

Blood ◽

10.1182/blood.v126.23.1188.1188 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1188-1188

Author(s):

Susanne Wingert ◽

Frederic B Thalheimer ◽

Nadine Haetscher ◽

Maike Rehage ◽

Hubert Serve ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Stem Cell ◽

Stem Cell Differentiation ◽

Genotoxic Stress ◽

Time Lapse ◽

Cellular Systems ◽

Hematopoietic Stem ◽

Time Lapse Microscopy ◽

Differentiation Induction

Abstract The growth arrest and DNA-damage-inducible 45 (Gadd45) protein family is encoded by three genes, Gadd45a, b and g. All members of the family are early responders of cellular stress with tumor-suppressive function. In leukemia, the Gadd45 genes are often epigenetically silenced. Lately, we identified the GADD45 Gamma as the molecular link of differentiation-promoting cytokines to induce differentiation in HSCs (1). Here, we unraveled the function of the genotoxic stress-induced family member GADD45 Alpha (GADD45A) in hematopoiesis. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains highly controversial. We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution using time-lapse microscopy-based HSC tracking. In contrast to other cellular systems, GADD45A expression neither caused a cell cycle arrest nor an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 h. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multi-lineage differentiation in vivo. The differentiation induction by GADD45A was transmitted by activating p38 MAPK signaling, and allowed the generation of megakaryocytic-erythroid, myeloid and lymphoid lineages. These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system. As the differentiation induction is conserved throughout the GADD45 family our study establishes this cell fate as an HSC-specific DNA-damage escape mechanism. Comparative analyses of the three proteins will further dissect the induced mechanisms at the molecular level. (1) Thalheimer, F.B., Wingert, S., De Giacomo, P., Haetscher, N., Rehage, M., Brill, B., Theis, F.J., Hennighausen, L., Schroeder, T., Rieger, M.A. Cytokine-Regulated GADD45G Induces Differentiation and Lineage Selection in Hematopoietic Stem Cells. Stem Cell Reports 3(1):34-43. (2014) Disclosures No relevant conflicts of interest to declare.

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