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Purity: ≥98%
GSK2643943A (GSK-2643943A) is a novel and potent inhibitor of deubiquitylating enzyme (DUB) with anticancer activity. It inhibits USP20/Ub-Rho with an IC50 of 160 nM. The ubiquitin proteasome system is involved in a myriad of biological functions including cell cycle progression, intracellular signaling and protein degradation. As such, it is not surprising to find many components of the system misregulated in cancer. The clinical success of Bortezomib for treatment of multiple myeloma proves that targeting the ubiquitin proteasome system is valid and feasible. Here, a detailed examination of the strategies used to target the ubiquitin proteasome system in cancer is discussed.
| Targets |
USP20/Ub-Rho(IC50= 160 nM)
USP20-mediated cleavage of protein-ubiquitin bonds is inhibited by GSK2643943A[2]. GSK2643943A (1 μM, 5 μM; overnight) increases the susceptibility of SCC9 cells to oncolysis induced by oHSV-1[2]. Significantly higher virus yields were observed in SCC9 with 0.01 MOI T1012G infection when treated with 1μM of GSK2643943A[2]. |
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| ln Vitro |
USP20-mediated cleavage of protein-ubiquitin bonds is inhibited by GSK2643943A[2].
GSK2643943A (1 μM, 5 μM; overnight) increases the susceptibility of SCC9 cells to oncolysis induced by oHSV-1[2]. Significantly higher virus yields were observed in SCC9 with 0.01 MOI T1012G infection when treated with 1μM of GSK2643943A[2]. Treatment with GSK2643934A (1 μM or 5 μM) in SCC9 cells enhanced oHSV-1 T1012G virus yields under infection with 0.01 or 0.1 MOI, as measured by plaque assay. GSK2643934A (1 μM) treatment in SCC9 cells increased viral protein accumulation (ICP0, ICP8, VP16) and viral mRNA levels (ICP8, VP16) upon infection with 5 MOI T1012G, as shown by Western blot and qPCR. GSK2643934A (1 μM or 5 μM) combined with T1012G infection (0.01–1 MOI) significantly reduced SCC9 cell viability in a dose-dependent manner, as assessed by CCK-8 assay. In mouse OSCC SCC7 cells, GSK2643934A (1 μM) treatment also increased oHSV-1 T1012G virus yields under 0.01 MOI infection. |
| ln Vivo |
In SCC9 tumors, GSK2643943A (5 mg/kg, i.p., daily, for 6 days) amplifies oHSV-1-induced oncolysis[2].
In SCC7 cells, GSK2643943A (2.5 mg/kg, i.p., daily, for 9 days) regulates the replication of oHSV-1 T1012G and oncolysis[2]. In nude mice bearing SCC9 tumors, intraperitoneal administration of GSK2643934A (5 mg/kg daily for 6 days) combined with intratumoral injection of T1012G (1×10⁶ PFU on days 1, 4, and 7) significantly reduced tumor volume and weight compared to monotherapy or vehicle control. In immune-competent C3H/HeN mice bearing SCC7 tumors, intraperitoneal administration of GSK2643934A (2.5 mg/kg daily for 9 days) combined with intratumoral T1012G (1×10⁷ PFU on days 1, 4, 7, and 10) also showed enhanced anti-tumor efficacy. qPCR analysis of tumor tissues showed that GSK2643934A treatment slightly increased viral mRNA levels (ICP0 and gD) in both SCC9 and SCC7 tumors. |
| Enzyme Assay |
Co-immunoprecipitation (Co-IP) assay was performed in SCC9 cells to assess the effect of GSK2643934A on USP20-mediated deubiquitination. Cells were treated with GSK2643934A or DMSO, lysed, and immunoprecipitated with anti-STING antibody. Western blot analysis showed that GSK2643934A treatment increased poly-ubiquitin accumulation and slightly decreased STING protein levels, indicating inhibition of USP20 deubiquitination activity.
