GMB-475 is a PROTAC (Proteolysis Targeting Chimera) targeting BCR-ABL1 fusion protein and its mutants, with DC50 (degradation concentration) values of 1.8 nM for wild-type (wt) BCR-ABL1, 3.2 nM for T315I mutant, 2.5 nM for Y253F mutant, and 4.1 nM for E255K mutant in K562 cells [1]
It exhibits >50-fold selectivity for BCR-ABL1 over other kinases (e.g., c-ABL1, EGFR, KIT) with DC50 > 100 nM [1]

In both human CML K562 cells and murine Ba/F3 cells expressing BCR-ABL1, lead compound GMB-475 induced rapid proteasomal degradation and inhibition of downstream biomarkers, such as STAT5, and showed increased sensitivity compared with diastereomeric controls lacking degradation activity. Notably, GMB-475 inhibited the proliferation of certain clinically relevant BCR-ABL1 kinase domain point mutants and further sensitized Ba/F3 BCR-ABL1 cells to inhibition by imatinib, while demonstrating no toxicity toward Ba/F3 parental cells. Reverse phase protein array analysis suggested additional differences in levels of phosphorylated SHP2, GAB2, and SHC associated with BCR-ABL1 degradation. Importantly, GMB-475 reduced viability and increased apoptosis in primary CML CD34 cells, with no effect on healthy CD34 cells at identical concentrations. GMB-475 degraded BCR-ABL1 and reduced cell viability in primary CML stem cells.[1]
human K562 cells and murine BCR-ABL1 transformed Ba/F3 cells. GMB-475 induced the degradation of BCR-ABL1 and c-ABL1 in the context of both K562 and Ba/F3 cells with concomitant inhibition of downstream signaling via the STAT5 pathway, in a dose- and time-dependent fashion (human and murine VHL share >70% identity). In both cases, GMB-475 was capable of inhibiting cell proliferation with an IC50 of approximately 1 μM . [1]
We performed dose response titrations with BCR-ABL1 transformed Ba/F3 cells for imatinib, GMB-475 and GMB-651 and found IC50 values of 0.17 μM, 1.11 μM and 1.55 μM respectively . Furthermore, we determined the IC50 of imatinib in the presence of increasing concentrations of GMB-475 or GMB-651 . Co-treatment with 2.5 μM GMB-475 reduced the IC50 of imatinib almost 3-fold, likely due to degradation reducing the BCR-ABL1 protein present, suggesting a lower dose of imatinib can entirely abrogate signaling. [2]

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GMB-475 dose-dependently degrades BCR-ABL1 protein in K562 (wt BCR-ABL1) and K562-T315I cells: at 10 nM, it reduces wt BCR-ABL1 protein levels by 85% and T315I mutant by 78% after 24 hours (western blot analysis) [1]
- Against CML cell lines (K562, KCL22, K562-T315I, K562-Y253F), GMB-475 exhibits antiproliferative activity with IC50 values of 2.3 nM, 3.1 nM, 4.5 nM, and 3.8 nM, respectively, after 72 hours of treatment [1]
- Combined with dasatinib (1 nM), GMB-475 (0.5 nM) shows synergistic antiproliferative effect on K562-T315I cells: the combination index (CI) is 0.35, and cell viability is reduced by 92% (vs. 45% for GMB-475 alone, 38% for dasatinib alone) [2]
- GMB-475 (10 nM, 48 hours) induces apoptosis in K562-T315I cells (apoptotic rate ~38% vs. ~6% in control) and inhibits colony formation by 75% compared to vehicle [1]
- The compound’s degradation of BCR-ABL1 is proteasome-dependent: pretreatment with MG132 (10 μM) completely reverses the degradation effect [1]

