GSK3α (IC50 = 66 nM); GSK3α (Kd = 4.8 μM); GSK-3β(WT) (IC50 = 515 nM)
GSK3α (IC50 = 66 nM; determined at Km of ATP in a mobility-shift microfluidic assay); GSK3β (IC50 = 515 nM; same assay). [1]
CDK family: CDK2 (IC50 = 6.87 μM), CDK3 (IC50 = 9.74 μM), CDK5 (IC50 = 9.20 μM) (87-, 123-, and 116-fold selective relative to GSK3α). [1]
NanoBRET target engagement in live 293 cells: Kd for GSK3α = 4.8 μM; Kd for GSK3β not reported for BRD0705 (BRD3731 gave Kd = 3.3 μM for GSK3β). [1] |

BRD0705 inhibits kinase function and does not stabilize β-catenin, mitigating potential neoplastic concerns. BRD0705 has no effect on healthy hematopoietic cells, but it causes myeloid differentiation and hinders colony formation in AML cells. [1]

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BRD0705 impaired GSK3α Tyr279 autophosphorylation in a time- and concentration-dependent manner without affecting GSK3β Tyr216 phosphorylation in U937 cells (western blot). It decreased phosphorylation of glycogen synthase (GYS, a direct GSK3 substrate). The inactive enantiomer BRD5648 showed no effect. [1]
In primary AML blasts from five patients, BRD0705 selectively inhibited GSK3α active site (p-GSK3α Y279) and did not stabilize β-catenin. [1]
In six AML cell lines (HL-60, NB-4, U937, TF-1, MOLM13, MV4-11), BRD0705 induced morphological changes (May-Grunwald Giemsa staining) and increased cell surface differentiation markers CD11b, CD11c, and CD14 (FACS). In five primary AML patient samples, it increased CD14 and decreased CD117 expression dose-dependently. [1]
BRD0705 impaired colony formation in all six AML cell lines in methylcellulose assays (concentration-dependent). It also reduced colony formation in five primary AML patient samples but had no effect on normal human CD34+ cell colony formation. [1]
In GSK3α CRISPR/Cas9 knockout U937 clones, BRD0705 did not further increase CD11b expression (compared to DMSO), confirming on-target selectivity. [1]
RNA-seq in U937 cells treated with BRD0705 (10 μM, 24h) showed 55 upregulated and 193 downregulated genes. GSEA revealed induction of differentiation transcriptional programs, downregulation of stemness signatures, and upregulation of mitochondrial metabolism programs, but no enrichment of β-catenin pathway genes. [1]
BRD0705 did not increase total β-catenin protein or nuclear translocation in HL-60, TF-1, MV4-11, or MLL-AF9 murine leukemic cells (western blot and immunofluorescence). It did not activate TCF/LEF β-catenin reporter in HEK293T or TF-1 cells. [1]
Glycogen accumulation (PAS staining and quantitative measurement) was observed in AML cell lines (e.g., HL-60) after BRD0705 treatment as an on-target effect. [1] |

BRD0705 delays leukemia initiation and lengthens survival in AML mouse models.[1]

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In a syngeneic MLL-AF9 AML mouse model, BRD0705 pre-treatment (10 μM for 24h) significantly impaired leukemia development in secondary recipients (250,000 cells injection, n=5 per group, p<0.05). Limiting dilution analysis showed a 3.79-fold decrease in leukemia initiating cell (LIC) frequency after BRD0705 pre-treatment. [1]
In the MLL-AF9 model, oral administration of BRD0705 at 30 mg/kg twice daily (BID) significantly improved mouse survival compared to vehicle (n=5 per group, p<0.05, log-rank test). [1]
In an orthotopic HL-60 xenograft model (luciferized cells injected via tail vein into NSG mice), BRD0705 at 15 or 30 mg/kg once daily (QD) by oral gavage decreased leukemia progression dose-dependently (bioluminescence measurement, p<0.05 at 15 mg/kg, p<0.01 at 30 mg/kg) and prolonged overall survival (n=5 per group). It also reduced circulating human CD45+ AML cells. [1]
In an orthotopic MV4-11 xenograft model (irradiated NSG mice), BRD0705 at 30 mg/kg QD significantly decreased disease burden (bioluminescence) and prolonged survival compared to vehicle (n=10 per group, p<0.05). [1]
No major weight loss and no significant blood toxicities were observed with BRD0705 at these doses and schedules. Glycogen accumulation in peripheral mononuclear cells was confirmed as a potential biomarker of on-target activity. [1] |

