HSV-1; GSK-3α (IC50 = 10 nM); GSK-3β (IC50 = 10 nM); CDK5/p35 (IC50 = 2.4 μM); CDK2/cyclin A (IC50 = 4.3 μM); CDK1/cyclin B (IC50 = 63 μM)

BIO-acetoxime (BIA; Compound 13) is a potent and selective GSK-3α/β inhibitor, with IC50s of both 10 nM. On CDK5/p35 (IC50, 2.4 M), CDK2/cyclin A (IC50, 4.3 μM), and CDK1/cyclin B (IC50, 63 μM), BIO-acetoxime (BIA) exhibits a negligible effect[1]. However, it has little impact on the morphology and viability of cells infected with mockin. BIO-acetoxime (BIA) (5 μM and 10 μM) increases the viability of HSV-1-infected cells. With an EC50 of 0.68 ± 0.28 μM, BIO-acetoxime (BIA) (0.625, 1.25, 2.5, 5, and 10 μM) also significantly lowers the release of HSV-1 particles in OC3 cells. Although BIO-acetoxime (BIA) may not affect the nuclear targeting of HSV-1 capsids, it does inhibit the expression of HSV-1 genes. Delay in adding BIO-acetoxime (BIA) also prevents HSV-1 infection[2].

BIO-acetoxime (BIA) shows anticonvulsant effects in the focal pilocarpine rat model at 0.5 mg/kg, and in 6-Hz fully kindled FVB/N mice when administered intravenously at doses of 0.5, 2.5, and 5 mg/kg[3].

Kinases activities are assayed in Buffer A or C, at 30 °C, at a final ATP concentration of 15 μM. Blank values are subtracted and activities calculated as pmoles of phosphate incorporated for a 10 min incubation. The activities are expressed in % of the maximal activity, i.e., in the absence of inhibitors. Controls are performed with appropriate dilutions of DMSO. GSK-3α/β is purified from porcine brain by affinity chromatography on immobilized axin. It is assayed, following a 1/100 dilution in 1 mg BSA/mL 10 mM DTT, with 5 μL 40 μM GS-1 peptide as a substrate, in buffer A, in the presence of 15 μM [γ-33P] ATP (3000 Ci/mmol; 1 mCi/mL) in a final volume of 30 μL. After 30 min incubation at 30 °C, 25 μL aliquots of supernatant are spotted onto 2.5 × 3 cm pieces of Whatman P81 phosphocellulose paper, and, 20 s later, the filters are washed five times (for at least 5 min each time) in a solution of 10 mL phosphoric acid/liter of water. The wet filters are counted in the presence of 1 mL of ACS scintillation fluid. CDK1/cyclin B is extracted in homogenization buffer from M phase starfish (Marthasterias glacialis) oocytes and purified by affinity chromatography on p9CKShs1-sepharose beads, from which it is eluted by free p9CKShs1. With 1 mg of histone H1 per milliliter of buffer C and 15 μM [γ-32P] ATP (3000 Ci/mmol; 1 mCi/mL) in a final volume of 30 μL, the kinase activity is measured. 25 μL aliquots of supernatant are spotted onto P81 phosphocellulose papers and subjected to the same treatment after 10 min of incubation at 30 °C. Recombinant mammalian CDK5 and p25, which were purified using affinity chromatography on glutathione-agarose and are both truncated versions of the 35 kDa CDK5 activator protein, are combined in equal amounts to create CDK5/p25. GST (Glutathione-S-Transferase) fusion proteins are expressed in E. coli. In the same manner as for CDK1/cyclin B, its activity is measured in buffer C.

For western blot analysis and RNA isolation, OC3 cells are plated in dishes with 3.5 and 6 cm in diameter, respectively. Unless otherwise noted, cells are mock treated or exposed to HSV-1 at a multiplicity of infection (m.o.i.) of 5. First, DMSO is dissolved to create a stock solution of the GSK-3 inhibitor BIO-acetoxime (BIA). As a solvent control, DMSO-containing medium is used. For 45 minutes prior to mock or HSV-1 infection, cells are pretreated with medium only, medium containing 0.1% DMSO, or BIO-acetoxime (BIA). BIO-acetoxime (BIA) is also present throughout the infection period. HSV-1 is directly exposed to medium containing 5 M BIO-acetoxime (BIA) for 1 hour at room temperature in order to study the direct effect on viruses. With an inverted microscope, cell morphology can be seen[2].

Mice: Male FVB/N mice are used to test the anticonvulsant effects of BIO-acetoxime in fully kindled mice. Mice are stimulated twice daily, excluding weekends, at a fixed subconvulsive threshold current with a minimum 4 hour interval between stimulations in order to reach the fully kindled state. To keep the mice fully kindled, stimulation is given to them only twice a day, two days a week. Nonkindled mice are kept at the original stimulation protocol for a maximum of another two to three weeks. The "epileptic" fully kindled mice are stimulated on Monday morning in order to study the anticonvulsant effect of BIO-acetoxime (BIA). Monday afternoon, 30 minutes before stimulation, the animals receive an intraperitoneal (i.p.) injection of vehicle. Mice are stimulated again on Tuesday morning. Mice are injected intraperitoneally (i.p.) with BIO-acetoxime (BIA) on Tuesday afternoon, and they are stimulated 30 minutes later. The Racine scale is used to gauge seizure severity. The pretreatment scores and the posttreatment scores—obtained following the treatment on Tuesday—are contrasted. The experiment is repeated with a one-week washout in order to confirm any effects that were noticed. To maintain the kindled state during this washout week, all mice are stimulated twice daily for 2 days. Compound testing over a longer time period has not yet been proven effective. This limits the number of doses of BIO-acetoxime that can be tested on a batch of lit mice.[3].

[1]. GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol. 2003 Dec;10(12):1255-66.

[2]. Antiherpetic potential of 6-bromoindirubin-3'-acetoxime (BIO-acetoxime) in human oral epithelial cells. Arch Virol. 2013 Jun;158(6):1287-96.

[3]. Identification of GSK-3 as a Potential Therapeutic Entry Point for Epilepsy. ACS Chem Neurosci. 2018 Nov 6.

C18H12BRN3O3

398.2102

397.0062

C, 54.29; H, 3.04; Br, 20.07; N, 10.55; O, 12.05

667463-85-6

(E/Z)-BIO-acetoxime;740841-15-0

Solid powder

O=C1NC2=C(C=CC(Br)=C2)/C1=C3NC4=C(C=CC=C4)C/3=N\OC(C)=O

MENDIXKFTXYTHJ-HPBPSMLHSA-N

InChI=1S/C18H12BrN3O3/c1-9(23)25-22-16-12-4-2-3-5-13(12)20-17(16)15-11-7-6-10(19)8-14(11)21-18(15)24/h2-8,20H,1H3,(H,21,24)/b17-15-,22-16+

(2Z,3E)-3-(acetoxyimino)-6'-bromo-[2,3'-biindolinylidene]-2'-one

BIA; GSK-3 Inhibitor X; 6-Bromoindirubin-3′-acetoxime; BIO-acetoxime; (2′Z,3′E)-6-Bromoindirubin-3′-acetoxime; 6-Bromoindirubin acetoxime; 6-Bromoindirubin-3′-acetoxime

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)

DMSO: ~19 mg/mL (47.7 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.22 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.

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Solubility in Formulation 2: 2.08 mg/mL (5.22 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.22 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly.

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