Art. -Nr.: HY-183393

NVP-BHS345

NVP-BHS345 is an ATP-competitive TORC1/TORC2 inhibitor with an IC₅₀ of 1 μM for both targets in yeast. NVP-BHS345 acutely inhibits TORC1/TORC2 kinase activity, even in genetically engineered TOR1^M2282T Saccharomyces cerevisiae strains. NVP-BHS345 also reduces the phosphorylation levels of Ser-232/Ser-233 sites in Sch9, Ypk1/Ypk2 and Rps6, and induces hyperphosphorylation of Slt2 and activation of the cell wall integrity pathway. NVP-BHS345 causes depolarization of the actin cytoskeleton, blocks fluid-phase endocytosis, prevents the accumulation of vacuolar lipophilic dyes, and regulates the phosphorylation levels of Ent1, Pan1, Prk1, Akl1 and other cellular proteins.

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CAS. Nr.: 1798292-13-3
Formel: C₂₅H₂₀N₆O
Molecular Weight:420.47

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

NVP-BHS345 Chemical Structure

CAS. Nr.: 1798292-13-3

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Biologische Aktivität
- Beschreibung
- IC₅₀ & Target
- In Vitro
Chemical Information
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Biologische Aktivität

Beschreibung

IC₅₀ & Target^[1]^[2]^[3]^[4]^[5]

TORC1

1 μM (IC₅₀)

TORC2

1 μM (IC₅₀)

