Three optimized assays for the evaluation of compounds that can rescue p53 mutants

Abstract

Summary Identifying drugs targeting p53 remains a major focus of precision oncology, with over twenty compounds that can rescue p53 mutants reported. Here, we suggest three easily accessible assays to determine the thermostability, protein folding, and transcriptional activity of p53 mutants—the go-to criteria for evaluating a rescue compound that acts by increasing p53 thermostability. Because of the diversity of p53 mutants, a compound that meets the criteria of one assay does not necessarily meet the criteria of the other assays. For complete details on the use and execution of this protocol, please refer to Chen et al. (2021) .

Decitabine activates type I interferon signaling to inhibit p53‐deficient myeloid malignant cells

“…In our AML/MDS Sample Bank at the Shanghai Institute of Hematology, five BM and PB samples were recorded as p53‐mutated at prognosis (Figure 4A ), with the samples from patients #4 and #5 receiving DAC treatment but experiencing disease progression at the time of sample collection. We confirmed these p53 mutants were functionally deficient by the classical luciferase reporter assay 9 (Figure 4B ). The cryopreserved primary BM mononuclear cells (BMMC) or PBMCs were thawed and cultured for 3 days with or without DAC.…”

Diverse rescue potencies of p53 mutations to ATO are predetermined by intrinsic mutational properties

“…The transactivation activity of each p53 mutant on the representative PUMA (p53 up-regulated modulator of apoptosis) promoter was evaluated individually using the classic luciferase reporter assay (Fig. 1D and table S2) ( 22 ). Cell growth inhibitory activities and mouse tumor–suppressive activities were evaluated by the decrease in abundance of each variant in the U937 cell library in cultured cells and xenografted mice, respectively, as determined by next-generation sequencing (NGS) (Fig.…”

“…For example, F212I and R213G mutations respectively occur on the epitopes of PAb1620 ( 32 ) and PAb240 ( 33 ), which may lead to an underestimate of antibody reactivity. In addition, the LSH mutation V272M may have a disproportional impact on PAb1620 reactivity because of DBD topology between the PAb1620 epitope and LSH motif through strand 10 ( 13 , 22 ). The R248Q and R280I mutations, which affect established DNA-contacting residues, caused a decrease in PAb1620 reactivity and an increase in PAb240 reactivity compared with wild-type p53 and the R273H mutant (fig.…”

Assessment of Thermal Stability of Mutant p53 Proteins via Differential Scanning Fluorimetry

“…When the dye binds to the hydrophobic core, this eliminates fluorescence quenching, and the fluorescence quantum yield increases dramatically. The optical signal is measured at each point and the thermal denaturation curve is generated, which allows for calculation of the melting temperature of a protein globule and, therefore, estimation of the protein thermal stability [ 18 , 19 ].…”

“…The full-length p53 mutants are known to be thermodynamically unstable, prone to aggregation and have a lot of disordered N- and C-terminal parts [ 19 ]. The p53 core domain is key to protein stability, and mutations in this domain directly affect relative stability of the whole protein [ 20 ].…”