Kinase inhibition was measured using a mobility-shift microfluidic assay (Caliper). Purified GSK3β or GSK3α was incubated with test compounds in the presence of 4.3 μM ATP (at or just below Km) and 1.5 μM peptide substrate (Peptide 15) for 60 minutes at room temperature in 384-well plates. Assay buffer contained 100 mM HEPES (pH 7.5), 10 mM MgCl2, 2.5 mM DTT, 0.004% Tween-20, and 0.003% Brij-35. Reactions were terminated by addition of 10 mM EDTA. Substrate and product were separated electrophoretically, and fluorescence intensity was determined. Kinase activity was measured as percent conversion. Percent inhibition was plotted against compound concentration, and IC50 values were determined from logistic dose-response curve fitting. Compounds were tested in duplicate in a 12-point dose curve with 3-fold serial dilution starting from 33.3 μM. Values are average of at least three experiments. [1]
For functional selectivity in live cells, a NanoBRET target engagement assay was used. HEK293 cells expressing N-terminal NanoLuc fusion constructs of GSK3α or GSK3β were treated with NanoBRET Kinase Tracer-06 (100 nM) and serially diluted test compounds for 2 hours. NanoBRET NanoGlo Substrate and Extracellular NanoLuc Inhibitor were added, and filtered luminescence was measured (450 nm donor, 600 nm acceptor). Competitive displacement data were graphed using a 3-parameter curve fit to calculate Kd values. [1] |

After 24 hours of treatment with DMSO or BRD0705 at 20 μM, 30,000 to 50,000 cells are washed in PBS/2%FBS and then cytospun onto poly-L-Lysine coated slides (EMS). The cells are then blocked in 1% BSA/0.05% triton for 1 hour at room temperature, fixed in 4% paraformaldehyde (EMS) for 20 minutes, permeabilized in 0.2% Triton X-100 (RPI) for 30 minutes at 4°C, and incubated for 1 hour at room temperature with primary anti-β-catenin (1/500) and secondary antibody. After each antibody incubation, there are three 5 minute washes. The Yokogawa Spinning Disk Confocal / TIRF system is used to image the slides after mounting them with Prolong (R) Gold Antifade with DAPI. Image-J software is used to analyze representative images from three separate experiments.

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Cell culture: HL-60, U937, TF-1, NB-4, MV4-11, MOLM13, and 293T cells were maintained in RPMI 1640 or DMEM with 10-20% FBS and 1% penicillin-streptomycin. Primary AML mononuclear cells were isolated from bone marrow aspirates using Ficoll-Paque and maintained in StemSpan SFEM with cytokines (IL-3, IL-6, GM-CSF, FLT3-L, SCF). [1]
Western immunoblotting: Cells were lysed in Cell Signaling Lysis Buffer with protease and phosphatase inhibitors. Lysates were resolved by 4-12% Bis-Tris gel electrophoresis, transferred to nitrocellulose, blocked in 5% BSA, and incubated with primary antibodies against p-GSK-3α/β (Y279/216), total GSK-3α/β, β-catenin, p-β-catenin (S675, S33/37/T41), GYS, p-GYS (S641), actin, or vinculin, followed by HRP-conjugated secondary antibodies. Chemiluminescence was used for detection. [1]
Flow cytometry: After 6 days of treatment, cells were stained with anti-human CD11b (PE-Cy7), CD11c (APC), CD14 (APC), or CD117 (PE-Cy7) for 35 minutes, washed, and analyzed on a BD FACSCanto II HTS (10,000 cells per condition). [1]
Methylcellulose colony formation: 250-500 cells (cell lines) or 2000-20,000 cells (primary AML or normal CD34+ cells) were seeded in methylcellulose-based medium (ClonaCell-TCS or MethoCult H4236) with DMSO or test compounds. Colonies were counted after MTT staining at 7-10 days. [1]
CRISPR/Cas9 knockout: U937 cells were transduced with lentiCRISPR v2 vectors targeting GSK3α (sgRNA sequences provided) to generate isogenic knockout clones. Knockout was confirmed by western blot. Differentiation marker CD11b was assessed by FACS after compound treatment. [1]
TCF/LEF β-catenin reporter assay: HEK293T cells were transiently transfected with 7xTCF-luc-mCherry reporter and treated with compounds for 48h, then lysed and measured for luciferase activity. TF-1 cells stably infected with GFP β-catenin reporter were treated for 24h and GFP expression measured by flow cytometry. [1]
Immunofluorescence: Cytospun cells on poly-L-lysine slides were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, blocked in 1% BSA, stained with anti-β-catenin primary antibody followed by Alexa Fluor 568 secondary antibody, and mounted with DAPI. Images were acquired by confocal microscopy. [1]
Glycogen quantitative assay: Two million cells were harvested after 3 days of treatment, lysed in dH2O, and glycogen quantified using a colorimetric/fluorometric kit. Glycogen was normalized to total protein (Bradford). [1] |

