The Fluorescent Two-Hybrid Assay to Screen for Protein–Protein Interaction Inhibitors in Live Cells: Targeting the Interaction of p53 with Mdm2 and Mdm4

Abstract

Protein–protein interactions (PPIs) are attractive but challenging targets for drug discovery. To overcome numerous limitations of the currently available cell-based PPI assays, we have recently established a fully reversible microscopy-assisted fluorescent two-hybrid (F2H) assay. The F2H assay offers a fast and straightforward readout: an interaction-dependent co-localization of two distinguishable fluorescent signals at a defined spot in the nucleus of mammalian cells. We developed two reversible F2H assays for the interactions between the tumor suppressor p53 and its negative regulators, Mdm2 and Mdm4. We then performed a pilot F2H screen with a subset of compounds, including small molecules (such as Nutlin-3) and stapled peptides. We identified five cell-penetrating compounds as potent p53–Mdm2 inhibitors. However, none exhibited intracellular activity on p53–Mdm4. Live cell data generated by the F2H assays enable the characterization of stapled peptides based on their ability to penetrate cells and disrupt p53–Mdm2 interaction as well as p53–Mdm4 interaction. Here, we show that the F2H assays enable side-by-side analysis of substances’ dual Mdm2–Mdm4 activity. In addition, they are suitable for testing various types of compounds (e.g., small molecules and peptidic inhibitors) and concurrently provide initial data on cellular toxicity. Furthermore, F2H assays readily allow real-time visualization of PPI dynamics in living cells.

Genetic visualization of protein interactions harnessing liquid phase transitions

“…3b). The IC50 value was 5.7 ± 0.6 μM (mean ± SD, n = 3), consistent with that determined by previous cell-based assays8. Similarly, the EC50 value of rapamycin for FRB-AG/PB1-FKBP was calculated to be 15.1 ± 1.7 nM (Supplementary Fig.…”

“…These methods are gaining popularity in the area of high-content analysis (HCA) because they are simple and compatible with recent advances in hardware and software8. However, the signal occurrence on a chromatin site, for example, is not sufficiently distinct to allow easy detection.…”

Avoiding drug resistance through extended drug target interfaces: a case for stapled peptides

“…Interaction of HDM2-C8 and the L34P, F55L, Y60C point mutants with p53 was further studied using the fluorescent two-hybrid assay (F2H) [54]. This assay facilitates real-time detection of p53-HDM2 interaction in living cells and perturbation by small molecule/peptidic antagonists.…”

Macrocyclic α-Helical Peptide Drug Discovery

“…Another example includes the design of p53 and structurally related phage display-derived stapled helical peptides having high binding affinity to both MDM2 and MDMX that has been successfully achieved. 42,57,58,65,66,72,79,81,82,87,94,95 In the case of BID BH3 stapled peptides, proof-of-concept studies demonstrating in vivo efficacy in a human leukemia xenograft model (10 mg kg À1 dosing intravenously qd for seven days) were achieved, subsequent to an iterative process to identify a molecule having enhanced proteolytic and serum stability, high binding affinity to anti-apoptotic protein Bcl-2, cell penetration and mitochondrial co-localization, and cytochrome C-driven cellular apoptosis. 43 Recent studies have further shown the successful development of BIM BH3 and BID BH3 stapled peptides which are capable of directly activating the pro-apoptotic protein BAX to affect apoptosis.…”

“…[19][20][21][22][23][24][27][28][29] and 62 for reviews) for the modulation a plethora of intracellular targets (i.e. Bcl2-family, 19,[22][23][24]43,47,48,58,[67][68][69][70][71]76,77,83,92,93,98 p53:MDM2/X, 42,57,65,66,72,79,81,82,87,89,94,95 MAML:Notch, 49 eIF4E, 158 ERa/ERb, 50 IRS1, 84 HIF-1:p300, 56,90 RAS:SOS, 59 Rab-GTPase, 96 b-catenin, 64,78,80 protein kinase-A, 91 RPA, 97 HIV-1 integrase, …”