Cat. No.: HY-10032A

PF-477736 hydrochloride

PF-477736 (hydrochloride) is an ATP-competitive Chk1 inhibitor (K_i 0.49 nM) that also inhibits CK2A1, PKCA, and Akt1, and is a broad-spectrum kinase inhibitor.PF-477736 induces apoptosis via PARP cleavage, caspase-3/7 activation, caspase-independent death, BAX and PUMA upregulation, H₂AX phosphorylation, DNA fragmentation, and Chk1 proteasomal degradation, while inhibiting proliferation and preventing mitotic entry.PF-477736 demonstrates selective synthetic lethality in LIMD1-deficient cells, efficacy in subcutaneous xenograft models of LIMD1-deficient tumors, and anti-proliferative activity against leukemia and lymphoma cell lines.PF-477736 induces DNA double-strand breaks and activates the ATM-p53-p21 axis, and sensitizes CHK1i-insensitive neuroblastoma cells to apoptosis with ATM or DNA-PK inhibitors.PF-477736 can be used for the research of non-small cell lung cancer, leukemia, lymphoma, and neuroblastoma.

For research use only. We do not sell to patients.

CAS No.: 1247874-19-6
Formula: C₂₂H₂₇Cl₂N₇O₂
Molecular Weight:492.40

Storage:

Please store the product under the recommended conditions in the Certificate of Analysis.

PF-477736 hydrochloride Chemical Structure

CAS No.: 1247874-19-6

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Strong Fluorescence Signal / Multi-Disciplinary Applications / High Fluorescence Specificity

Biological Activity
- Description
- In Vitro
- In Vivo
Chemical Information
Purity & Documentation
References
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Biological Activity

Description

PF-477736 (hydrochloride) is an ATP-competitive Chk1 inhibitor (K_i 0.49 nM) that also inhibits CK2A1, PKCA, and Akt1, and is a broad-spectrum kinase inhibitor^[1]^[2].PF-477736 induces apoptosis via PARP cleavage, caspase-3/7 activation, caspase-independent death, BAX and PUMA upregulation, H₂AX phosphorylation, DNA fragmentation, and Chk1 proteasomal degradation, while inhibiting proliferation and preventing mitotic entry^[1]^[2].PF-477736 demonstrates selective synthetic lethality in LIMD1-deficient cells, efficacy in subcutaneous xenograft models of LIMD1-deficient tumors, and anti-proliferative activity against leukemia and lymphoma cell lines^[1].PF-477736 induces DNA double-strand breaks and activates the ATM-p53-p21 axis, and sensitizes CHK1i-insensitive neuroblastoma cells to apoptosis with ATM or DNA-PK inhibitors^[2].PF-477736 can be used for the research of non-small cell lung cancer, leukemia, lymphoma, and neuroblastoma^[1]^[2].

All 14-3-3 Protein Isoforms :

In Vitro

Anti-cancerApoptosisOthersCell Cycle Regulation

Anti-cancer

PF-477736 (1 μM; 4 days) selectively kills LIMD1^−/− HeLa cells with ~2-fold selectivity over LIMD1^+/+ controls^[1].
PF-477736 (10 days) significantly decreases the clonogenic potential of LIMD1^−/− A549 and HeLa cells compared to LIMD1^+/+ controls^[1].
PF-477736 (1 μM) more potently inhibits the proliferation of LIMD1^−/− HeLa cells (~7-fold reduction in AUC) compared to LIMD1^+/+ cells (~1.6-fold reduction in AUC)^[1].
PF-477736 (4 days) selectively kills LIMD1^−/− A549 cells with ~2-fold selectivity over LIMD1^+/+ controls^[1].
PF-477736 selectively targets RCC48 cells with RNAi-driven LIMD1 deficiency^[1].
PF-477736 (10-point titration; 72 h) selectively inhibits the proliferation of leukemia and lymphoma cell lines (average GI₅₀ 0.28 μM) compared to solid tumor cell lines.

Apoptosis

Anti-cancer

PF-477736 (1 μM; 48 h) selectively induces apoptosis in LIMD1^−/− HeLa and A549 cells^[1].

