The molecular mechanism of action of methylene quinuclidinone and its effects on the structure of p53 mutants

Abstract

One of the most important tumor suppressor proteins in eukaryotic cells is the transcription factor called p53. The importance of this protein in cells comes from the fact that it regulates a wide variety of cellular processes including the cell cycle, metabolism, DNA repair, senescence and apoptosis. In cancer cells, p53 is a major target as the most mutated protein, which has led to the search for potential activators of the mutant protein. Currently, the only mutated-p53 activator in clinical trials is a small molecule called APR-246. There is evidence that the active metabolite of APR-246 binds covalently to mutant p53 and restores its wild-type (wt) activity. In this work, we created atomistic in silico models of the wt, mutant and drugged mutant p53 proteins each in complex with DNA. Using molecular dynamics simulations we generated equilibrated models of the complexes. Detailed analysis revealed that the binding of the APR-246 active metabolite to the mutant proteins alters their interaction with DNA. In particular, the binding of the molecule at loop L1 of the protein allows the loop to anchor the protein to DNA similarly to wt p53. Several important p53-DNA interactions lost due to mutation were also restored in the drugged mutants. These findings, not only provide a possible mechanism of action of this drug, but also criteria to use in virtual screening campaigns for other p53 activators.

Role of Reactivating Mutant p53 Protein in Suppressing Growth and Metastasis of Triple-Negative Breast Cancer

“… 6 , 7 , 11 Much of the functionality of APR-246 can be attributed to its ability to reactivate mtp53 protein by modifying the DNA-binding domain, promoting the capacity of reconfigured p53 to arrest the cell cycle and induce apoptosis. 6 , 14 , 15 It is believed that alkylation of these cysteine residues may prevent aggregation caused by the thiol groups, possibly increasing the fraction of the protein able to bind to DNA and regulate gene transcription. 7 …”

“… 6 , 19 Although Cys124 is not a DNA binding residue, this cysteine residue is responsible for interactions with DNA via Loop 1, anchoring p53 to DNA. 11 , 14 In addition to Cys124, Cys277 has been identified as a binding target that is essential to MQ-mediated thermostabilization of the wtp53 protein core domain. 12 …”

Tumor-Associated Antigen xCT and Mutant-p53 as Molecular Targets for New Combinatorial Antitumor Strategies

“…Indeed, the antitumor activity of APR-246 has mainly been described as relating to mut-p53 reactivation due to the covalent binding of MQ to thiol groups on cysteine 124 and 277 residues in the core domain of mut-p53. This drives a conformational change resulting in the reactivation of p53 pro-apoptotic functions [ 107 ]. The administration of these drugs results in the refolding of p53 into the wild-type conformation and the activation of p53-downstream pathways.…”

Ascorbate Inhibits Proliferation and Promotes Myeloid Differentiation in TP53-Mutant Leukemia

“…Prima-1 Met is a pro-drug that is converted to methylene quinuclidinone (MQ), which can act as a Michael acceptor, rendering it susceptible to nucleophilic attack from the sulfhydryl moiety of cysteine residues. MQ has been shown to bind Cys124 and Cys277 in p53 to promote the reactivation of R175H and R273H p53 mutants by stabilizing the protein and shifting the equilibrium in favor of an active conformation ( 58 , 59 ). In addition to p53-dependent effects, MQ targets the cellular redox balance via binding to glutathione and thioredoxin reductase, causing the depletion of glutathione and the inhibition of thioredoxin reductase ( 30 , 41 ).…”