Targeting the Prion-like Aggregation of Mutant p53 to Combat Cancer

ConspectusPrion-like behavior of several amyloidogenic proteins has been demonstrated in recent years. Despite having functional roles in some cases, irregular aggregation can have devastating consequences. The most commonly known amyloid diseases are Alzheimer’s, Parkinson’s, and Transmissible Spongiform Encephalopathies (TSEs). The pathophysiology of prion-like diseases involves the structural transformation of wild-type (wt) proteins to transmissible forms that can convert healthy proteins, generating aggregates. The mutant form of tumor suppressor protein, p53, has recently been shown to exhibit prion-like properties. Within the context of p53 aggregation and the search for ways to avert it, this review emphasizes discoveries, approaches, and research from our laboratory and others.Although its standard functions are strongly connected to tumor suppression, p53 mutants and aggregates are involved in cancer progression. p53 aggregates are heterogeneous assemblies composed of amorphous aggregates, oligomers, and amyloid-like fibrils. Evidence of these structures in tumor tissues, the in vitro capability for p53 mutants to coaggregate with wt protein, and the detection of cell-to-cell transmission indicate that cancer has the basic characteristics of prion and prion-like diseases.Various approaches aim to restore p53 functions in cancer. Methods include the use of small-molecule and peptide stabilizers of mutant p53, zinc administration, gene therapy, alkylating and DNA intercalators, and blockage of p53–MDM2 interaction. A primary challenge in developing small-molecule inhibitors of p53 aggregation is the large number of p53 mutations. Another issue is the inability to recover p53 function by dissociating mature fibrils. Consequently, efforts have emerged to target the intermediate species of the aggregation reaction. Φ-value analysis has been used to characterize the kinetics of the early phases of p53 aggregation. Our experiments using high hydrostatic pressure (HHP) and chemical denaturants have helped to clarify excited conformers of p53 that are prone to aggregation. Molecular dynamics (MD) and phasor analysis of single Trp fluorescence signals point toward the presence of preamyloidogenic conformations of p53, which are not observed for p63 or p73.Exploring the features of competent preamyloidogenic states of wt and different p53 mutants may provide a framework for designing personalized drugs for the restoration of p53 function. Protection of backbone hydrogen bonds (BHBs) has been shown to be an important factor for the stability of amyloidogenic proteins and was employed to identify and stabilize the structural defect resulting from the p53 Y220C mutation. Using MD simulations, we compared BHB protection factors between p53 family members to determine the donor–acceptor pairs in p53 that exhibit lower protection. The identification of structurally vulnerable sites in p53 should provide new insights into rational designs that can rapidly be screened using our experimental methodology. Through continued and combined efforts, the outlook is positive for the development of strategies for regulating p53 amyloid transformation

Plot of experimental pIC₅₀ values vs. values calculated by QSAR equation (1).

<p>Blue rhombi: calibration data, red squares: leave one out cross validation predictions. Highlighted by ellipse: Data points for compounds <b>7</b> and <b>32</b> which are not well explained by the equation.</p

Antileishmanial and cytotoxicity effects of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones.

<p>na: not active; nd: not determined; IC₅₀: 50% inhibitory concentration; CC₅₀: 50% cytotoxic concentration; 95% C.I.: 95% confidence interval; S.I.: selectivity index (CC₅₀ mammalian cells/IC₅₀ amastigotes).</p><p>Antileishmanial and cytotoxicity effects of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones.</p

Insight into and Computational Studies of the Selective Synthesis of 6<i>H</i>‑Dibenzo[<i>b</i>,<i>h</i>]xanthenes

Starting from 2-hydroxy-1,4-naphthoquinone (lawsone), we synthesized eight new 6<i>H</i>-dibenzo­[<i>b</i>,<i>h</i>]­xanthene derivatives selectively under solvent-free conditions. Spectroscopic investigations confirmed that only the isomer 6<i>H</i>-dibenzo­[<i>b</i>,<i>h</i>]­xanthene was obtained in all eight cases. Computational studies provide a rationalization for the selective appearance of these isomers having as an intermediate an addition product

CYP450 Metabolism of a Semisynthetic Naphthoquinone, an Anticancer Drug Candidate, by Human Liver Microsomes

CNFD (6b,7-dihydro-5H-cyclopenta[b]naphtho[2,1-d]furan-5,6(9aH)-dione) is a semisynthetic naphthoquinone derived from lawsone that has cytotoxic action in different tumor lines and anticancer activity in vivo. Therefore, this molecule is a relevant candidate for drug development, but there is still no information on its human metabolism and systemic elimination. This study aimed to investigate the in vitro metabolism of this naphthoquinone by human liver microsomes. Initially, in order to determine the in vitro enzymatic kinetic parameters, a high performance liquid chromatography (HPLC) method to quantify the CNFD was developed and validated. In addition, the enzymatic kinetic data, the predicted pharmacokinetic in vivo parameters and the phenotyping study were presented. The main metabolism sites and metabolites have been suggested in silico. The developed HPLC method was linear, reproducible, selective, accurate, and stable. The enzymatic kinetic parameters revealed a sigmoidal profile. In vitro to in vivo extrapolation hepatic metabolic clearance was 10.39 mL min-1 kg-1 protein and the liver extraction rate was 51%. The clearance in vivo associated with a hepatic extraction ratio indicates that the hepatic metabolism is the main route of elimination. Although all cytochrome P450 enzymes evaluated metabolized CNFD, CYP2C9 and CYP3A4 showed higher metabolic capacity. For the first time, metabolism studies of CNFD were demonstrated.</div

