Chk1 (Ki = 0.9 nM); Chk1 (IC50 <1 nM); Chk2 (IC50 = 8 nM)

Prexasertib (LY2606368) inhibits ARK5 (IC50=64 nM), BRSK2 (IC50=48 nM), SIK (IC50=42 nM), and MELK (IC50=38 nM). DNA deterioration caused by LY2606368 requires both CDK2 and CDC25A[1].
Prexasertib (8-250 nM; pre-treated for 15 minutes) causes damage to DNA during the S-phase in HT-29 cells[1].
Prexasertib (4 nM; 24 hours) causes a significant change in cell cycle populations from G1 and G2-M to S-phase, along with an increase in H2AX phosphorylation[1].
Prexasertib (33 nM; for 12 hours) causes the fragmentation of chromosomes in HeLa cells. Replication stress is induced by prexasertib Mesylate Hydrate (100 nM; 0.5 to 9 hours), which also reduces the amount of RPA2 that is available for DNA binding[1].

Prexasertib (LY2606368; 1-10 mg/kg; SC; twice daily for 3 days, rest 4 days; for three cycles) inhibits the growth of tumor xenografts[1].
Prexasertib (15 mg/kg; SC) phosphorylates RPA2 (S4/S8) and H2AX (S139), inhibiting CHK1 in the blood[1].

Prexasertib (LY2606368) inhibits CHK1 and CHK2 with IC50 values less than 1 nM and 8 nM, respectively, with a strong and specific potency. For CHK1 activity via serine 296 autophosphorylation, LY2606368 has an EC50 of 1 nM, and for HT-29 CHK2 autophosphorylation, it is <31 nM (S516). With an EC50 of 9 nM, LY2606368 potently inhibits the G2-M checkpoint that doxorubicin has activated in p53-deficient HeLa cells. Still, 100 nM Instead of weakly inhibiting PMA-stimulated RSK, LY2606368 slightly increases the phosphorylation of S6 on serines 235/236. LY2606368 exhibits broad antiproliferative activity against U-2 OS, Calu-6, HT-29, HeLa, and NCI-H460 cell lines, exhibiting IC50 values of 3 nM, 3 nM, 10 nM, 37 nM, and 68 nM, respectively. Induction of H2AX phosphorylation and a significant shift in cell-cycle populations from G1 and G2-M to S-phase are both brought about by LY2606368 (4 nM) in U-2 OS cells. The anti-proliferative properties of AGS and MKN1 cells are demonstrated by LY2606368 (25 μM). HR repair capacity in DR-GFP cells is inhibited by LY2606368 (20 nM). When combined with the PARP inhibitor BMN673, LY2606368 (5 nM) exhibits synergistic anticancer effects in gastric cancer cells.

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siRNA knockdown[1]
Transfection of U-2 OS cells with siRNAs followed the Lipofectamine RNAiMAX reverse-transfection protocol. Cells were plated with the transfection mixtures and treated with Prexasertib (LY2606368) or DMSO 48 hours later. The siRNA used as a control was the ON-TARGETplus non-targeting pool, whereas the CDK2; and CDC25A targeted siRNAs were obtained commercially. The final concentration of siRNA used for each transfection was 20 nmol/L.

The MTS Cell Proliferation Colorimetric Assay Kit measures the anticancer effects of BMN673 and LY2606368, the proliferation inhibition effect of CHK1 ablation, and IR sensitivity. After seeding cells into 96-well cell culture plates, each well is treated according to the experiment conditions specified. After two hours of incubation, the cell viability of each well is measured using a microplate reader set to detect wavelengths of 490 nM.

