Ranking the Binding Energies of p53 Mutant Activators and Their ADMET Properties.

The guardian of the genome, p53, is the most mutated protein found in all cancer cells. Restoration of wild-type activity to mutant p53 offers promise to eradicate cancer cells using novel pharmacological agents. Several molecules have already been found to activate mutant p53. While the exact mechanism of action of these compounds has not been fully understood, a transiently open pocket has been identified in some mutants. In our study, we docked twelve known activators to p53 into the open pocket to further understand their mechanism of action and rank the best binders. In addition, we predicted the absorption, distribution, metabolism, excretion and toxicity properties of these compounds to assess their pharmaceutical usefulness. Our studies showed that alkylating ligands do not all bind at the same position, probably due to their varying sizes. In addition, we found that non-alkylating ligands are capable of binding at the same pocket and directly interacting with Cys124. The comparison of the different ligands demonstrates that stictic acid has a great potential as a p53 activator in terms of less adverse effects although it has poorer pharmacokinetic properties.

Degradation of 2-(3-aminopropylamino)-ethanethiol (WR-1065) by Cu-dependent amine oxidases and influence on glutathione status of Chinese hamster ovary cells.

The radioprotective drug 2-(3-aminopropylamino)ethanethiol (WR-1065) can be degraded when incubated in cell culture medium in vitro. The degradation reaction consumes oxygen and results from the action of Cu-dependent amine oxidases present in the serum content of the medium. Analysis of the degradation products of WR-1065 demonstrates the formation of cysteamine, acrolein and H2O2. WR-2721, the inactive prodrug of WR-1065, is not a substrate for these enzymes. Extracellular degradation of WR-1065 by Cu-dependent amine oxidases leads to an intracellular depletion of glutathione (GSH) in Chinese hamster ovary (CHO) cells and to reduction of clonogenic cell survival. Addition of aminoguanidine, an inhibitor of Cu-dependent amine oxidases, protects CHO cells from the toxic effects of WR-1065 and under these conditions an increase of intracellular GSH levels occurs. These data demonstrate that WR-1065 can be degraded to toxic compounds by the presence of Cu-dependent amine oxidases which might have further implications for the clinical use of WR-2721.

Mechanisms for the oxygen radical-mediated toxicity of various thiol-containing compounds in cultured mammalian cells.

When Chinese hamster V79 cells are exposed to various thiol compounds in phosphate-buffered saline (PBS), some compounds cause toxicity (loss of colony formation), although the dependence on drug concentration and the magnitude of the cell killing vary between the different thiols. For example: dithiothreitol (DTT) and WR-1065 cause a biphasic toxicity whereby cell killing occurs at about 0.2 to 1.0 mM thiol, but is not seen at higher or lower drug concentrations; N-acetylcysteine (NAC) is toxic only at concentrations > or = 2 mM and shows no biphasic pattern; and glutathione (GSH) and penicillamine are only minimally toxic at all concentrations. The effect of the addition of 1 microM Cu2+ to the thiol also depends on the particular thiol: e.g., Cu2+ increases cell killing in the biphasic pattern with WR-1065; it increases the toxicity of NAC only at high thiol concentrations; and it elicits a slight toxicity in the biphasic pattern by GSH and penicillamine. In all cases tested, if the thiol is toxic, the cell killing can be decreased or prevented by addition of catalase, consistent with the hypothesis that the toxicity is mediated through H2O2 produced during the thiol oxidation. However, when the oxidation rates of the various thiols in PBS without and with Cu2+ were measured, the data did not show a simple correlation between the toxicity of the various thiols and their oxidation rates. The rate of the reaction of the various thiols with H2O2 was also determined and showed a better, but still not good, correlation with toxicity. However, cell killing by the various thiols correlated better with the ratio between the half-lives for thiol oxidation and reaction of thiol with H2O2 than with either reaction rate alone. This suggests that the toxicity pattern and magnitude of cell killing in V79 cells by various thiols depend on the interplay between the rate of thiol oxidation and the rate of reaction between the thiol and the H2O2 produced in the thiol oxidation.

Protection against radiation-induced mutagenesis at the hprt locus by spermine and N,N"-(dithiodi-2,1-ethanediyl)bis-1,3-propanediamine (WR-33278).

The polyamine spermine and the disulfide N,N"-(dithiodi-2,1-ethanediyl)bis-1,3-propanediamine (WR-33278) are structurally similar agents capable of binding to DNA. WR-33278 is the disulfide moiety of the clinically studied radioprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). Because of their reported structural and functional similarities, it was of interest to compare their effects on cell survival and mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in Chinese hamster AA8 cells. WR-33278 and spermine (at concentrations of 0.01 and 0.001 mM) were electroporated into cells. Electroporation, 300 V and 125 microF, was performed either 30 min prior to or 3 h following exposure of cells to 750 cGy of ionizing radiation. Electroporation alone reduced cell survival to 75% but had no effect on hprt mutation frequency. The electroporation of either spermine or WR-33278 at concentrations greater than 0.01 mM was extremely toxic. The exposure of cells to both electroporation and irradiation gave rise to enhanced cell killing and mutation induction, with the sequence of irradiation followed 3 h later by electroporation being the more toxic protocol. Cell survival was only enhanced following electroporation of 0.01 mM of spermine and WR-33278 30 min prior to irradiation. Protection against radiation-induced hprt mutations was observed for both spermine and WR-33278 under all experimental conditions tested. Spermine at exposure concentrations of 0.01 and 0.001 mM administered 30 min before or 3 h after irradiation reduced mutation frequencies by factors of 2.2, 1.2, 1.9 and 2.2, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

[Decrease of toxic effects of aminothiol radiation-protective agents and increase of chemical protection action against ionizing radiation by the use of unithiol]. / Snizhenie toksicheskogo deistviia aminotiolovykh radioprotektorov i povyshenie éffekta khimicheskoi zashchity ot ioniziruiushchei radiatsii s pomoshchiu unitiola.

