Discovery of a small-molecule inhibitor of specific serine residue BAD phosphorylation

Abstract

Human BCL-2–associated death promoter (hBAD) is an apoptosis-regulatory protein mediating survival signals to carcinoma cells upon phosphorylation of Ser99, among other residues. Herein, we screened multiple small-molecule databases queried in a Laplacian-modified naive Bayesian-based cheminformatics platform and identified a Petasis reaction product as a site-specific inhibitor for hBAD phosphorylation. Based on apoptotic efficacy against mammary carcinoma cells, N-cyclopentyl-3-((4-(2,3-dichlorophenyl) piperazin-1-yl) (2-hydroxyphenyl) methyl) benzamide (NPB) was identified as a potential lead compound. In vitro biochemical analyses demonstrated that NPB inhibited the phosphorylation of hBAD specifically on Ser99. NPB was observed to exert this effect independently of AKT and other kinase activities despite the demonstration of AKT-mediated BAD-Ser99 phosphorylation. Using a structure-based bioinformatics platform, we observed that NPB exhibited predicted interactions with hBAD in silico and verified the same by direct binding kinetics. NPB reduced phosphorylation of BAD-Ser99 and enhanced caspase 3/7 activity with associated loss of cell viability in various human cancer cell lines derived from mammary, endometrial, ovarian, hepatocellular, colon, prostatic, and pancreatic carcinoma. Furthermore, by use of a xenograft model, it was observed that NPB, as a single agent, markedly diminished BAD phosphorylation in tumor tissue and significantly inhibited tumor growth. Similar doses of NPB utilized in acute toxicity studies in mice did not exhibit significant effects. Hence, we report a site-specific inhibitor of BAD phosphorylation with efficacy in tumor models.

Piperlongumine Inhibits Akt Phosphorylation to Reverse Resistance to Cisplatin in Human Non-Small Cell Lung Cancer Cells via ROS Regulation

“…These results suggest that PL might promote drug efflux through the Akt/FoxO3/NRF2/P-gp signaling pathway. Akt suppresses apoptosis by phosphorylating its substrate BAD at Ser-75 and Ser-99 (Pandey et al, 2018). To further characterize the mechanisms underlying PL-induced apoptosis, Ser-75 and Ser-99 phosphorylation levels on BAD were examined ( Figures 3A, B ) and found to be significantly decreased after PL treatment in A549/Cis cells.…”

The IκB Kinase Inhibitor ACHP Targets the STAT3 Signaling Pathway in Human Non-Small Cell Lung Carcinoma Cells

“…The protein concentration was estimated in cell lysates and equal concentrations of proteins were resolved on 8-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by their transfer to a nitrocellulose membrane as reported earlier [44][45][46]. The membranes were treated with 5% skim milk and incubated with the desired antibodies at 4 • C overnight.…”

“…The apoptosis-inducing effect of ACHP on A549 and H1299 cells was evaluated using an Annexin assay as described previously [46,50]. The A549 and H1299 cells were exposed to ACHP (10 µM) for indicated time points and collected using trypsin (1%) in phosphate-buffered saline, followed by giving a single wash with cold phosphate-buffered saline.…”

Current Advance of Therapeutic Agents in Clinical Trials Potentially Targeting Tumor Plasticity

“…In addition, abnormal expression of protein kinase B (PKB/Akt) is related to many cancers (97). GSK-690693 (83), ARQ-751 (84), and TAS-117 (85) that can effectively treat solid tumors through inhibiting PKB/Akt are being evaluated in phase I and II clinical studies. Ipatasertib has been combined with other antitumor drugs to treat prostate cancer and breast cancer and is undergoing an investigation in a phase III clinical trial (86).…”