Fig. 2: pBADSer99 inhibitor NCK demonstrates improved potency over NPB. | npj Precision Oncology

Fig. 2: pBADSer99 inhibitor NCK demonstrates improved potency over NPB.

From: Vertical pathway inhibition of receptor tyrosine kinases and BAD with synergistic efficacy in triple negative breast cancer

Fig. 2

a Petasis reaction, a three component boronic Mannich-type reaction which utilizes boronic acids as a potential nucleophilic species, salicylaldehyde, and substituted piperazines to form the new C–C bond of the formula I compound, was utilized to synthesize NCK (C22H21Cl2N3O). b 3D surface and enlarged view of the docked compounds NPB (red) & NCK (black) with the BAD protein (dim grey). The yellow color indicates the site of the Serine 99 residue. c 2D structure representation of NCK interacting with BAD protein residues. d Sensorgrams obtained by SPR analysis of NCK with the BAD protein. BAD protein was immobilized on the surface of a CM5 sensor chip. A solution of NCK at variable concentrations (1.25–160 μM) was injected to generate the binding responses (RU) recorded as a function of time (s). The results were analyzed using BIA evaluation 4.1. e Western Blot analysis was used to assess the level of BAD phosphorylation at Ser99, Ser75 and Ser118 in TNBC cells after treatment with NCK and NPB. β-ACTIN was used as input control for cell lysate. The sizes of detected bands in kDa are shown on the left. f Dose-dependent effect of NCK and NPB in 2D and 3D culture on MDA-MB-231 and BT549 TNBC cells measured by using total cell number and AlamarBlue assay respectively (n = 3). g Western Blot analysis was used to assess the level of BAD phosphorylation at Ser99 in TNBC cells after transfection with siRNA-BAD. β-ACTIN was used as input control for cell lysate. The sizes of detected bands in kDa are shown on the left. h CASPASE3/7 activities of MDA-MB-231 cells after transfecting with siRNA targeting BAD transcript or scrambled control and treated with 5 μM NCK were evaluated using the Biovision Caspase 3/7 DEVD Assay Kit. Data represent means ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. i Cell survival of MDA-MB-231 cells after transfecting with siRNA targeting BAD transcript or scrambled control and treated with 5 μM NCK. Data represent means ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001.

Back to article page