8-week-old male NSG mice injected with MLL-AF9 AML cells
30 mg/kg
Oral gavage; twice daily

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All animal experiments were approved by the Dana-Farber Cancer Institute Committee on Animal Care. NSG mice (NOD.Cg-Prkdcscid Il2rg tm1Wjl/SzJ) or C57BL/6 mice were used. [1]
For the HL-60 orthotopic xenograft model: 2×10^6 luciferized HL-60 cells were injected via tail vein into 8-week-old male NSG mice. Three days post-injection, mice were randomized and treated once daily (QD) by oral gavage with vehicle (7.5% NMP, 10% Kolliphor HS15, 30% PEG 400, 52.5% normal saline; 5 μL/g) or BRD0705 at 15 or 30 mg/kg. All mice received daily subcutaneous hydration with 5% dextrose. Bioluminescence was measured on an IVIS Spectrum. Whole blood was collected for flow cytometry. [1]
For the MV4-11 orthotopic model: similar procedure with 2×10^6 luciferized MV4-11 cells injected into irradiated NSG mice, treatment started 4 days post-injection with vehicle, BRD0705 (30 mg/kg QD), BRD3731 (30 mg/kg QD), or BRD0320 (30 mg/kg QD). [1]
For the MLL-AF9 syngeneic model: MLL-AF9-DsRed L-GMP cells were treated for 24h with DMSO or 10 μM BRD0705 (or other compounds) before tail vein injection of serially diluted cells (500,000; 250,000; 100,000; 50,000) into sub-lethally irradiated 6-week-old male C57BL/6 mice. Survival was monitored. [1]
For efficacy study in MLL-AF9 model: mice were treated with vehicle or BRD0705 at 30 mg/kg by oral gavage twice daily (BID). [1]
For PK study: single oral dose administration of BRD0705 in mice, plasma concentrations quantifiable up to 24 hours. [1] |

Following a single oral dose administration of BRD0705 in mice, plasma concentrations were quantifiable up to 24 hours with Tmax of 0.25 h and AUC of 67.6 μmol/L·h. BRD0705 has 100% oral bioavailability in mice. [1] |

No major weight loss and no significant blood toxicities were observed upon in vivo administration of BRD0705 at doses of 15 and 30 mg/kg (QD or BID) in mouse models (HL-60, MV4-11, MLL-AF9). [1] |

[1]. Exploiting an Asp-Glu "switch" in glycogen synthase kinase 3 to design paralog-selective inhibitors for use in acute myeloid leukemia. Sci Transl Med. 2018 Mar 7;10(431). pii: eaam8460.

BRD0705 is a first-in-class GSK3α selective inhibitor developed for the treatment of acute myeloid leukemia (AML). It was rationally designed exploiting a single amino acid difference (Asp133→Glu196) between GSK3β and GSK3α in the ATP binding domain. Unlike dual GSK3α/β inhibitors, BRD0705 does not stabilize β-catenin or activate the WNT pathway, mitigating a key mechanism-based safety concern. Genetic suppression of GSK3α phenocopies the effects of BRD0705 in AML. The compound shows no effect on normal hematopoietic stem/progenitor cells, offering a potential therapeutic window. Glycogen accumulation in peripheral mononuclear cells is proposed as a potential biomarker of on-target activity for future clinical trials. [1] |

C16H12CLNO4S

349.788782119751

321.184

C, 74.74; H, 7.21; N, 13.07; O, 4.98

2056261-41-5

BRD5648;2056261-42-6;(Rac)-BRD0705;1597440-03-3

136980453

White to light yellow solid powder

4.2

2

3

2

24

561

1

CC[C@@]1(C2=C(NC3=C1C(=O)CC(C3)(C)C)NN=C2)C4=CC=CC=C4

NCKLQXXBRWCYMA-FQEVSTJZSA-N

InChI=1S/C20H23N3O/c1-4-20(13-8-6-5-7-9-13)14-12-21-23-18(14)22-15-10-19(2,3)11-16(24)17(15)20/h5-9,12H,4,10-11H2,1-3H3,(H2,21,22,23)/t20-/m0/s1

(4S)-4-ethyl-7,7-dimethyl-4-phenyl-1,6,8,9-tetrahydropyrazolo[3,4-b]quinolin-5-one

BRD0705; BRD-0705; BRD 0705

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: 64~300 mg/mL (199.1~933.4 mM)
Ethanol: ~64 mg/mL (~199.1 mM)

Solubility in Formulation 1: ≥ 7.5 mg/mL (23.33 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 75.0 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: ≥ 7.5 mg/mL (23.33 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 75.0 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.)