Prexasertib (LY2606368) is distinguished as a highly selective and ATP-competitive second-generation inhibitor of checkpoint kinase 1 (CHK1), possessing a Ki value of 0.9 nM and an IC50 of less than 1 nM. Its ability to inhibit CHK1 with such potency underlines its mechanism of action, which involves causing double-stranded DNA breaks and replication catastrophe, leading to apoptosis. Beyond CHK1, Prexasertib exhibits inhibition against CHK2 (IC50=8 nM) and RSK1 (IC50=9 nM), demonstrating its broad spectrum of action against other kinases involved in cell cycle regulation and tumor growth[1].[2].
体内研究
In vivo, Prexasertib demonstrates significant anti-tumor efficacy in tumor xenograft models, with dosing regimens leading to growth inhibition. Specifically, when administered subcutaneously at doses ranging from 1-10 mg/kg twice daily for three days with rest periods, it effectively inhibits tumor growth.
Additionally, a dose of 15 mg/kg results in CHK1 inhibition in the blood and increased phosphorylation of H2AX (S139) and RPA2 (S4/S8), markers associated with DNA damage and stress responses[1].
体外研究
Additionally, Prexasertib shows inhibition against a range of other kinases, including MELK (IC50=38 nM), SIK (IC50=42 nM), BRSK2 (IC50=48 nM), and ARK5 (IC50=64 nM), indicating its multi-targeted approach in cancer therapy. It has been noted that the efficacy of LY2606368 in causing DNA damage is dependent on the presence of CDC25A and CDK2, key regulators of cell cycle progression[1].
In cellular assays, Prexasertib has demonstrated significant impact on DNA integrity and cell cycle dynamics. For instance, treatment with Prexasertib results in DNA damage during the S-phase in HeLa cells and inhibits CHK1 and CHK2 autophosphorylation in HT-29 cells, affecting the cells' ability to respond to DNA damage and replication stress[1].
Furthermore, at a concentration of 4 nM for 24 h, it induces a notable shift in cell-cycle populations from G1 and G2-M to S-phase in U-2 OS cells, coupled with the induction of H2AX phosphorylation, a marker of DNA damage[1].
Prexasertib 细胞实验
Cell Line
Concentration
Treated Time
Description
References
OVCAR8
5.4 nM
48 hours
Determine the IC50 of Prexasertib on OVCAR8 cells
Oncogene. 2020 Aug;39(33):5520-5535.
OVCAR5
7.5 nM
48 hours
Determine the IC50 of Prexasertib on OVCAR5 cells
Oncogene. 2020 Aug;39(33):5520-5535.
OVCAR8R
3 µM
48 hours
Determine the IC50 of Prexasertib on resistant OVCAR8R cells
Oncogene. 2020 Aug;39(33):5520-5535.
OVCAR5R
3 µM
48 hours
Determine the IC50 of Prexasertib on resistant OVCAR5R cells
Oncogene. 2020 Aug;39(33):5520-5535.
CRC-SCs (colorectal cancer stem cells)
10 nM, 50 nM, 100 nM
1 hour, 4 hours, 9 hours, 24 hours
Assess the impact of LY2606368 on ATM phosphorylation via RPPA analysis, showing LY2606368 induces ATM phosphorylation in sensitive CRC-SCs.
Gut. 2018 May;67(5):903-917.
SCLC cell lines
300 nM
120 hours
Evaluate the antiproliferative effect of LY2606368 on SCLC cell lines, with IC50 <100 nmol/L in half of the human SCLC cell lines
Cancer Res. 2017 Jul 15;77(14):3870-3884.
H460
50 nM
19 days
Screen for sgRNAs that increase or decrease cell killing by the CHK1 inhibitor prexasertib
Mol Cell. 2020 Nov 5;80(3):410-422.e6.
A549
100 nM
19 days
Screen for sgRNAs that increase or decrease cell killing by the CHK1 inhibitor prexasertib
Mol Cell. 2020 Nov 5;80(3):410-422.e6.
