Surface plasmon resonance and cytotoxicity assays of drug efficacies predicted computationally to inhibit p53/MDM2 interaction.

Docking on the p53-binding site of murine double minute 2 (MDM2) by small molecules restores p53's tumor-suppressor function. We previously assessed 3244 FDA-approved drugs via "computational conformer selection" for inhibiting MDM2 and p53 interaction. Here, we developed a surface plasmon resonance method to experimentally confirm the inhibitory effects of the known MDM2 inhibitor, nutlin-3a, and two drug candidates predicted by our computational method. This p53/MDM2 interaction displayed a dosage-dependent weakening when MDM2 is pre-mixed with drug candidates. The inhibition efficiency order is nutlin-3a (IC50 = 97 nM) > bepridil (206 nM) > azelastine (307 nM). Furthermore, we verified their anti-proliferation effects on SJSA-1 (wild-type p53 and overexpressed MDM2), SW480 (mutated p53), and SaOs-2 (deleted p53) cancer cell lines. The inhibitory order towards SJSA-1 cell line is nutlin-3a (IC50 = 0.8 µM) > bepridil (23 µM) > azelastine (25 µM). Our experimental results are in line with the computational prediction, and the higher IC50 values from the cell-based assays are due to the requirement of higher drug concentrations to penetrate cell membranes. The anti-proliferation effects of bepridil and azelastine on the cell lines with mutated and deleted p53 implied some p53-independent anti-proliferation effects.

t-Darpp is an elongated monomer that binds calcium and is phosphorylated by cyclin-dependent kinases 1 and 5.

t-Darpp (truncated isoform of dopamine- and cAMP-regulated phosphoprotein) is a protein encoded by the PPP1R1B gene and is expressed in breast, colon, esophageal, gastric, and prostate cancers, as well as in normal adult brain striatal cells. Overexpression of t-Darpp in cultured cells leads to increased protein kinase A activity and increased phosphorylation of AKT (protein kinase B). In HER2+ breast cancer cells, t-Darpp confers resistance to the chemotherapeutic agent trastuzumab. To shed light on t-Darpp function, we studied its secondary structure, oligomerization status, metal-binding properties, and phosphorylation by cyclin-dependent kinases 1 and 5. t-Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn, and 35% random coil structures. It binds calcium, but not other metals commonly found in biological systems. The T39 site, critical for t-Darpp activation of the AKT signaling pathway, is a substrate for phosphorylation by cyclin-dependent kinase 1 and cyclin-dependent kinase 5. Gel filtration chromatography, sedimentation equilibrium analysis, blue native gel electrophoresis, and glutaraldehyde-mediated cross-linking experiments demonstrate that the majority of t-Darpp exists as a monomer, but forms low levels (< 3%) of hetero-oligomers with its longer isoform Darpp-32. t-Darpp has a large Stokes radius of 4.4 nm relative to its mass of 19 kDa, indicating that it has an elongated structure.