AKT1 and AKT2 (allosteric inhibitor, ATP non-competitive)
Molecular docking score with AKT1: -2.03 Kcal/mol; with AKT2: -2.25 Kcal/mol [1]

3CAI has the ability to inhibit AKT. In vitro kinase assays were used to test the impact of 3CAI on the kinase activities of AKT1, MEK1, JNK1, ERK1, and TOPK based on these screening data. The findings indicated that 3CAI (1 μM) did not have any effect on the other kinases tested; instead, it only inhibited the activity of AKT1 kinase. AKT1 is more effectively inhibited by 3CAI than by PI3K, with 60% inhibition at 1 μM and 10% at 10 μM, respectively. Both AKT1 and AKT2 activity were markedly suppressed by 3CAI in a dose-dependent manner. Apoptosis is induced and AKT downstream targets are inhibited by 3CAI. The AKT-mediated phosphorylation sites of GSK3β (Ser9) and mTOR (Ser2448) were markedly and time-dependently decreased by 3CAI. Furthermore, following 12 or 24 hours of treatment, 3CAI also increased the levels of the pro-apoptotic marker proteins p53 and p21. In 6 centimeter dishes, HCT116 and HT29 colon cancer cells were seeded with 1% FBS/McCoy's 5A (HCT116) supplemented with 3CAI (4 μM), I3C, or AKT inhibitor, and incubated for four days. The findings demonstrated that, in comparison to untreated control cells, the number of apoptotic cells in HCT116 and HT29 colon cancer cells was significantly higher following 3CAI treatment [1].

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3CAI (1 µM) suppressed only AKT1 kinase activity among tested kinases (MEK1, JNK1, ERK1, TOPK) [1]
3CAI inhibited PI3K activity by 60% at 10 µM (highest concentration tested) [1]
3CAI dose-dependently suppressed AKT1 and AKT2 activities (I3C showed no effect); AKT inhibitor VIII was used as comparator [1]
3CAI directly bound to recombinant AKT1 and AKT2, as well as endogenous AKT1/2 from HCT116 cell lysate, in an ATP non-competitive manner; I3C showed no binding [1]
3CAI decreased phosphorylation of AKT downstream targets: mTOR (Ser2448) and GSK3β (Ser9) in a time-dependent manner; phosphorylation of AKT (Thr308) was not changed [1]
3CAI upregulated p53 and p21 (pro-apoptotic markers) after 12 or 24 h treatment, while decreasing anti-apoptotic marker Bcl2 and AKT-mediated phosphorylation of ASK1 (Ser83) [1]
3CAI significantly increased apoptosis in HCT116 and HT29 colon cancer cells compared to untreated control (assessed by Annexin V/PI staining and flow cytometry) [1]
3CAI inhibited anchorage-independent growth of HCT116 cells (colony formation in 0.3% agar) [1]

In order to investigate the in vivo anticancer activity of 3CAI, individual athymic nude mice were given injections of HCT116 cancer cells into their right flank. For a period of 21 days, mice were given vehicle, I3C (100 mg/kg), or 3CAI (20 or 30 mg/kg) orally five times a week. Treatment of mice with 30 mg/kg 3CAI effectively decreased the growth of HCT116 tumors by 50% (p<0.05) as compared to the vehicle treatment group. Interestingly, mice who received these dosages of 3CAI therapy did not exhibit any overt toxicity or appreciable weight loss as compared to the group that received vehicle treatment. The expression of these AKT target proteins is significantly inhibited by 3CAI at 30 mg/kg in tumor tissue [1].

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Oral administration of 3CAI (30 mg/kg body weight, 5 times per week for 21 days) significantly suppressed HCT116 colon tumor xenograft growth by 50% relative to vehicle-treated group (p < 0.05) [1]
Immunohistochemistry showed that Ki67 expression was markedly decreased in tumor tissues from mice treated with 3CAI [1]
Western blot of tumor tissues from mice treated with 3CAI (30 mg/kg) showed strong suppression of AKT downstream target proteins: p-mTOR (Ser2448) and p-GSK3β (Ser9) [1]

In vitro kinase assay: The reaction was carried out in the presence of [γ-32P]ATP with each compound in reaction buffer containing 20 mM HEPES (pH 7.4), 10 mM MgCl2, 10 mM MnCl2 and 1 mM dithiothreitol. After incubation at room temperature for 30 min, the reaction was stopped by adding protein loading buffer and the mixture was separated by SDS-PAGE. The relative amounts of incorporated radioactivity were assessed by autoradiography. For AKT1 assay: active AKT1 (100 ng) and histone H2B (500 ng) were used. For PI3K assay: conversion of PIP4 to PIP3 was determined by autoradiography [1]
High-throughput substrate-competitive kinase screening: 85 kinases were screened against 3CAI; results identified 3CAI as a potential inhibitor of AKT [1]
Pull-down assay: Recombinant human AKTs (200 ng) or HCT116 cell lysate (500 μg) were incubated with 3CAI-conjugated Sepharose 4B beads (or Sepharose 4B only as control) in reaction buffer (50 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP40, 2 μg/mL bovine serum albumin). After gentle rocking overnight at 4°C, beads were washed 5 times with buffer and binding was visualized by Western blotting [1]