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In Vitro

NVP-BHS345 (0.1-200 μM; 30 h) inhibits growth of wild-type Saccharomyces cerevisiae by targeting TORC1 and TORC2, with co-expression of TOR1^M2282T and TOR2^M2286T conferring complete resistance to concentrations up to 200 μM^[1].
NVP-BHS345 (10 μM; 12 min) acutely inhibits both TORC1 and TORC2 activity in wild-type Saccharomyces cerevisiae, with TOR1^M2282T conferring partial TORC1 resistance and TOR2^M2286T conferring robust TORC2 resistance^[1].
NVP-BHS345 (15 μM; 90 min) acutely arrests fluid-phase endocytosis in Saccharomyces cerevisiae TOR1^M2282T cells via a Ypk1-dependent mechanism^[1].
NVP-BHS345 (15 μM; 30 min pre-incubation, followed by 30 min MM4-64 incubation with drug present) blocks lipid endocytosis in Saccharomyces cerevisiae TOR1^M2282T cells via a Ypk1-dependent mechanism, preventing vacuolar membrane accumulation of MM4-64^[1].
NVP-BHS345 (15 μM; 30-90 min) induces rapid, time-dependent actin depolarization in Saccharomyces cerevisiae TOR1^M2282T cells via a Ypk1-dependent mechanism, with half-maximal effect observed by 30 min^[1].
NVP-BHS345 (15 μM; 30-180 min) acutely inhibits TORC2 and activates the CWI pathway in Saccharomyces cerevisiae TOR1^M2282T cells, indicating actin depolarization induced by NVP-BHS345 is not caused by CWI pathway inactivation^[1].
NVP-BHS345 (15 μM; 30-90 min (actin polarization); 90 min (Lucifer Yellow uptake); 30 min pre-incubation followed by 30 min MM4-64 incubation with drug present (lipid endocytosis)) induced actin depolarization and endocytosis arrest that was largely suppressed by deletion of FPK1/2 in Saccharomyces cerevisiae TOR1^M2282T cells, identifying Fpk1/2 as major mediators of TORC2-dependent regulation of these processes^[1].
NVP-BHS345 (15 μM; 30 min) induces Ypk1-dependent, Fpk1/2-independent dephosphorylation of endocytic proteins Ent1 and Pan1 in Saccharomyces cerevisiae TOR1^M2282T cells, identifying a direct TORC2 signaling pathway to the endocytic machinery^[1].
NVP-BHS345 (100 μM; 15-60 min) inhibits both TORC1 and TORC2 in wild-type Saccharomyces cerevisiae cells, leading to complete dephosphorylation of Rps6 on Ser-232 and Ser-233 within 60 minutes of treatment with 100 μM^[2].
NVP-BHS345 (100 μM; 30-60 min)-induced Rps6 dephosphorylation is blocked in glc7-127 Saccharomyces cerevisiae cells, indicating the PP1 phosphatase Glc7 is required for this dephosphorylation event^[2].
NVP-BHS345 (100 μM; 30-60 min)-induced Rps6 dephosphorylation is blocked in shp1Δ Saccharomyces cerevisiae cells, indicating the Glc7 regulatory subunit Shp1 is required for this dephosphorylation event^[2].
NVP-BHS345 (10 μM; 70 min) enhances Zeocin-induced yeast chromosome shattering in wild-type Saccharomyces cerevisiae cells with Pan1 depletion, resulting in a B/A signal intensity ratio of 17^[3].
NVP-BHS345 (5-10 μM; 70 min) induces massive chromosome fragmentation in exponentially growing wild-type Saccharomyces cerevisiae when combined with Zeocin (25-75 μg/mL; 70 min), with full fragmentation occurring at 10 μM NVP-BHS345 and 75 μg/mL Zeocin, and this effect is partially dependent on functional base excision repair glycosylases^[4].
NVP-BHS345 potently inhibits the kinase activity of purified budding yeast Saccharomyces cerevisiae TORC1 and TORC2 complexes with an IC₅₀ of 1 μM^[5].
NVP-BHS345 (0.01-100 μM) does not significantly inhibit the kinase activity of immunoprecipitated budding yeast Mec1-Ddc2 complexes at concentrations up to 10 μM, with only partial inhibition observed at 100 μM^[5].
NVP-BHS345 (5 μM; 3 days) weakly enhances sgs1^Δ budding yeast sensitivity to HU and Camptothecin (HY-16560), while profoundly and uniformly sensitizing W303 wild-type, sgs1^Δ, and mec1-100 budding yeast strains to Zeocin and IR, reducing colony formation by ~1000-fold^[5].
NVP-BHS345 (10 μM; 2 hr) causes persistent hyperphosphorylation of the DNA damage checkpoint kinase Rad53 in Zeocin-treated budding yeast wild-type (GA-1981) cells, indicating unimpaired checkpoint activation^[5].
NVP-BHS345 (10 μM; 1 hr/ continuous exposure) drastically increases the percentage of budding yeast wild-type (GA-1981) cells with Rad52 foci (a marker of DSB repair) when combined with Zeocin or IR, indicating elevated or persistent DNA double-strand breaks^[5].
NVP-BHS345 (1-100 μM; 0-60 min) causes dose-dependent inhibition of TORC1 (measured via Sch9 phosphorylation) and complex dose-dependent effects on TORC2 activity (measured via Ypk1 phosphorylation) in budding yeast MS113 cells, with rapid inhibition of Ypk1 phosphorylation at 100 μM^[5].
NVP-BHS345 (50 μM; 2 days) inhibits growth of budding yeast wild-type cells, with partial resistance conferred by TORC1 or TORC2 bypass mutations and full resistance conferred by combined TORC1&TORC2 bypass mutations, indicating it targets both TORC1 and TORC2^[5].

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

Cell Proliferation Assay^[1]

Cell Line:

engineered Saccharomyces cerevisiae strains (WT, TOR1M2282T, TOR2M2286T, TOR1M2282T TOR2M2286T)
Concentration:

0.1-200 μM
Incubation Time:

30 h
Result:

Inhibited growth of WT yeast strains, with a sharp reduction in A600 observed at concentrations ≥4 μM.
Did not confer resistance to NVP-BHS345 via expression of TOR1M2282T alone.
Partially restored growth at concentrations up to 20 μM via expression of TOR2M2286T.
Robustly restored growth across all tested concentrations, with minimal inhibition even at 200 μM via co-expression of TOR1M2282T and TOR2M2286T.