Female CD-1 nu-/nu- mice (26-28 g) with Calu-6 cells[1]
1, 3.3, or 10 mg/kg
SC; twice daily for 3 days, rest 4 days; for three cycles

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Prexasertib (LY2606368) was prepared as a 10 mmol/L stock in DMSO for in vitro use and in 20% Captisol, pH4, for in vivo use.
In vivo biochemistry and tumor growth inhibition[1]
Female CD-1 nu-/nu- mice (26–28 g) from Charles River Labs were used for this study. Tumor growth was initiated by subcutaneous injection of 1 × 106 Calu-6 cells in a 1:1 mixture of serum-free growth medium and Matrigel in the rear flank of each subject animal. When tumor volumes reached approximately 150 mm3 in size, the animals were randomized by tumor size and body weight, and placed into their respective treatment groups. Vehicle consisting of 20% Captisol pH4 or Prexasertib (LY2606368) was administered by subcutaneous injection in a volume of 200 μL. Four, eight, 12, 24, and 48 hours after drug administration, blood for plasma drug exposure was extracted via cardiac puncture and assayed on a Sciex API 4000 LC/MS-MS system. The xenograft tissue was promptly removed and prepared as previously described. Lysates were analyzed by immunoblot analysis for protein phosphorylation levels. Group means, SEs and P values were calculated using Kronos.[1]
To measure xenograft tumor growth inhibition, tumors were implanted, established, and the animals randomized as above. Eight animals were used in each treatment group. Vehicle alone or Prexasertib (LY2606368) was administered BIDx3, followed by 4 days of rest and repeated for an additional two cycles. Tumor size and body weight were recorded biweekly and compared between vehicle- and drug-treated groups.

Forty-five patients were treated; seven experienced dose-limiting toxicities (all hematologic). The maximum-tolerated doses (MTDs) were 40 mg/m(2) (schedule 1) and 105 mg/m(2) (schedule 2). The most common related grade 3 or 4 treatment-emergent adverse events were neutropenia, leukopenia, anemia, thrombocytopenia, and fatigue. Grade 4 neutropenia occurred in 73.3% of patients and was transient (typically < 5 days). Febrile neutropenia incidence was low (7%). The LY2606368 exposure over the first 72 hours (area under the curve from 0 to 72 hours) at the MTD for each schedule coincided with the exposure in mouse xenografts that resulted in maximal tumor responses. Minor intra- and intercycle accumulation of LY2606368 was observed at the MTDs for both schedules. Two patients (4.4%) had a partial response; one had squamous cell carcinoma (SCC) of the anus and one had SCC of the head and neck. Fifteen patients (33.3%) had a best overall response of stable disease (range, 1.2 to 6.7 months), six of whom had SCC. Conclusion: An LY2606368 dose of 105 mg/m(2) once every 14 days is being evaluated as the recommended phase II dose in dose-expansion cohorts for patients with SCC.

[1]. LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms. Mol Cancer Ther. 2015 Sep;14(9):2004-1.

[2]. Chk1 inhibition potentiates the therapeutic efficacy of PARP inhibitor BMN673 in gastric cancer. Am J Cancer Res. 2017 Mar 1;7(3):473-483.

Prexasertib has been used in trials studying the treatment and basic science of mCRPC, Leukemia, Neoplasm, breast cancer, and Ovarian Cancer, among others.
Prexasertib is an inhibitor of checkpoint kinase 1 (chk1) with potential antineoplastic activity. Upon administration, prexasertib selectively binds to chk1, thereby preventing activity of chk1 and abrogating the repair of damaged DNA. This may lead to an accumulation of damaged DNA and may promote genomic instability and apoptosis. Prexasertib may potentiate the cytotoxicity of DNA-damaging agents and reverse tumor cell resistance to chemotherapeutic agents. Chk1, a serine/threonine kinase, mediates cell cycle checkpoint control and is essential for DNA repair and plays a key role in resistance to chemotherapeutic agents.

