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. 2018 May 1;9(5):439.
doi: 10.1038/s41419-018-0463-7.

APR-246 reactivates mutant p53 by targeting cysteines 124 and 277

Qiang Zhang  1 , Vladimir J N Bykov  1 , Klas G Wiman  2 , Joanna Zawacka-Pankau  3
Affiliations

APR-246 reactivates mutant p53 by targeting cysteines 124 and 277

Qiang Zhang et al. Cell Death Dis. .

"V体育安卓版" Erratum in

Abstract

The TP53 tumor suppressor gene is frequently inactivated in human tumors by missense mutations in the DNA binding domain. TP53 mutations lead to protein unfolding, decreased thermostability and loss of DNA binding and transcription factor function. Pharmacological targeting of mutant p53 to restore its tumor suppressor function is a promising strategy for cancer therapy. The mutant p53 reactivating compound APR-246 (PRIMA-1Met) has been successfully tested in a phase I/IIa clinical trial. APR-246 is converted to the reactive electrophile methylene quinuclidinone (MQ), which binds covalently to p53 core domain. We identified cysteine 277 as a prime binding target for MQ in p53. Cys277 is also essential for MQ-mediated thermostabilization of wild-type, R175H and R273H mutant p53, while both Cys124 and Cys277 are required for APR-246-mediated functional restoration of R175H mutant p53 in living tumor cells. These findings may open opportunities for rational design of novel mutant p53-targeting compounds VSports手机版. .

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Conflict of interest statement

K. G. W. and V. J. N. B. are co-founders and shareholders of Aprea Therapeutics AB, a company that develops p53-based cancer therapy including APR-246. K V体育安卓版. G. W. is a member of its Clinical Advisory Board. Research in the K. G. W. lab has received financial support from Aprea Therapeutics AB. K. G. W. has received a salary from Aprea Therapeutics AB.

Figures

Fig. 1
Fig. 1. MQ binds to cysteine residues in wild-type and mutant p53 core domains in a dose-dependent manner.
Mass measurement of wild-type, R273H and R175H p53 core domains by LTQ-MS. a mass spectra of p53 core domains. b–d Reaction titration with MQ or MQ-H. p53 core domains were incubated with MQ at 50–200 µM (wt and R273H) or 10–50 µM (R175H) concentration ranges. One MQ adduct increased the molecular mass of p53 core domains by 137 Da. e Structure of MQ and MQ-H.
Fig. 2
Fig. 2. MQ modification of cysteine residues enhances p53 core domain thermostability.
Melting curves of wt and R273H and R175H mutant p53 core domains as assessed by DSF (a) and CD at 218 nm (b). Changes in Tm after MQ or MQ-H modification as assessed by DSF (c) and CD at 218 nm (d) (mean ± SD, n = 3). All proteins were thermostabilized by MQ modification (red bars), but not by MQ-H (brown bars). * p < 0.05 (student t-test).
Fig. 3
Fig. 3. Cys277 is a prime binding site for MQ in the p53 core domain.
Mass measurement of p53 core domains by LTQ-MS. a–c Indicated p53 core domain proteins were incubated with MQ at increasing concentrations and mass shift was assessed. Proportion of MQ-modified proteins was calculated based on relative intensity of each detected mass. d Modification of Cys124 and Cys275/277 in the R273H mutant p53 core domain after treatment with 50 µM MQ as assessed by LC-MS after tryptic digestion of MQ-treated protein (mean ± SE, n = 3).
Fig. 4
Fig. 4. Cys277 is critical for MQ-mediated p53 thermostabilization.
C277A substitution completely abolished MQ-mediated thermostabilization whereas other substitutions had little or no effect on wt a, R273H b and R175H c core domains at 2 mM concentration. Higher concentrations of MQ (1, 2 or 4 mM) did not thermostabilize the indicated p53 core domain proteins with C277A substitution d.
Fig. 5
Fig. 5. APR-246 and MQ enhance wild-type p53 conformation-specific PAb1620 epitope in tumor cells carrying R175H mutant p53.
a Immunofluorescence staining of TOV-112D cells treated with APR-246, MQ or MQ-H using wild-type p53 conformation-specific antibody PAb1620 and co-immunostaining with general p53 antibody FL-393. b Immunofluorescence staining of TOV-112D cells treated with APR-246, MQ or MQ-H using the mutant p53 conformation-specific antibody HO3.5 and co-immunostaining with general p53 antibody FL-393. c Quantification of PAb1620 immunostaining. d Quantification of HO3.5 immunostaining. PAb1620 or HO3.5-positive cells were counted in 15 fields from 3 independent experiments. The number of PAb1620 or HO3.5-positive cells was divided by the total cell number to obtain percentage of positive cells.
Fig. 6
Fig. 6. Cys124 and Cys277 are crucial for APR-246/MQ-mediated R175H reactivation in living cells.
H1299 cells expressing corresponding p53 mutant proteins were stained with Annexin V, p21, Fas and Bax and examined by flow cytometry. Both C124A and C277A abolished APR-246-induced apoptosis a, and upregulation of p53 targets p21 b, Bax c and Fas d
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