GSK2643943A is a deubiquitinating enzyme (DUB) inhibitor targeting USP20. GSK2643943A has affinity for USP20/Ub-Rho with IC50 of 160 nM. GSK2643943A has anti-tumor effects and may be utilized in the study of oral squamous cell carcinoma (OSCC).

Physicochemical Properties

Molecular Formula	C17H12FN3
Molecular Weight	277.30
Exact Mass	277.101
CAS #	2449301-27-1
PubChem CID	137319705
Appearance	Light yellow to yellow solid powder
Density	1.33±0.1 g/cm3(Predicted)
Boiling Point	540.4±50.0 °C(Predicted)
LogP	4.3
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	2
Heavy Atom Count	21
Complexity	441
Defined Atom Stereocenter Count	0
SMILES	C1=CC(=CC(=C1)F)/C=C/C2=CC3=C(C=C2)C(=C(N3)N)C#N
InChi Key	CGXBPMZRTMXEIA-SNAWJCMRSA-N
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+
Chemical Name	2-amino-6-[(E)-2-(3-fluorophenyl)ethenyl]-1H-indole-3-carbonitrile
HS Tariff Code	2934.99.9001
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)

Biological Activity

Targets	IC50: 160 nM (USP20/Ub-Rho)[1]
ln Vitro	USP20-mediated breaking of protein-ubiquitin bonds is inhibited by GSK2643943A [2]. GSK2643943A sensitizes SCC9 cells to oHSV-1-induced oncolysis at 1 μM and 5 μM over night [2]. When 0.01 MOI T1012G was added to SCC9, the generation of virus increased significantly in response to GSK2643943A (1 μM) [2].
ln Vivo	In SCC9 tumors, GSK2643943A (5 mg/kg, ip, daily for 6 days) improves oHSV-1-induced oncolysis[2]. In SCC7 cells, GSK2643943A (2.5 mg/kg, ip, daily for 9 days) regulates the replication of oHSV-1 T1012G and oncolysis[2].
Cell Assay	Cell Viability Assay[2] Cell Types: SCC9 cells Tested Concentrations: 1 μM, 5 μM (GSK+0.01 MOI T1012) 1 μM (GSK+0.01 MOI/ 1 MOI T1012) Incubation Duration:overnight Experimental Results: Displayed a significant drop in viability ( R50%) (5 μM GSK+0.01 MOI T1012 infection) and 50% loss of SCC9 viability (1 μM GSK+0.01 MOI T1012 infection) . Remarkably diminished the viability of SCC9 upon exposure to 1 MOI T1012G infection. Western Blot Analysis[2] Cell Types: SCC9 cells Tested Concentrations: 1 μM Incubation Duration: 3h, 9 h and 20 h Experimental Results: Generally up-regulated the expression of viral proteins at various phases. RT-PCR[2] Cell Types: SCC9 cells Tested Concentrations: 1 μM Incubation Duration: 9 h Experimental Results: Dramatically increased the accumulation of viral ICP8 and VP16 mRNA in SCC9 cells.
Animal Protocol	Animal/Disease Models: The subcutaneous (sc) xenograft model[2]. (SCC9 or SCC7 cells (8×106 cells or 1×106 cells), 5weeks old, female, BALB/c nude mice or C3H/HeN mice, four groups, n = 6-7, per group)[2] Doses: 5 mg/kg Route of Administration: GSK2643943A (alone): intraperitoneal (ip) administration, daily, for 6 days. GSK2643943A (combination): intraperitoneal (ip) administration, daily for 6 days + intratumoral injection with 50 mL of 1×106 PFU T1012G in PBS on day 1, day 4, and day 7. Experimental Results: Caused a visible drop of tumor volumes and Dramatically decreased the tumor volumes in mice with combined treatment of GSK2643943A and oHSV-1 T1012G. Increased slightly viral ICP0 and gD mRNA accumulation in SCC9 tumors. Animal/Disease Models: The SCC7 mouse model[2]. Doses: 2.5 mg/kg Route of Administration: GSK2643943A (alone): intraperitoneal (ip) administration, daily, for 9 days. GSK2643943A (combination): intraperitoneal (ip) administration, daily, for 9 days + intratumoral injection, with 50 mL of 1×107 PFU T1012G in PBS on days 1, 4, 7, and 10.
References	[1]. Targeting the Ubiquitin Proteasome System in Cancer. Shahzad, Hafiz Naveed (2018). Neoplasm Targeting the Ubiquitin Proteasome System in Cancer. , 10.5772/intechopen.69560(Chapter 8). [2]. USP18 and USP20 restrict oHSV-1 replication in resistant human oral squamous carcinoma cell line SCC9 and affect the viability of SCC9 cells. Mol Ther Oncolytics. 2021 Nov 11;23:477-487.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 125 mg/mL (450.78 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 20.8 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. Solubility in Formulation 2: 2.08 mg/mL (7.50 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.6062 mL	18.0310 mL	36.0620 mL
	5 mM	0.7212 mL	3.6062 mL	7.2124 mL
	10 mM	0.3606 mL	1.8031 mL	3.6062 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.