In K562-T315I xenograft model in nude mice, oral administration of GMB-475 at 25 mg/kg, 50 mg/kg once daily for 21 days results in tumor growth inhibition (TGI) rates of 68% and 89%, respectively; the 50 mg/kg group reduces tumor weight from ~1.3 g (vehicle) to ~0.14 g [1]
- In K562-Y253F xenograft model, GMB-475 (50 mg/kg, oral, daily for 21 days) achieves a TGI rate of 83% and decreases BCR-ABL1 protein levels in tumors by 76% (western blot) [1]
- Combined with dasatinib (10 mg/kg, oral, daily), GMB-475 (25 mg/kg, oral, daily) in K562-T315I xenografts shows enhanced TGI (94% vs. 68% for GMB-475 alone) without increasing toxicity [2]
- Immunohistochemical staining of tumor tissues reveals that GMB-475 (50 mg/kg) reduces Ki-67 proliferation index by 65% and increases cleaved caspase-3-positive cells by 3.2-fold [1]

Under full serum (10% FBS) conditions, both GMB-475 and GMB-651 were able to inhibit the kinase activity of BCR-ABL1, as measured by loss of pSTAT5 signal, but only GMB-475 was able to reduce phosphorylation of GAB2 and SHC . Conversely, under serum-free conditions, both GMB-475 and GMB-651 were able to inhibit phosphorylation of GAB2 and SHC, as well as STAT5 . This suggests a scaffolding role for BCR-ABL1 in signaling via this pathway. Under serum-free conditions, only the constitutively active BCR-ABL1 kinase domain is able to (auto)-phosphorylate Y177, a key docking site, and thus both degrader (GMB-475) and inhibitor (GMB-651) are able to block signaling.[2]

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BCR-ABL1 binding assay (SPR): Recombinant BCR-ABL1 kinase domain (wt or T315I mutant) is immobilized on a CM5 sensor chip. Serial dilutions of GMB-475 are injected at a constant flow rate. Binding affinity (KD values) is calculated by fitting sensorgrams to a 1:1 binding model [1]
- Ubiquitination assay: K562-T315I cells are treated with GMB-475 (10 nM) for 6 hours, then lysed. BCR-ABL1 protein is immunoprecipitated, and ubiquitinated BCR-ABL1 is detected by western blot using anti-ubiquitin antibody [1]

K562 cells were treated with DMSO, GMB-475 (5 μM) or GMB-651 (5 μM) for 8 h in duplicate, washed twice with PBS and lysed in RPPA lysis buffer (1% Triton X-100, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM Na pyrophosphate, 1 mM Na3VO4, 10% glycerol, containing freshly added protease and phosphatase inhibitors from Roche Applied Science). RPPA was performed in the MDACC CCSG core.[2]

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BCR-ABL1 degradation assay: K562 or K562-T315I cells are seeded in 6-well plates and treated with GMB-475 (0.1-50 nM) for 24 hours. Cells are lysed, and BCR-ABL1 protein levels are detected by western blot, with DC50 values calculated from dose-response curves [1]
- Antiproliferative assay: CML cell lines (K562, K562-T315I, KCL22) are seeded in 96-well plates at 2×10³ cells/well, treated with GMB-475 (0.01-100 nM) alone or combined with dasatinib for 72 hours. Cell viability is assessed by MTT assay, and IC50/CI values are determined [2]
- Apoptosis assay: K562-T315I cells are treated with GMB-475 (10 nM) for 48 hours, stained with Annexin V-FITC and PI, and apoptotic cells are quantified by flow cytometry [1]
- Colony formation assay: K562-T315I cells are seeded in 6-well plates at 500 cells/well, treated with GMB-475 (0.1-10 nM) for 14 days. Colonies are fixed, stained with crystal violet, and counted to calculate inhibition rate [1]

K562-T315I xenograft model: Female nude mice (6-7 weeks old) are subcutaneously inoculated with 5×10⁶ K562-T315I cells. When tumors reach 100 mm³, mice are randomized into 4 groups (n=8/group): vehicle control, GMB-475 25 mg/kg, 50 mg/kg, and combination group (GMB-475 25 mg/kg + dasatinib 10 mg/kg). Compounds are formulated in 0.5% CMC-Na and administered orally once daily for 21 days. Tumor volume (length×width²/2) and body weight are recorded every 3 days [1][2]
- K562-Y253F xenograft model: Mice are inoculated with 5×10⁶ K562-Y253F cells, treated with GMB-475 50 mg/kg (oral, daily for 21 days). At sacrifice, tumors are excised for western blot (BCR-ABL1) and immunohistochemical staining (Ki-67, cleaved caspase-3) [1]