It has been shown that unithiol diminishes toxic action of cysteamine, AET, and disulfide of WR-1065 on mice. This permits to enhance protection of animals against X-rays by increasing of protector doses. The effect of unithiol on cysteamine action in rats was the same. Antitoxic effect of unithiol on cysteamine was shown both at i.p. and p.o. protector administration. The effect was also revealed in Chinese hamster V-79 cell culture. Combined disulfide of cysteamine and unithiol was synthetized, which ensures effective prolonged protection against ionizing radiation.

Potentiation of bleomycin by the aminothiol WR-1065 in assays for chromosomal damage in G0 human lymphocytes.

The aminothiol radioprotector WR-1065 potentiates the induction of chromosome aberrations and micronuclei by the chemotherapy drug bleomycin in G(0) human lymphocytes. Potentiation by 5 mM WR-1065 was observed at bleomycin concentrations from 0.1 to 100 micrograms/ml in a 2-h treatment. The frequencies of micronuclei induced by bleomycin in the presence of WR-1065 reached that of 500-fold higher concentrations of bleomycin alone. The potential therapeutic implications of these findings are discussed.

Comparative intestinal and testes toxicity of four aminothiols in irradiated and nonirradiated mice.

Intestinal and testicular toxicity in groups of nonirradiated and irradiated mice were investigated after intraperitoneal injection of aminothiol compounds or saline. Four aminothiols were studied. Three were prodrugs: WR-2721, WR-3689, and WR-151327 and one was the active form of WR-2721: WR-1065. Thirty minutes after injection, the mice were sham-irradiated or bilaterally exposed (whole body) to 60Co gamma-irradiation at a dose rate of 1 Gy per min to a total dose of 15 Gy. Four days after injection, mice were euthanised, and the intestines and testes were removed and histologically examined. The intestinal crypt cell number was increased in all the irradiated mice given WR-compounds compared to controls (P < 0.05). Interestingly, the crypt cell number in nonirradiated mice given WR-1065 was also greater than control or WR-2721 (P < 0.05) treated mice. Germinal cell numbers from testes of mice administered aminothiols prior to radiation decreased or did not change. Some swelling of the seminiferous tubules was also observed. The germinal cell numbers in sham-irradiated mice were also less than the controls. Thus, aminothiol addition can provide limited protection to intestinal crypt cells but not to germinal cells of the testes in response to gamma-irradiation. There is also evidence that aminothiols are toxic to the germinal cell layer of the seminiferous tubules when given to sham-irradiated mice.

Effect of thiols on micronucleus frequency in gamma-irradiated mammalian cells.

The effects of the thiols cysteamine, WR-1065, and WR-255591 on radiation-induced micronucleus (MN) frequency and cell killing were compared in cultured Chinese hamster ovary cells. MN were measured using the cytochalasin B assay of Fenech and Morley (1985), which minimizes the effect of cytokinetic perturbations on MN expression. The dose-response curves for MN induction were curvilinear both for control cells at doses between 1.5 and 4.5 Gy and for thiol-treated cells at doses between 3 and 9 Gy. Protection against MN induction by each thiol was independent of radiation dose. Furthermore, there was a close correlation between the degree of modification of MN induction and cell survival by each thiol, i.e., the MN frequency closely predicted the survival level regardless of the presence of absence of the thiols. A similar predictive relationship has also been reported by us for cell survival and DNA double-strand break (DSB) induction in this cell line following treatment with these same thiols. Collectively, these data support the hypothesis that, for DNA-repair-proficient mammalian cells treated with radiomodifying agents that do not alter DNA-repair processes, MN and DSB induction are predictive of the level of radiation lethality and of each other.

S-[2-[(2'-carbamoylethyl)amino]ethyl] phosphorothioate and related compounds as potential antiradiation agents.

A reinvestigation of the radiation protection activity of S-[2-[(2'-carbamylethyl)amino]ethyl] lithium hydrogen phosphorothioate (4a) revealed that this compound possessed good (70% protection at a dose of 600 mg/kg) activity. The thione and imino bioisosteres of 4, S-[2-(2'-thiocarbamylethylamino)ethyl] lithium hydrogen phosphorothioate (13a) and S-[2-(2'-amidinoethylamino)ethyl] phosphorothioic acid (18b) showed 100% protection at doses of 300 and 150 mg/kg, respectively. The N-methyl (4b) and tert-butyl (4c) analogues of amide 4a, the N-methyl (13b) analogue of the thioamide 13a, the N-methyl (18a) analogue of amidine (18b), and the cyclic amidine S-[2-[[2'-(4,5-dihydroimidozoyl)ethyl]amino]ethyl] lithium hydrogen phosphorothioate (21) all showed 80% protection at the highest dose tested.