Others

PF-477736 (1 μM; 4 days) selectively targets LIMD1^−/− SAEC-Bmi1 cells and its efficacy across LUAD cell lines positively correlates with LIMD1 protein expression^[1].
PF-477736 (3 μM) is a broad-spectrum kinase inhibitor that inhibits 303 out of 468 tested kinases by over 50% at 3 μM^[1].
PF-477736 (1 μM; 1 h) treatment elicits significant phosphorylation changes and inhibits CK2A1, PKCA, and Akt1 specifically in LIMD1^−/− HeLa cells but not in LIMD1^+/+ cells^[1].

Anti-cancer

Apoptosis

Cell Cycle Regulation

PF-477736 (1 µM; 3 days) is highly effective against CHP134 and SMS-SAN cells, whereas SK-N-BE and NB-39-nu cells are relatively insensitive^[2].
PF-477736 (1 µM) induces differential expression of p53 target genes in SMS-SAN and NB-39-nu cells, with SMS-SAN cells upregulating BAX and PUMA and NB-39-nu cells upregulating p21 and MDM2^[2].
PF-477736 (1 µM; 24 h)-induced upregulation of p21 and MDM2 in NB-39-nu cells is p53-dependent^[2].
PF-477736 (5-10 µM) induces apoptosis in NB-39-nu cells^[2].
PF-477736 (0.175 µM; 6-7 days) and an ATM inhibitor overcomes CHK1i insensitivity and induces mitotic catastrophe and apoptosis in NB-39-nu cells^[2].
PF-477736 (0.175 µM; 6 days) inhibition of DNA-PK or ATM can overcome PF-477736 insensitivity and inhibit the growth of MYCN-amplified neuroblastoma cells^[2].

Anti-cancer

Cell Cycle Regulation

Increased p53 protein levels may be associated with reduced sensitivity to CHK1 inhibitors in MYCN-amplified neuroblastoma cells^[2].
PF-477736 (1 µM; 24 h) in CHK1i-insensitive NB-39-nu cells stabilizes p53 through activation of the ATM-p53-p21 axis, whereas the sensitive SMS-SAN cells show a lesser response^[2].
PF-477736 (1-10 µM) induces DNA double-strand breaks and activates the ATM-p53 signaling axis in a dose-dependent manner in NB-39-nu cells^[2].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Cell Viability Assay^[1]

Cell Line:

HeLa (LIMD1+/+ and LIMD1−/−)
Concentration:

1 μM
Incubation Time:

4 days
Result:

Selectively decreased cell viability in LIMD1−/− cells compared to LIMD1+/+ cells with ~2-fold selectivity.

Apoptosis Analysis^[1]

Cell Line:

HeLa and A549 (LIMD1+/+ and LIMD1−/−)
Concentration:

1 μM
Incubation Time:

48 h
Result:

Induced 'blebbing-like' structures of cell membranes in LIMD1−/− cells.
Increased PARP cleavage and caspase activation in LIMD1−/− clones compared to LIMD1+/+ controls.
Increased early and late apoptotic cell populations in treated LIMD1−/− cells.

Cell Viability Assay^[1]

Cell Line:

LUAD cell lines and SAEC-Bmi1
Concentration:

1 μM
Incubation Time:

4 days
Result:

In SAEC-Bmi1 cells, ~2-4-fold selectivity for LIMD1−/− cells was observed.
A significant positive correlation (Pearson’s correlation coefficient = 0.579) was found between LIMD1 protein expression and surviving fraction across a panel of LUAD cell lines.

Cell Cytotoxicity Assay^[2]

Cell Line:

HL60, Jurkat, K562, MV4-11, Raji, U937, and solid tumor cell lines (one lung and six colon cancer cell lines)
Concentration:

10-point titration
Incubation Time:

72 h
Result:

Inhibited proliferation of leukemia and lymphoma cell lines with an average GI50 of 0.28 μM.
Achieved GI50 values of 0.73 μM (HL60), 0.12 μM (Jurkat), 1.9 μM (K562), 0.13 μM (MV4-11), 0.33 μM (Raji), and 0.099 μM (U937).
Inhibited solid tumor cell lines with an average GI50 of 1.7 μM.
Correlated with V158411 sensitivity with an R² of 0.829.

