Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology

Abstract

The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.

Keywords: cell death; mitochondria; molecular modeling; molecular pharmacology; nucleoside/nucleotide biosynthesis; nucleoside/nucleotide transport; p53; tumor cell biology.

Figure 1

Tenovins can inhibit DHODH.A, a dose titration of tenovin 1 in the enzymatic assay. Curves show the color reduction in DCIP over time for illustrative purposes. B, values obtained using a kinetic DHODH enzyme assay. Values correspond to the average of three independent repeats ± S.D. with three technical repeats each. C, thermal denaturation curve of DHODH incubated with various tenovins (200 μm) and brequinar (200 μm) as a positive control. D, co-crystal structure of tenovin 6 in complex with the enzyme DHODH. DHODH is shown as a cartoon (gray color), and the cofactor FMN (cyan carbons), the substrate DHO (yellow carbons), and bound tenovin 6 (green carbon) are shown as thick lines. The tenovin 6 binding pocket residues are denoted as thin lines, and the hydrogen bonds between DHODH and tenovin 6 are shown as dashed lines (magenta). Details of the crystal structure can be found in Table S1.

Figure 2

Modeling the interaction between the tenovins and DHODH. Shown is the predicted binding mode of tenovin 1 (A) and tenovin 6 (B) in complex with DHODH. The enzyme DHODH is shown as a cartoon (gray color), and the cofactor FMN (cyan carbon), the substrate DHO (yellow carbon), and bound tenovins (green carbon) are shown as thick lines. The tenovin-binding pocket residues are shown as thin lines, and the hydrogen bonds between DHODH and tenovins 1 and 6 are shown as dashed lines (magenta). C, time evolution of the DHODH–tenovin interactions. Distances between the atom pairs are calculated for the conformations sampled during the second half of the simulations. D, computed free energies differences between the binding of tenovins to DHODH in the membrane relative to the binding of tenovin 6 to DHODH in the membrane (negative value implies tighter binding of a particular tenovin relative to tenovin 6); the FEP/MBAR method was used. E, the free energies required to pull tenovin 1, 6, or 39OH from the binding pocket on DHODH. Positions of the three tenovins relative to DHODH are shown in Fig. S1C.

Figure 3

Analysis of p53 dependence in the mechanism of action of the tenovins.A, Western blotting of p53 levels in ARN8 cells upon treatment with a 5 μm concentration of the indicated tenovins for either 2, 4, 6, or 8 h. B, SKNSH cells with normal WT p53 or stably expressing ddp53 were treated with a dose titration of the indicated tenovins for 72 h. Results are a single representative experiment with three technical replicates ± S.D. A total of three biological replicates were conducted. C, ARN8, HCT116 p53 WT, or p53 KO cells were treated with a fixed 5 μm dose of each tenovin and monitored for the confluence of the culture over 72 h by IncuCyte. Results are a single representative experiment with three technical replicates ± S.D. A total of three biological replicates were conducted. D, HCT116 p53 WT or p53 KO cells were treated with a 5 μm concentration of the indicated tenovins for 24 h prior to blotting for p53 and p53 targets. The total protein loading control for these blots is shown in Fig. S4.

Figure 4

Phenotypic analysis of tenovins upon supplementation with nucleotides.A, p53 transcriptional activity assay (CPRG) in ARN8 cells treated with tenovins for 18 h supplemented with 100 μm uridine, 1 mm DHO, or 1 mm OA. Results are a single representative experiment with three technical replicates ± S.D. A total of three biological replicates were conducted. B, Western blotting of p53 and p53 targets in ARN8 cells treated for 24 h with a 5 μm concentration of the indicated tenovins supplemented with 100 μm uridine, 1 mm DHO, or 1 mm OA. The total protein loading for these blots is shown in Fig. S4. C, SRB assay in ARN8 cells treated with the indicated tenovins for 72 h supplemented with 100 μm uridine, 1 mm DHO, or 1 mm OA. Results are a single representative experiment with three technical replicates ± S.D. A total of three biological replicates were conducted.

Figure 5

Cell viability and cell cycle effects of tenovins upon supplementation with nucleotides.A–G, ARN8 cells treated for 72 h with compound or treated for 72 h followed by compound wash-out and recovery in fresh medium for 24 h. H, ARN8 cells treated for 48 h with the indicated compounds at 10 μm (tenovins 1 and 33) or 200 nm (brequinar) with or without supplementation with 100 μm uridine, 1 mm DHO, or 1 mm OA.

Figure 6

Inhibition of uridine uptake by tenovins.A, ARN8 or U2OS cells treated for 15 min with a 5 μm concentration of the indicated compounds, with uptake of ³H uridine measured by a scintillation counter. All data are normalized to protein levels prior to normalization to DMSO control (DMSO = 100). Error bars, S.D. of three technical replicates. B, Western blotting of U2OS cells treated for 24 h with a 5 μm concentration of the indicated compounds. C, U2OS cells treated for 24 h with the indicated compounds, with uptake of [³H]uridine measured by a scintillation counter. All data are normalized to protein levels for each individual compound as a proxy for cell number and then normalized to the DMSO control (DMSO = 100). Error bars, S.D. of three technical replicates.