Figure 2.
AXL inhibitor SLC-391 stabilizes the AXL kinase domain, binds to its active site, and inhibits the growth of AML cells in vitro. (A-B) Chemical structures of 1,3,4-oxadiazolyl-2-aminopyridine (A) and SLC-391 [3-(5-(cyclopropylmethyl)-1,3,4-oxadiazole-2-yl)-5-(1-(piperidine-4-yl)-1H-pyrazole-4-yl)pyridine-2-amine] (B). (C) Differential scanning fluorimetry analysis determined the melting temperatures to be 36.7°C (±1.5) and 52.7°C (±0.1) for AXL kinase and the AXL kinase-SLC-391 complex, respectively. (D) Electrostatic surface representations of AXL kinase–SLC-391 complex model obtained from in silico docking and previously reported crystal structure of AXL kinase–macrocyclic inhibitor complex (PDB code: 5U6B). (E) Cartoon representations of the modeled AXL kinase–SLC-391 complex model and the AXL kinase–macrocyclic inhibitor complex crystal structure showed that SLC-391 potentially blocks the kinase active site. (F) Several AML cell lines were treated with SLC-391 (10 nM to 1 µM) for 72 hours, and viable cells were counted. IC50 values were calculated based on drug concentrations that result in 50% cell viability compared with DMSO-treated control cells (left panel). Comparison of SLC-391 IC50 values in MLL (n = 3) vs non-MLL (n = 5) AML cell lines (right panel). (G) Western blotting analysis of THP-1 cells treated with SLC-391 for 3 hours at the indicated concentrations. GAS6 was added 15 minutes before harvesting the cells. p-AXL(Y779) and p-AKT(S473) levels were quantified, normalized to actin, and compared with no drug treatment control, as indicated. (H) MV4-11 cells were treated with SLC-391 (SLC; 0.1 or 0.25 µM) and venetoclax (VEN; 5 or 10 nM), alone or in combination, for 72 hours, and viable cells were counted. CI values were calculated based on serial dilutions of drug concentrations by CompuSyn software. CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively. (I) The same treated cells as in (H) were subjected to Annexin V/propidium iodide staining and flow cytometry analysis. Annexin V+ cells were graphed as apoptotic cells. (J) MV4-11 cells treated as in panel H were lysed, and whole-cell lysates were subjected to western blotting and probed with the indicated antibodies. Data shown are the mean ± SD of measurements for ≥3 experimental replicates. The P values were calculated using a 2-tailed, unpaired Student t test.

AXL inhibitor SLC-391 stabilizes the AXL kinase domain, binds to its active site, and inhibits the growth of AML cells in vitro. (A-B) Chemical structures of 1,3,4-oxadiazolyl-2-aminopyridine (A) and SLC-391 [3-(5-(cyclopropylmethyl)-1,3,4-oxadiazole-2-yl)-5-(1-(piperidine-4-yl)-1H-pyrazole-4-yl)pyridine-2-amine] (B). (C) Differential scanning fluorimetry analysis determined the melting temperatures to be 36.7°C (±1.5) and 52.7°C (±0.1) for AXL kinase and the AXL kinase-SLC-391 complex, respectively. (D) Electrostatic surface representations of AXL kinase–SLC-391 complex model obtained from in silico docking and previously reported crystal structure of AXL kinase–macrocyclic inhibitor complex (PDB code: 5U6B). (E) Cartoon representations of the modeled AXL kinase–SLC-391 complex model and the AXL kinase–macrocyclic inhibitor complex crystal structure showed that SLC-391 potentially blocks the kinase active site. (F) Several AML cell lines were treated with SLC-391 (10 nM to 1 µM) for 72 hours, and viable cells were counted. IC50 values were calculated based on drug concentrations that result in 50% cell viability compared with DMSO-treated control cells (left panel). Comparison of SLC-391 IC50 values in MLL (n = 3) vs non-MLL (n = 5) AML cell lines (right panel). (G) Western blotting analysis of THP-1 cells treated with SLC-391 for 3 hours at the indicated concentrations. GAS6 was added 15 minutes before harvesting the cells. p-AXL(Y779) and p-AKT(S473) levels were quantified, normalized to actin, and compared with no drug treatment control, as indicated. (H) MV4-11 cells were treated with SLC-391 (SLC; 0.1 or 0.25 µM) and venetoclax (VEN; 5 or 10 nM), alone or in combination, for 72 hours, and viable cells were counted. CI values were calculated based on serial dilutions of drug concentrations by CompuSyn software. CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively. (I) The same treated cells as in (H) were subjected to Annexin V/propidium iodide staining and flow cytometry analysis. Annexin V+ cells were graphed as apoptotic cells. (J) MV4-11 cells treated as in panel H were lysed, and whole-cell lysates were subjected to western blotting and probed with the indicated antibodies. Data shown are the mean ± SD of measurements for ≥3 experimental replicates. The P values were calculated using a 2-tailed, unpaired Student t test.

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