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CHK1 is a multifunctional protein kinase integral to both the cellular response to DNA damage and control of the number of active replication forks. CHK1 inhibitors are currently under investigation as chemopotentiating agents due to CHK1's role in establishing DNA damage checkpoints in the cell cycle. Here, we describe the characterization of a novel CHK1 inhibitor, LY2606368, which as a single agent causes double-stranded DNA breakage while simultaneously removing the protection of the DNA damage checkpoints. The action of LY2606368 is dependent upon inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Loss of the CHK1-dependent DNA damage checkpoints permits cells with damaged DNA to proceed into early mitosis and die. The majority of treated mitotic nuclei consist of extensively fragmented chromosomes. Inhibition of apoptosis by the caspase inhibitor Z-VAD-FMK had no effect on chromosome fragmentation, indicating that LY2606368 causes replication catastrophe. Changes in the ratio of RPA2 to phosphorylated H2AX following LY2606368 treatment further support replication catastrophe as the mechanism of DNA damage. LY2606368 shows similar activity in xenograft tumor models, which results in significant tumor growth inhibition. LY2606368 is a potent representative of a novel class of drugs for the treatment of cancer that acts through replication catastrophe.[2]

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CHEK1 encodes the serine/threonine kinase CHK1, a central component of the DNA damage response. CHK1 regulates cell cycle checkpoints following genotoxic stress to prevent the entry of cells with damaged DNA into mitosis and coordinates various aspects of DNA repair. Accordingly, CHK1 has become a target of considerable interest in oncology. CHK1 inhibitors potentiate the efficacy of DNA-damaging chemotherapeutics by abrogating CHK1-mediated cell cycle arrest and preventing repair of damaged DNA. In addition, CHK1 inhibitors interfere with the biological role of CHK1 as a principal regulator of the cell cycle that controls the initiation of DNA replication, stabilizes replication forks, and coordinates mitosis. Since these functions of CHK1 facilitate progression through an unperturbed cell cycle, CHK1 inhibitors are being developed not only as chemopotentiators, but also as single-agent therapies. This review is intended to provide information on the current progress of CHK1 inhibitors in pre-clinical and clinical development and will focus on mechanisms of single-agent activity and potential strategies for patient tailoring and combinations with non-genotoxic agents.[3]

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The primary objective was to determine safety, toxicity, and a recommended phase II dose regimen of LY2606368, an inhibitor of checkpoint kinase 1, as monotherapy. Patients and methods: This phase I, nonrandomized, open-label, dose-escalation trial used a 3 + 3 dose-escalation scheme and included patients with advanced solid tumors. Intravenous LY2606368 was dose escalated from 10 to 50 mg/m(2) on schedule 1 (days 1 to 3 every 14 days) or from 40 to 130 mg/m(2) on schedule 2 (day 1 every 14 days). Safety measures and pharmacokinetics were assessed, and pharmacodynamics were measured in blood, hair follicles, and circulating tumor cells. Conclusion: An LY2606368 dose of 105 mg/m(2) once every 14 days is being evaluated as the recommended phase II dose in dose-expansion cohorts for patients with SCC.[1]

C18H19N7O2

365.39

365.16

C, 59.17; H, 5.24; N, 26.83; O, 8.76

1234015-52-1

Prexasertib dihydrochloride;1234015-54-3;Prexasertib dimesylate;1234015-58-7;Prexasertib Mesylate Hydrate;1234015-57-6;Prexasertib mesylate;1234015-55-4

46700756

Light yellow to brown solid powder

1.4±0.1 g/cm3

608.5±55.0 °C at 760 mmHg

321.8±31.5 °C

0.0±1.7 mmHg at 25°C

1.655

2.03

3

8

8

27

499

0

Cl[H].O(C([H])([H])C([H])([H])C([H])([H])N([H])[H])C1=C([H])C([H])=C([H])C(=C1C1=C([H])C(N([H])C2C([H])=NC(C#N)=C([H])N=2)=NN1[H])OC([H])([H])[H]

DOTGPNHGTYJDEP-UHFFFAOYSA-N

InChI=1S/C18H19N7O2/c1-26-14-4-2-5-15(27-7-3-6-19)18(14)13-8-16(25-24-13)23-17-11-21-12(9-20)10-22-17/h2,4-5,8,10-11H,3,6-7,19H2,1H3,(H2,22,23,24,25)

5-[[5-[2-(3-aminopropoxy)-6-methoxyphenyl]-1H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 1.67 mg/mL (4.57 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)