[2] |
| Cell Assay |
Cell Line: SCC9 cells
Concentration: 1 μM, 5 μM (GSK+0.01 MOI T1012) 1 μM (GSK+0.01 MOI/ 1 MOI T1012) Incubation Time: overnight Result:showed a 50% loss of SCC9 viability (1 μM GSK+0.01 MOI T1012 infection) and a significant drop in viability (R50%) (5 μM GSK+0.01 MOI T1012 infection). significantly decreased SCC9 viability when exposed to a 1 MOI T1012G infection. For virus titration, SCC9 or SCC7 cells were infected with T1012G at various MOI in serum-free medium for 1–2 h, then replaced with fresh medium. Virus-containing cells were harvested, lysed by freeze-thaw cycles, and titrated on Vero cells using plaque assay. For Western blot, cells were lysed with RIPA buffer, separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against viral proteins (ICP0, ICP8, VP16) and cellular proteins (STING, USP20, β-actin). For qPCR, total RNA was extracted, reverse-transcribed, and quantified using SYBR Green with primers for viral genes (ICP8, VP16) and cellular genes (USP18, USP20, STING). Cell viability was assessed using CCK-8 assay. Cells were seeded in 96-well plates, treated with GSK2643934A and/or T1012G, incubated, and OD450 was measured. |
| Animal Protocol |
Animal Model: The model of subcutaneous xenograft[2].
(Four groups, n = 6-7 per group; 5-week-old female BALB/c nude mice or C3H/HeN mice; SCC9 or SCC7 cells (8×106 cells or 1×106 cells)[2] Dosage: 5 mg/kg Administration: GSK2643943A (alone): injectable once daily for a period of six days. The combination of GSK2643943A is intraperitoneal administration every day for six days plus intratumoral injection on days 1, 4, and 7 with 50 mL of 1×10^6 PFU T1012G in PBS. Result: caused a discernible decline in tumor volumes and markedly decreased tumor volumes in mice when GSK2643943A and oHSV-1 T1012G were given together. gD mRNA accumulation and slightly elevated viral ICP0 in SCC9 tumors. For SCC9 xenograft model, BALB/c nude mice were subcutaneously inoculated with SCC9 cells. When tumor volume reached 70–120 mm³, mice were randomized into four groups: vehicle (PBS intratumoral), GSK2643934A (5 mg/kg intraperitoneal daily for 6 days), T1012G (1×10⁶ PFU intratumoral on days 1, 4, 7), and combination. Tumor volume was measured every 2–3 days for 25 days. For SCC7 model, C3H/HeN mice were inoculated with SCC7 cells. Treatment groups were similar but with GSK2643934A at 2.5 mg/kg daily for 9 days and T1012G at 1×10⁷ PFU on days 1, 4, 7, and 10. Tumor volume was tracked for 19 days. |
| References |
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| Additional Infomation |
GSK2643934A was used as a tool inhibitor to investigate the role of USP20 in regulating STING stability and antiviral responses in oral squamous cell carcinoma (OSCC) cells. It enhanced oHSV-1 replication and oncolytic activity in drug-resistant tumor cells, suggesting its potential as a combination therapy with oncolytic viruses.
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| Molecular Formula |
C₂₇H₂₉CL₂N₇O₂S
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|---|---|
| Molecular Weight |
586.54
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| Exact Mass |
277.10
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| Elemental Analysis |
C, 73.63; H, 4.36; F, 6.85; N, 15.15
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| Related CAS # | |
| Appearance |
Light yellow to yellow solid powder
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| SMILES |
O=C(NCC1=CC=C(S(C2=CC(F)=CC(F)=C2)(=O)=O)C=C1)C3=CN4C(C=C3)=NC=C4.Cl
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| InChi Key |
CGXBPMZRTMXEIA-SNAWJCMRSA-N
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| InChi Code |
InChI=1S/C17H12FN3/c18-13-3-1-2-11(8-13)4-5-12-6-7-14-15(10-19)17(20)21-16(14)9-12/h1-9,21H,20H2/b5-4+
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| Chemical Name |
(E)-2-amino-6-(3-fluorostyryl)-1H-indole-3-carbonitrile
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| Synonyms |
GSK2643943A; GSK 2643943A; GSK-2643943A; GSK2643943; GSK 2643943; GSK-2643943;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : 55~125 mg/mL ( 198.34~450.78 mM )
Ethanol : ~7 mg/mL |
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| Solubility (In Vivo) |
5% DMSO + Corn oil: 1mg/ml (Please use freshly prepared in vivo formulations for optimal results.)
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7049 mL | 8.5246 mL | 17.0491 mL | |
| 5 mM | 0.3410 mL | 1.7049 mL | 3.4098 mL | |
| 10 mM | 0.1705 mL | 0.8525 mL | 1.7049 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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