OVCAR8
1-40 nM
24 hours
Increased PARylation was observed even after 24 h exposure at low concentrations
Cell Death Discov. 2024 Jun 11;10(1):278.
Rh41 (alveolar RMS)
1 µM
24 hours
Evaluate the effect of Prexasertib on cell proliferation, results showed Prexasertib significantly inhibited cell proliferation and activated DNA damage response.
Clin Cancer Res. 2019 Apr 1;25(7):2278-2289.
RD (embryonal RMS)
1 µM
24 hours
Evaluate the effect of Prexasertib on cell proliferation, results showed Prexasertib significantly inhibited cell proliferation and activated DNA damage response.
Clin Cancer Res. 2019 Apr 1;25(7):2278-2289.
SJCRH30 (alveolar rhabdomyosarcoma)
1 µM
24 hours
Evaluate the effect of Prexasertib on cell proliferation, results showed Prexasertib significantly inhibited cell proliferation and activated DNA damage response.
Clin Cancer Res. 2019 Apr 1;25(7):2278-2289.
MG-63 (osteosarcoma)
1 µM
24 hours
Evaluate the effect of Prexasertib on cell proliferation, results showed Prexasertib significantly inhibited cell proliferation and activated DNA damage response.
Clin Cancer Res. 2019 Apr 1;25(7):2278-2289.
A673 (Ewing’s sarcoma)
1 µM
24 hours
Evaluate the effect of Prexasertib on cell proliferation, results showed Prexasertib significantly inhibited cell proliferation and activated DNA damage response.
Clin Cancer Res. 2019 Apr 1;25(7):2278-2289.
H69, H446, and RPP/mTmG cells
1 µM
24 hours
Evaluated prexasertib-induced cytogenetic stress. Treatment led to a significant increase in MN frequency (p<0.001).
Cancer Discov. 2019 May;9(5):646-661.
ML-1
10 nM
24 hours
Induced apoptosis, including DNA fragmentation, phosphatidylserine exposure, and cleavage of caspase substrates
Cancer Res. 2021 May 15;81(10):2666-2678.
THP.1
10 nM
24 hours
Induced apoptosis, including DNA fragmentation, phosphatidylserine exposure, and cleavage of caspase substrates
Cancer Res. 2021 May 15;81(10):2666-2678.
U937
10 nM
24 hours
Induced apoptosis, including DNA fragmentation, phosphatidylserine exposure, and cleavage of caspase substrates
Cancer Res. 2021 May 15;81(10):2666-2678.
SKN
100 nM
24 hours
To evaluate the effect of Prexasertib on the cell cycle, results showed that 100 nmol/L Prexasertib significantly reduced the number of G1 phase cells and increased the number of S and G2–M phase cells.
Clin Cancer Res. 2022 May 13;28(10):2147-2159.
SK-LMS-1
100 nM
24 hours
To evaluate the effect of Prexasertib on the cell cycle, results showed that 100 nmol/L Prexasertib significantly reduced the number of G1 phase cells and increased the number of S and G2–M phase cells.
Clin Cancer Res. 2022 May 13;28(10):2147-2159.
SK-UT-1
10 nM
24 hours
To evaluate the effect of Prexasertib on the cell cycle, results showed that 10 nmol/L Prexasertib was sufficient to induce cell-cycle arrest.
Clin Cancer Res. 2022 May 13;28(10):2147-2159.
NCI-H460
50-100 nM
24 hours
To evaluate the effect of CHK1 inhibition on replication stress markers
Cell Rep. 2021 Mar 2;34(9):108808.
A549
50-100 nM
24 hours
To evaluate the effect of CHK1 inhibition on replication stress markers
Cell Rep. 2021 Mar 2;34(9):108808.
Huh-7 cells
5 nM
24 hours
To investigate the effect of Prexasertib on apoptosis in Huh-7 cells, results showed that Prexasertib inhibited CHK1 protein levels and promoted cleaved PARP expression, indicating induction of apoptosis.