Cell Viability Assay^[3]

Cell Line:

CHP134, NB-39-nu, SK-N-BE, SMS-SAN
Concentration:

1 µM
Incubation Time:

3 days
Result:

Induced much higher sensitivity in CHP134 and SMS-SAN cells compared with SK-N-BE and NB-39-nu cells.

Real Time qPCR^[3]

Cell Line:

NB-39-nu
Concentration:

1 µM
Incubation Time:

24 h
Result:

Increased p21 and MDM2 mRNA levels, but the upregulation was significantly blunted by p53 knockdown.

Western Blot Analysis^[3]

Cell Line:

NB-39-nu, SMS-SAN
Concentration:

1 µM
Incubation Time:

24 h
Result:

Dramatically elevated levels of active p53 phosphorylated on Ser15 in NB-39-nu cells, and markedly upregulated p21 and MDM2 protein levels.
Accompanied these events by increasing levels of active p-ATM-Ser1981.
Showed a lesser extent of increasing levels of p-p53-Ser15 and p21 in SMS-SAN cells compared to NB-39-nu, and observed decreased levels of active p-ATM-Ser1981.

Cell Viability Assay^[3]

Cell Line:

NB-39-nu
Concentration:

0.175 µM
Incubation Time:

6-7 days
Result:

Had modest effects on cell viability as a single agent.
Significantly decreased cell viability in combination with ATMi compared with either single agent alone.
Upregulated levels of apoptosis markers cleaved caspase-3 and cleaved PARP to a much greater extent in combination with ATMi.
Detected expression of the mitosis marker p-histone H3 only in cells exposed to both PF-477736 and ATMi.
Observed apparent cell shrinkage and disruption of cell-cell contact only under the condition of combination exposure.
Had minimal effects on DSBs as a single agent, whereas the same dose in combination with ATMi caused a marked increase in DSBs.

Cell Viability Assay^[3]

Cell Line:

NB-39-nu, SK-N-BE
Concentration:

0.175 µM
Incubation Time:

6 days
Result:

Significantly decreased cell viability when combined with DNA-PK (NU7441) compared with either single agent alone, and had a greater growth-inhibitory effect than the combination with ATMi.
Significantly reduced the viability of SK-N-BE cells to a greater extent than did the single agents alone when combined with ATMi or DNA-PKi.

In Vivo

Anti-cancerApoptosis

Anti-cancer

Apoptosis

PF-477736 (7.5 mg/kg; twice a day with a 6-hour difference; once a week) selectively inhibits tumour growth in LIMD1-deficient lung xenografts in vivo^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Model:

NOD/SCID (female, six weeks old)^[1]
Dosage:

7.5 mg/kg
Administration:

twice a day with a 6-hour difference; once a week
Result:

Had no significant effect on LIMD1+/+ tumours.
Significantly decreased tumour growth in LIMD1−/− tumours.
Selectively inhibited proliferation in LIMD1-deficient lung xenografts.
Increased apoptosis within LIMD1-deficient tumours.

Related Protocols :

Chemical Information

CAS No. 1247874-19-6
Molecular Weight 492.40
Formula C₂₂H₂₇Cl₂N₇O₂
SMILES

O=C1C2=C3C(C=NN1)=C(C4=CN(C)N=C4)NC3=CC(NC([C@@H](C5CCCCC5)N)=O)=C2.Cl.Cl
Shipping

Room temperature in continental US; may vary elsewhere.
Storage

Please store the product under the recommended conditions in the Certificate of Analysis.

Purity & Documentation

Handling Instructions (2659 KB)

References

[1]. Davidson K, et al. Targeted therapy for LIMD1-deficient non-small cell lung cancer subtypes. Cell death & disease. 2021 Nov 11;12(11):1075.

[2]. Ando K, et al. Co-Inhibition of the DNA Damage Response and CHK1 Enhances Apoptosis of Neuroblastoma Cells. International journal of molecular sciences. 2019 Jul 29;20(15):3700.

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PF-477736 hydrochloride

All 14-3-3 Protein Isoforms