Western Blot Analysis^[1]

Cell Line:

engineered Saccharomyces cerevisiae strains (WT, TOR1M2282T, TOR2M2286T, TOR1M2282T TOR2M2286T)
Concentration:

10 μM
Incubation Time:

12 min
Result:

Abolished Sch9Thr-737 and Ypk1Thr-662 phosphorylation in WT cells.
Partially restored Sch9Thr-737 phosphorylation but did not restore Ypk1Thr-662 phosphorylation via expression of TOR1M2282T.
Partially restored Sch9Thr-737 phosphorylation and robustly restored Ypk1Thr-662 phosphorylation via expression of TOR2M2286T.
Fully restored phosphorylation of both Sch9Thr-737 and Ypk1Thr-662 via co-expression of TOR1M2282T and TOR2M2286T.

Western Blot Analysis^[1]

Cell Line:

Saccharomyces cerevisiae TOR1M2282T strain
Concentration:

15 μM
Incubation Time:

30, 60, 90, 120, 150, or 180 min
Result:

Caused rapid dephosphorylation of Ypk1Thr-662 (detectable by 30 min).
Induced progressive hyperphosphorylation of Slt2Thr-190/Tyr-192, with peak phosphorylation observed at 180 min.

Western Blot Analysis^[1]

Cell Line:

Saccharomyces cerevisiae TOR1M2282T strain
Concentration:

15 μM
Incubation Time:

30 min
Result:

Induced dephosphorylation of Ent1 and Pan1, which was suppressed in cells expressing Ypk1D242A.
Did not trigger dephosphorylation of Ent1 and Pan1 via myriocin or aureobasidin A treatment.
Caused dephosphorylation of Ent1 and Pan1 similarly in TOR1M2282T and TOR1M2282T Δfpk1/2 cells.

Western Blot Analysis^[2]

Cell Line:

wild-type Saccharomyces cerevisiae
Concentration:

100 μM
Incubation Time:

15 min, 30 min, 45 min, 60 min
Result:

Triggered rapid dephosphorylation of both Ser-232 and Ser-233 on Rps6.
Decreased the ratio of phosphorylated Rps6 to total Rps6 (Rps6-PP/Rps6) from 1.0 at 0 min to 0.0 at 60 min.
Decreased the ratio of RXXS*/T*-phosphorylated Rps6 to total Rps6 from 1.0 at 0 min to 0.2 at 60 min.

Western Blot Analysis^[2]

Cell Line:

Saccharomyces cerevisiae glc7-127 mutant cells
Concentration:

100 μM
Incubation Time:

30 min, 60 min
Result:

Suppressed NVP-BHS345-induced dephosphorylation of Rps6.
Maintained the ratio of phosphorylated Rps6 to total Rps6 (Rps6-PP/Rps6) at 2.2 after 60 min of treatment, compared to wild-type cells where this ratio dropped to 0.0.
Maintained the ratio of RXXS*/T*-phosphorylated Rps6 to total Rps6 at 2.5 after 60 min of treatment.

Western Blot Analysis^[2]

Cell Line:

Saccharomyces cerevisiae shp1Δ mutant cells
Concentration:

100 μM
Incubation Time:

30 min, 60 min
Result:

Suppressed NVP-BHS345-induced dephosphorylation of Rps6.
Maintained the ratio of phosphorylated Rps6 to total Rps6 (Rps6-PP/Rps6) at 0.9 after 60 min of treatment, compared to wild-type cells where this ratio dropped to 0.0.

Cell Proliferation Assay^[5]

Cell Line:

budding yeast W303 wild-type, sgs1Δ, mec1-100 strains
Concentration:

5 μM (in combination with 10 mM HU, 5 μg/mL camptothecin, 30 μg/mL Zeocin, or 200 Gy IR)
Incubation Time:

3 days
Result:

Weakly enhanced the sensitivity of sgs1Δ cells to HU or camptothecin.
Profoundly sensitized all three strains to Zeocin and IR, reducing colony formation to roughly 1/1000th of DMSO control levels for all strains treated with Zeocin + NVP-BHS345.

