Identification of a Novel p53 Modulator Endowed with Antitumoural and Antibacterial Activity through a Scaffold Repurposing Approach

Abstract

Intracellular pathogens, such as Chlamydia trachomatis, have been recently shown to induce degradation of p53 during infection, thus impairing the protective response of the host cells. Therefore, p53 reactivation by disruption of the p53-MDM2 complex could reduce infection and restore pro-apoptotic effect of p53. Here, we report the identification of a novel MDM2 inhibitor with potential antitumoural and antibacterial activity able to reactivate p53. A virtual screening was performed on an in-house chemical library, previously synthesised for other targets, and led to the identification of a hit compound with a benzo[a]dihydrocarbazole structure, RM37. This compound induced p53 up-regulation in U343MG glioblastoma cells by blocking MDM2-p53 interaction and reduced tumour cell growth. NMR studies confirmed its ability to dissociate the MDM2-p53 complex. Notably, RM37 reduced Chlamydia infection in HeLa cells in a concentration-dependent manner and ameliorated the inflammatory status associated with infection.

Keywords: Chlamydia trachomatis; antibacterial activity; glioblastoma multiforme; p53 modulator; p53–MDM2 complex; virtual screening.

Figure 1

On the top and bottom of the chart, chemical structures of five known potent MDM2 inhibitors are shown. Chemical features shared among these inhibitors and some of database molecules, e.g., carbazoles (in the centre) are highlighted in red lines.

Figure 2

Binding modes obtained by docking results of compounds RM58 (a) and RM37 (b) into MDM2 binding site. Protein is shown as cyano surface, whereas RM58 and RM37 are shown as magenta and golden sticks, respectively. Leu26, Trp23, and Phe19 subpockets are shown with green, yellow, and pink dashed lines, respectively.

Figure 3

One-dimensional proton spectrum of side chains of tryptophans (W) of p53–MDM2 complex (a), p53–MDM2 complex after addition of RM37 (b), or Nutlin-3a (c).

Figure 4

Effects of RM37 and RM58 on U343MG glioblastoma cells. U343MG cells were treated with different concentrations of RM37 and RM58 for 48h. The number of viable cells (a) and percentage of dead cells (b) were reported. Significance of the differences was determined with a one-way ANOVA with Bonferroni post-test * p < 0.05; *** p < 0.001 versus CTRL. (c) RM37 was incubated with GBM cell lysates containing the native MDM2–p53 complex, and levels of MDM2–p53 complex were quantified using HRP-conjugated antibody and TMB substrate. Data represent mean ± SEM of three independent experiments.

Figure 5

p53 protein accumulation in U343MG cells. U343MG cells were challenged with DMSO (control) or 20 µM RM37 for 4h, 8h, or 10h. Lysates were subjected to Western blot analysis using an antibody specific for p53. One representative Western blot is shown (a). Full screen of Western blot assay was provided in a separate file. Quantitative analysis was performed using ImageJ and shown in panel (b). Data are expressed as fold of optical density (OD) of the immunoreactive band relative to that of control, set to 1, and are mean values ± SEM of three different experiments. ** p < 0.01, *** p < 0.001 versus control.

Figure 6

Degree of interaction of p53 with MDM2 in GBM cells. (a) U343MG cells were treated with 20 µM RM37 for 6 or 16h. Relative quantification of p53 mRNA was performed by real-time RT-PCR. Data are expressed as fold of change versus control cells, set to 1. (b,c) U343MG cells were treated with 20 µM RM37 for 8h. After incubation, immunoprecipitation assay was performed. Representative immunoblot was shown. (b) Full screen of Western blot assay was provided in separate file. Data are expressed as fold of optical density (OD) of immunoreactive band relative to that of control, set to 1, and are mean values ± SEM of three different experiments. *** p < 0.01 versus control.

Figure 7

p53 functional reactivation in U343MG cells. (a,b) U343MG cells were challenged with 20 µM RM37 or 10 µM Nutlin-3a for 24 h. Relative mRNA quantification of p53 target genes (Bax, p21, and MDM2) was performed by real-time RT-PCR, as described in the Methods section. Data are expressed as fold of change versus control cells, set to 1. (b,c) U343MG cells were treated with DMSO (CTRL) or different concentrations of RM37 (1–20 µM) for 48 h. In the end, the number of apoptotic cells (c) and cell cycle progression (d) were evaluated. Data are mean values ± SEM of three different experiments. ** p < 0.01; *** p < 0.001 versus respective CTRL.

Figure 8

Effects of RM37 on different glioblastoma cells. (a) U87MG live cells; (b) U87MG dead cells; (c) T98G live cells; (d) T98G dead cells following treatment with different concentrations of RM37 (1–20 µM) for 48h. Data are mean values ± SEM of three different experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 versus CTRL.

Figure 9

Quantification of Chlamydia trachomatis on HeLa-infected cells. HeLa cells were infected with Chlamydia trachomatis and then treated with Nutlin-3a, RM37, and RM53. (a) Following reinfection, real-time PCR analysis was performed to quantify Chlamydia trachomatis copies on untreated (Reinfection) and treated infected cells. Data are expressed as the number of copies/µL in each condition. (b–d) Following reinfection, supernatants of HeLa cells were used to quantify the indicated cytokines and chemokines by Luminex assay. Data are expressed as pg/mL in each condition, versus those of control cells, and are mean ± SEM of three different experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus reinfection. (e) Chemical structures of hit compound RM37 and its derivative RM53.

Figure 10

Effects on p53 pathway’s reactivation upon C. trachomatis infection. (a,b) HeLa cells were inoculated with C. trachomatis serovar D (ATCC VR-885) at multiplicity of infection (MOI) 0.5 and treated with RM37. Cell lysates were subjected to Western blot analysis using specific antibody for p53. (a) A representative image is shown. Full screen of Western blot assay was provided in a separate file. (b) Data are shown as arbitrary optical density (OD) (mean values ± SEM of three different experiments). ** p < 0.01, *** p < 0.001 versus control.