New Families of Hetero-tri-spin 2p−3d−4f Complexes: Synthesis, Crystal Structures, and Magnetic Properties

In this work we report the synthesis, crystal structures, and magnetic behavior of 2p–3d–4f heterospin systems containing the nitroxide radical 4-azido-2,2,6,6-tetramethylpiperidine-1-oxyl radical (N₃tempo). These compounds were synthesized through a one-pot reaction by using [Cu­(hfac)₂], [Ln­(hfac)₃] (hfac = hexafluoroacetylacetonate, Ln = Dy^III, Tb^III or Gd^III), and the N₃tempo radical. Depending on the stoichiometric ratio used, the synthesis leads to penta- or trimetallic compounds, with molecular formulas [Cu₃Ln₂(hfac)₈­(OH)₄(N₃tempo)] (Ln = Gd, Tb, Dy) and [CuLn₂(hfac)₈­(N₃tempo)₂(H₂O)₂] (Ln = Gd, Dy). The magnetic properties of all compounds were investigated by direct current (dc) and alternating current (ac) measurements. The ac magnetic susceptibility measurements of Tb^III- and Dy^III-containing compounds of both families revealed slow relaxation of the magnetization, with magnetic quantum tunneling in zero field

Inhibition of the ATPase activity of full-length NS3 by the compounds 9b and 9c.

<p>Increasing concentrations of naphthoquinones 9b and 9c were tested for their activity against ATPase site. The reaction was performed in buffer containing 40 mM Tris-HCl (pH 7.5), 5 mM DTT, 100 mM KCl, and 5 mM MgCl₂. The compounds were pre-incubated with 600nM NS3FL for 10 min at 30 °C followed by the addition of 1 mM ATP. Each point represents the average of three independent replicates in different concentrations of the compounds.</p

Inhibition of the ATPase activity of purified NS3 by naphthoquinones.

<p>(A) NaphSDS-Page analysis of the purification full-length NS3 (NS3FL) from <i>E coli</i>. Lane 1, molecular weight marker; lane 2, 10xHis-NS3FL (10xHis-Ubiquitin site) after refolding by dialysis; lane 3 and 4, NS3FL after 10xHis-Ubiquitin site tag excision by YUH in ratio 1:5 and 1:1, respectively; and lane 5, purified NS3FL protein following His-TRAP column chromatography. The gel was stained with Comassie brilhant blue G. (B) A range of pyran naphthoquinones was tested for their activity against ATPase site. The screening was performed in buffer containing 40 mM Tris-HCl (pH 7.5), 5 mM DTT, 100 mM KCl, and 5 mM MgCl₂. The compounds were pre-incubated with 600nM NS3FL for 10 min at 30 °C followed by the addition of 1 mM ATP. The screening was carried out in duplicate and the unpaired t-test was used for the statistical analysis. Those compounds showing statistical difference from the control (p < 0.05) were labeled with an asterisk.</p

Insight into and Computational Studies of the Selective Synthesis of 6<i>H</i>‑Dibenzo[<i>b</i>,<i>h</i>]xanthenes

Starting from 2-hydroxy-1,4-naphthoquinone (lawsone), we synthesized eight new 6<i>H</i>-dibenzo­[<i>b</i>,<i>h</i>]­xanthene derivatives selectively under solvent-free conditions. Spectroscopic investigations confirmed that only the isomer 6<i>H</i>-dibenzo­[<i>b</i>,<i>h</i>]­xanthene was obtained in all eight cases. Computational studies provide a rationalization for the selective appearance of these isomers having as an intermediate an addition product

Dose-response curves and citotoxicity of 1,4-pyran naphthoquinones 9b and 9c in DENV-2-infected Vero cells.

<p>Vero cells were infected with DENV-2 at an m.o.i of 0.1 in the presence of increasing concentrations of the 1,4-pyran naphthoquinones 9b and 9c. 72 hours after infection the cell-free supernatants were harvested and processed for the quantification of produced viral progeny by qPCR. (A) The percentage of inhibition was calculated using the ΔΔCT values. (B) The concentration of each compound that inhibit 50% of the viral replication (IC₅₀) was determined by a Hill’s regression curve after logarithmic interpolation of the data using the GraphPad 6 software. (C) The potential cytotoxic effect of each compound was evaluated in uninfected cells by the vital neutral red staying and represented as percentage of the untreated control. Data represent mean values ± standard deviations (SD) for three independent experiments.</p