Western Blot Analysis^[5]

Cell Line:

budding yeast wild-type (GA-1981) cells
Concentration:

10 μM (in combination with 75 μg/mL Zeocin)
Incubation Time:

2 hr
Result:

Caused persistent hyperphosphorylation of the activated phosphorylated form of Rad53 (Rad53) during and after treatment, unlike the dephosphorylation observed with Zeocin alone.

Immunofluorescence^[5]

Cell Line:

budding yeast wild-type (GA-1981) cells bearing a Rad52-YFP plasmid
Concentration:

10 μM (in combination with 60 μg/mL Zeocin; in combination with 200 Gy IR)
Incubation Time:

1 hr (Zeocin treatment); continuous exposure (IR treatment)
Result:

Resulted in 79.6% of cells having Rad52 foci, with 48.2% of cells having multiple foci when combined with Zeocin (versus 9.8% with Zeocin alone).
Resulted in 71.2% of cells having multiple Rad52 foci when combined with IR.

Western Blot Analysis^[5]

Cell Line:

budding yeast MS113 (W303a URA3::HA-SCH9) cells
Concentration:

1-100 μM
Incubation Time:

0-60 min (100 μM treatment)
Result:

Caused a dose-dependent loss of Sch9 phosphorylation (TORC1 activity marker), with significant reduction observed at 25 μM.
Caused a dose-dependent increase in Ypk1 phosphorylation at lower concentrations (1-10 μM) followed by a reduction at higher concentrations (25-100 μM).
Caused a rapid loss of Ypk1 phosphorylation at 100 μM, with levels dropping to ~23% of control by 2 min and remaining suppressed over 60 min.

Cell Proliferation Assay^[5]

Cell Line:

budding yeast wild-type (GA-5743), TORC1 bypass (GA-5744), TORC2 bypass (GA-5745), TORC1&TORC2 bypass (GA-5746) strains
Concentration:

50 μM
Incubation Time:

2 days
Result:

Impaired growth of wild-type and TORC2 bypass strains on NVP-BHS345 plates.
Conferred partial resistance to TORC1 bypass strains.
Conferred the strongest resistance to TORC1&TORC2 bypass strains.

Chemical Information

CAS. Nr. 1798292-13-3
Molecular Weight 420.47
Formel C₂₅H₂₀N₆O
SMILES

N#CC(C)(C)C1=CC=C(N2C(N(C)C3=C2C4=CC(C5=CN=CN=C5)=CC=C4N=C3)=O)C=C1
Versand

Room temperature in continental US; may vary elsewhere.
Speicherung

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

Reinheit & Dokumentation

Handling Instructions (2659 KB)

Verweise

[1]. Rispal D, et al. Target of Rapamycin Complex 2 Regulates Actin Polarization and Endocytosis via Multiple Pathways. The Journal of biological chemistry. 2015 Jun 12;290(24):14963-78. [Content Brief]

[2]. Yerlikaya S, et al. TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae. Molecular biology of the cell. 2016 Jan 15;27(2):397-409. [Content Brief]

[3]. Hurst V, et al. Loss of cytoplasmic actin filaments raises nuclear actin levels to drive INO80C-dependent chromosome fragmentation. Nature communications. 2024 Nov 15;15(1):9910. [Content Brief]

[4]. Shimada K, et al. TORC2 inhibition triggers yeast chromosome fragmentation through misregulated Base Excision Repair of clustered oxidation events. Nature communications. 2024 Nov 15;15(1):9908. [Content Brief]

[5]. Shimada K, et al. TORC2 signaling pathway guarantees genome stability in the face of DNA strand breaks. Molecular cell. 2013 Sep 26;51(6):829-39. [Content Brief]

Calculators

Molaritätsrechner

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Molecular Weight Molecular Weight

Verdünnungsrechner

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Konzentration _{(Stammlösung)} Konzentration _{(Stammlösung)}

Volumen _{(Stammlösung)} Volumen _{(Stammlösung)}

Konzentration _{(Ziellösung)} Konzentration _{(Ziellösung)}

Volumen _{(Ziellösung)} Volumen _{(Ziellösung)}

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NVP-BHS345

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