Br J Cancer. 2021 Jul;125(1):101-111.
Hepa1-6 cells
1 µM
24 hours
To investigate the effect of Prexasertib on apoptosis in Hepa1-6 cells, results showed that Prexasertib inhibited CHK1 protein levels and promoted cleaved PARP expression, indicating induction of apoptosis.
Br J Cancer. 2021 Jul;125(1):101-111.
SKOV3
1 µM
30 min to 8 hours
Prexasertib treatment caused increased PARylation of proteins, indicating PARP1/2 activation
Cell Death Discov. 2024 Jun 11;10(1):278.
OVCAR8
1 µM
30 min to 8 hours
Prexasertib treatment caused increased PARylation of proteins, indicating PARP1/2 activation
Cell Death Discov. 2024 Jun 11;10(1):278.
TOV112D
1–10 nM
3–6 days
To evaluate the cytotoxicity of Prexasertib in ovarian cancer cell lines, results showed that most cell lines were sensitive to Prexasertib with IC50 values of 1–10 nM.
Clin Cancer Res. 2019 Oct 15;25(20):6127-6140.
ES2
1–10 nM
3–6 days
To evaluate the cytotoxicity of Prexasertib in ovarian cancer cell lines, results showed that most cell lines were sensitive to Prexasertib with IC50 values of 1–10 nM.
Clin Cancer Res. 2019 Oct 15;25(20):6127-6140.
UWB1.289
1–10 nM
3–6 days
To evaluate the cytotoxicity of Prexasertib in ovarian cancer cell lines, results showed that most cell lines were sensitive to Prexasertib with IC50 values of 1–10 nM.
Clin Cancer Res. 2019 Oct 15;25(20):6127-6140.
OVCAR3
1–10 nM
3–6 days
To evaluate the cytotoxicity of Prexasertib in ovarian cancer cell lines, results showed that most cell lines were sensitive to Prexasertib with IC50 values of 1–10 nM.
Clin Cancer Res. 2019 Oct 15;25(20):6127-6140.
Retinal ganglion cells (RGCs)
10 µM
4 days
Prexasertib significantly improved RGC survival and promoted neurite outgrowth.
Sci Adv. 2022 Sep 16;8(37):eabq2611.
Adult rat dorsal root ganglion neurons (DRGN)
10 µM
4 days
Prexasertib significantly improved DRGN survival (from 40% to 90%) and promoted neurite outgrowth (neurite length increased from 180 μm to 520 μm).
Sci Adv. 2022 Sep 16;8(37):eabq2611.
CCRF-CEM SLFN11-del and parental cells
1 µM
4 hours
CHK1i treatment for 4 hours induced SLFN11 binding to chromatin and increased the chromatin binding of CDC45 in both parental and SLNF11-del cells
Mol Cell. 2018 Feb 1;69(3):371-384.e6.
CCRF-CEM cells
100 nM
6 hours
To investigate the effect of SLFN11 on chromatin accessibility and IEG transcriptional activation under replication stress. Results showed that SLFN11 enhanced chromatin accessibility and selectively activated IEG transcription.
Cell Rep. 2020 Mar 24;30(12):4137-4151.e6.
A673
1 nM
6 hours
To evaluate the effect of Prexasertib on CHK1 protein levels and protein synthesis. Results showed that Prexasertib reduced CHK1 protein levels and inhibited protein synthesis.
Mol Cancer Ther. 2018 Dec;17(12):2676-2688.
TC71
1 nM
6 hours
To evaluate the effect of Prexasertib on CHK1 protein levels and protein synthesis. Results showed that Prexasertib reduced CHK1 protein levels and inhibited protein synthesis.
Mol Cancer Ther. 2018 Dec;17(12):2676-2688.
EW8
1 nM
6 hours
To evaluate the effect of Prexasertib on CHK1 protein levels and protein synthesis. Results showed that Prexasertib reduced CHK1 protein levels and inhibited protein synthesis.