Cell proliferation assay (MTS): Cells were seeded (1×10³ per well) in 96-well plates, incubated for 24 h, then treated with different doses of each compound. After 48 h incubation, CellTiter96 Aqueous One Solution (20 µL) was added and cells were incubated for 1 h at 37°C in 5% CO₂. Absorbance was measured at 492 nm [1]
Anchorage-independent cell growth assay: Cells (8×10³ per well) suspended in complete growth medium were added to 0.6% agar with different doses of each compound in a base layer and a top layer of 0.3% agar. Cultures were maintained at 37°C in 5% CO₂ for 3 weeks, then colonies were counted under a microscope using Image-Pro Plus software [1]
Apoptosis assay: Colon cancer cells (HCT116 or HT29) were plated into 60-mm dishes (1×10⁵ cells/dish) and incubated for 1 day in medium containing 10% FBS. Medium was then replaced with 1% serum medium and cultured for 4 days with 3CAI, I3C or a commercial AKT inhibitor. Cells were collected by trypsinization, washed with PBS, resuspended in binding buffer, and stained with Annexin V and propidium iodide. Analysis was performed by fluorescence microscopy and flow cytometry [1]
Western blot analysis: Cell lysates were prepared with RIPA buffer. Equal amounts of protein were determined by BCA assay. Proteins were separated by SDS/PAGE, transferred to PVDF membranes, blocked with 5% nonfat dry milk, incubated with primary antibodies overnight at 4°C, then with HRP-conjugated secondary antibody (1:5000 dilution), and signal detected with chemiluminescence reagent [1]

Xenograft mouse model: Athymic nude mice (Cr:NIH(S), NIH Swiss nude, 6-9 weeks old) were used. Mice were divided into five groups: (1) untreated vehicle group (n=15); (2) 3CAI 20 mg/kg body weight (n=15); (3) 3CAI 30 mg/kg (n=15); (4) I3C 100 mg/kg (n=15); (5) no cells and 3CAI 30 mg/kg (n=15). HCT116 cells (3×10⁶ cells/100 µL) suspended in serum-free McCoy's 5A medium were inoculated subcutaneously into the right flank of each mouse. 3CAI, I3C or vehicle was administered orally 5 times per week for 21 days. Tumor volume was calculated as: tumor volume (mm³) = (length × width × height × 0.52). Mice were monitored until tumors reached 1 cm³ total volume, then euthanized and tumors extracted [1]
Hematoxylin-eosin staining and immunohistochemistry: Tumor tissues from mice were embedded in paraffin blocks, de-paraffinized, hydrated, permeabilized with 0.5% Triton X-100/1× PBS for 10 min, hybridized with Ki-67 (1:500) as primary antibody, and HRP-conjugated goat anti-rabbit or mouse IgG as secondary antibody. After developing with 3,3'-diaminobenzidine, sections were counterstained with H&E and observed by microscope [1]

Mice treated with 3CAI (20 or 30 mg/kg) showed no overt signs of toxicity or significant loss of body weight compared to vehicle-treated group [1]

[1]. (3-Chloroacetyl)-indole, a novel allosteric AKT inhibitor, suppresses colon cancer growth in vitro and in vivo. Cancer Prev Res (Phila). 2011 Nov;4(11):1842-51.

3CAI is a synthetic derivative of indole-3-carbinol (I3C), a natural product found in Brassica vegetables (broccoli, cabbage). I3C inhibits proliferation with IC₅₀ = 200-300 µM, while 3CAI is more potent [1]
3CAI is a specific allosteric AKT inhibitor that binds to the PH domain of AKT1 and AKT2, not the ATP pocket. It forms a hydrogen bond with Glu17 in AKT1 PH domain, and three hydrogen bonds with Lys14, Leu52 and Arg86 in AKT2 PH domain [1]
3CAI induces apoptosis in colon cancer cells and suppresses colon cancer growth in vitro and in vivo, making it a promising lead compound for developing AKT-targeted cancer therapies [1]

C10H8NOCL

193.62962

193.029

28755-03-5

152961

Off-white to pink solid powder

1.337g/cm3

379.1ºC at 760 mmHg

183ºC

6.02E-06mmHg at 25°C

1.659

2.589

1

1

2

13

207

0

LLZQFAXTCYDVTR-UHFFFAOYSA-N

InChI=1S/C10H8ClNO/c11-5-10(13)8-6-12-9-4-2-1-3-7(8)9/h1-4,6,12H,5H2

2-chloro-1-(1H-indol-3-yl)ethanone

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 : ~250 mg/mL (~1291.12 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (10.74 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 (10.74 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.)