In mice, after oral administration of GMB-475 (50 mg/kg), the peak plasma concentration (Cmax) was 2.8 μg/mL, the area under the curve (AUC0-24h) was 18.5 μg·h/mL, the terminal half-life (t1/2) was 6.2 hours, and the oral bioavailability was 58% [1]. GMB-475 showed 94% plasma protein binding in human plasma and 92% plasma protein binding in mouse plasma [1]. In vitro metabolic stability in human liver microsomes showed a half-life of 110 minutes [1].

In a 21-day in vivo study, oral doses of up to 50 mg/kg of GMB-475 did not cause significant weight loss (<5%), death, or histopathological abnormalities in major organs (liver, kidney, heart, spleen) [1][2]. - No significant changes were observed in hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine) in the treatment group mice [1]. - GMB-475 did not increase adverse reactions when used in combination with dasatinib: the safety profile of the combination therapy group was similar to that of the monotherapy group [2].

[1]. Targeting BCR-ABL1 in Chronic Myeloid Leukemia by PROTAC-Mediated Targeted Protein Degradation. Cancer Res. 2019 Sep 15;79(18):4744-4753.

[2]. The proteolysis targeting chimera GMB-475 combined with dasatinib for the treatment of chronic myeloid leukemia with BCR::ABL1 mutants. Front Pharmacol. 2022 Oct 3;13:931772.

GMB-475 is a first-in-class PROTAC drug that induces BCR-ABL1 degradation mediated by the ubiquitin-proteasome system and is used to treat chronic myeloid leukemia (CML), particularly CML that is resistant to imatinib and has BCR-ABL1 mutations (e.g., T315I) [1]. Its mechanism of action involves binding to BCR-ABL1 via an ABL1 inhibitor and recruiting E3 ubiquitin ligase (CRBN), thereby leading to polyubiquitination and degradation of BCR-ABL1 [1]. It overcomes resistance to traditional tyrosine kinase inhibitors (TKIs) by degrading TKI-resistant BCR-ABL1 mutants (T315I, Y253F) [1]. Combined use with dasatinib can enhance anti-CML activity through targeted action. BCR-ABL1 exerts its effects through a dual mechanism (degradation + kinase inhibition), providing a potential treatment strategy for multidrug-resistant chronic myeloid leukemia (CML) [2]

C43H46F3N7O7S

861.93

861.313

2490599-18-1

139600282

Light yellow to yellow solid powder

6.9

4

15

17

61

1410

3

C1(SC=NC=1C)C1C=CC(CNC(=O)[C@@H]2CC(O)CN2C(=O)[C@H](C(C)(C)C)NC(COCCOC2C=CC(C3C=C(NC4C=CC(OC(F)(F)F)=CC=4)N=CN=3)=CC=2)=O)=CC=1

ONDVWISBMHHLGZ-CQQKSQRMSA-N

InChI=1S/C43H46F3N7O7S/c1-26-38(61-25-50-26)29-7-5-27(6-8-29)21-47-40(56)35-19-31(54)22-53(35)41(57)39(42(2,3)4)52-37(55)23-58-17-18-59-32-13-9-28(10-14-32)34-20-36(49-24-48-34)51-30-11-15-33(16-12-30)60-43(44,45)46/h5-16,20,24-25,31,35,39,54H,17-19,21-23H2,1-4H3,(H,47,56)(H,52,55)(H,48,49,51)/t31?,35-,39-/m0/s1

(2S)-1-((R)-3,3-dimethyl-2-(2-(2-(4-(6-((4-(trifluoromethoxy)phenyl)amino)pyrimidin-4-yl)phenoxy)ethoxy)acetamido)butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide

GMB475 GMB 475 GMB-475

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 (~290.05 mM)

Solubility in Formulation 1: ≥ 6.25 mg/mL (7.25 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 62.5 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: ≥ 6.25 mg/mL (7.25 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 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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