Analyzed PD-L1 protein expression by RPPA, immunoblot, and flow cytometry. DDR targeting significantly increased the total level of PD-L1 protein in all cell lines tested with the greatest PD-L1 fold change (up to 5 fold) with prexasertib.
Cancer Discov. 2019 May;9(5):646-661.
H82
30 nM
72 hours
To evaluate the cytotoxic effects of Prexasertib in combination with cisplatin on SCLC cell lines, results showed that Prexasertib significantly enhanced cisplatin cytotoxicity.
J Thorac Oncol. 2019 Jun;14(6):1032-1045.
GLC4
30 nM
72 hours
To evaluate the cytotoxic effects of Prexasertib in combination with cisplatin on SCLC cell lines, results showed that Prexasertib significantly enhanced cisplatin cytotoxicity.
J Thorac Oncol. 2019 Jun;14(6):1032-1045.
D425
15 nM
72 hours
Evaluate the synergistic effect of Prexasertib with DNA-damaging chemotherapies, results showed Prexasertib significantly enhanced the cytotoxicity of chemotherapies
Sci Transl Med. 2021 Jan 20;13(577):eaba7401.
D283
12 nM
72 hours
Evaluate the synergistic effect of Prexasertib with DNA-damaging chemotherapies, results showed Prexasertib significantly enhanced the cytotoxicity of chemotherapies
Sci Transl Med. 2021 Jan 20;13(577):eaba7401.
NSCLC cell lines
>0.8 µM
Evaluate the antiproliferative effect of LY2606368 on NSCLC cell lines, which were resistant to LY2606368
Cancer Res. 2017 Jul 15;77(14):3870-3884.
OSKG
6.5 nM
Prexasertib alone resulted in strongly reduced clonogenic survival, with IC50 of 6.5 nM and IC100 of 20 nM.
Int J Cancer. 2020 Aug 15;147(4):1059-1070.
OSRH-2011/5
2 nM
Prexasertib alone resulted in strongly reduced clonogenic survival, with IC50 of 2 nM and IC100 of 7 nM.
Int J Cancer. 2020 Aug 15;147(4):1059-1070.
SAOS-2
3 nM
Prexasertib alone resulted in strongly reduced clonogenic survival, with IC50 of 3 nM and IC100 of 10 nM.
Int J Cancer. 2020 Aug 15;147(4):1059-1070.
KHOS-240S
1.7 nM
Prexasertib alone resulted in strongly reduced clonogenic survival, with IC50 of 1.7 nM and IC100 of 7 nM.
Int J Cancer. 2020 Aug 15;147(4):1059-1070.
Prexasertib 动物实验
Species
Animal Model
Administration
Dosage
Frequency
Description
References
CB-17 SCID beige mice
Cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models
Subcutaneous injection
10 mg/kg
Twice daily for 3 days followed by 4 days rest for 3-4 weeks
Evaluate the anti-tumor effects of Prexasertib as a monotherapy or in combination with chemotherapy in multiple preclinical models of pediatric cancer. Results showed Prexasertib exhibited significant anti-tumor activity, including complete and partial regressions.
Clin Cancer Res. 2019 Apr 1;25(7):2278-2289.
Mice
Immunocompetent SCLC model (RPP/mTmG flank tumors)
Subcutaneous injection
10 mg/kg
2 days per week, twice daily, for 3 weeks
Evaluated the anti-tumor effect of prexasertib in combination with anti-PD-L1 antibody. Remarkable tumor regression was observed in the combination treated group within 1 week, with complete response in 6/10 mice.
Cancer Discov. 2019 May;9(5):646-661.
C57BL/6 mice
Subcutaneous Hepa1-6 xenograft model
Subcutaneous injection
10 mg/kg
Twice daily for 2 days followed by 5 days rest, repeated weekly for 3 weeks
To investigate the anti-tumor effects of Prexasertib on Hepa1-6 subcutaneous xenografts, results showed that Prexasertib inhibited tumor growth, increased tumor cell apoptosis, and promoted NK cell infiltration.
Br J Cancer. 2021 Jul;125(1):101-111.
NCr mice
Ewing sarcoma xenograft model
Subcutaneous injection
10 mg/kg
Twice daily for 3 days, repeated after an 11-day interval
To evaluate the effect of Prexasertib alone or in combination with gemcitabine on tumor growth and mouse survival. Results showed that Prexasertib alone caused tumor regression, and the combination with gemcitabine significantly prolonged mouse survival.
Mol Cancer Ther. 2018 Dec;17(12):2676-2688.
NSGS mice
SF3B1/RUNX1-mutant MDS patient iPSC-derived HSPCs
Subcutaneous injection
10 mg/kg
Twice daily for 3 consecutive days followed by 4 days of rest, repeated for 3 weeks
Prexasertib selectively eradicated SF3B1-mutant HSPCs, significantly reducing the proportion of engrafted SF3B1-mutant cells
Blood Cancer Discov. 2024 Sep 3;5(5):353-370
Nude mice
AML xenograft model
Subcutaneous injection
10 mg/kg or 15 mg/kg
Every 12 hours on days 1–3, 8–10, 15–17 and 22–24
Evaluated the antileukemic effects of Prexasertib in vivo, showing dose-dependent inhibition of xenograft growth and enhanced host survival
Cancer Res. 2021 May 15;81(10):2666-2678.
Nude mice
SCLC syngeneic model
Subcutaneous injection
10 mg/kg, twice daily
3 days per week for 3 weeks
Evaluate the antitumor effect of LY2606368 monotherapy in SCLC models, with 6 mice showing complete response and 3 mice showing >75% tumor reduction
Cancer Res. 2017 Jul 15;77(14):3870-3884.
Rats
T8 dorsal column (DC) crush injury model
Intrathecal injection
2 μg
Once weekly for 6 weeks
Prexasertib significantly suppressed Chk2 phosphorylation, promoted axon regeneration/sprouting, and restored sensory and locomotor function after spinal cord injury.
Sci Adv. 2022 Sep 16;8(37):eabq2611.
Mice
D425 MB orthotopic xenografts
Intravenous injection
20 mg/kg
Once weekly for 6 weeks
Evaluate the anti-tumor effect of Prexasertib combined with cyclophosphamide, results showed the combination therapy significantly extended the survival of mice
Sci Transl Med. 2021 Jan 20;13(577):eaba7401.
BALB/C nude mice
SK-UT-1 tumor model
Intraperitoneal injection
3 mg/kg Prexasertib + 3 mg/kg Cisplatin
Twice a week for four weeks
To evaluate the antitumor effect of Prexasertib in combination with cisplatin, results showed that the combination therapy significantly inhibited tumor proliferation and prolonged the time to tumor progression.
Clin Cancer Res. 2022 May 13;28(10):2147-2159.
Nude mice
SCLC xenograft model
Subcutaneous injection
5 mg/kg
Twice daily for 3 days followed by 4 days of rest for a total of 3 cycles
To evaluate the antitumor activity of Prexasertib alone or in combination with cisplatin in SCLC xenograft models, results showed that Prexasertib significantly enhanced cisplatin antitumor activity and overcame cisplatin resistance.
J Thorac Oncol. 2019 Jun;14(6):1032-1045.
Immunocompromised mice
CRC-SCs xenograft model
Subcutaneous injection
5 mg/kg or 10 mg/kg
Three cycles of three consecutive days, twice daily, followed by 4 days of rest
Evaluate the inhibitory effect of LY2606368 on the growth of CRC-SCs xenografts, showing LY2606368 significantly inhibits tumor growth in sensitive CRC-SCs.
Gut. 2018 May;67(5):903-917.
NSG mice
HGSOC PDX models
Subcutaneous injection
8 mg/kg
Twice daily for 3 days, followed by four days of rest and repeated for two additional cycles
To evaluate the anti-tumor activity of Prexasertib in HGSOC PDX models, results showed that Prexasertib monotherapy demonstrated significant anti-tumor activity across all models.
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