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. 2019 Oct;11(19):2491-2504.
doi: 10.4155/fmc-2019-0181.

A structure-guided molecular chaperone approach for restoring the transcriptional activity of the p53 cancer mutant Y220C

Matthias R Bauer  1 , Rhiannon N Jones  2 , Raysa K Tareque  2 , Bradley Springett  2 , Felix A Dingler  1 , Lorena Verduci  1 , Ketan J Patel  1 , Alan R Fersht  1 , Andreas C Joerger  1   3   4 , John Spencer  2
Affiliations

A structure-guided molecular chaperone approach for restoring the transcriptional activity of the p53 cancer mutant Y220C

Matthias R Bauer et al. Future Med Chem. 2019 Oct.

Erratum in

  • Erratum.
    [No authors listed] [No authors listed] Future Med Chem. 2021 Mar;13(6):593-594. doi: 10.4155/fmc-2019-0181e1. Epub 2021 Feb 12. Future Med Chem. 2021. PMID: 33573418 Free PMC article. No abstract available.

Abstract

Aim: The p53 cancer mutation Y220C creates a conformationally unstable protein with a unique elongated surface crevice that can be targeted by molecular chaperones. We report the structure-guided optimization of the carbazole-based stabilizer PK083. Materials & methods: Biophysical, cellular and x-ray crystallographic techniques have been employed to elucidate the mode of action of the carbazole scaffolds. Results: Targeting an unoccupied subsite of the surface crevice with heterocycle-substituted PK083 analogs resulted in a 70-fold affinity increase to single-digit micromolar levels, increased thermal stability and decreased rate of aggregation of the mutant protein. PK9318, one of the most potent binders, restored p53 signaling in the liver cancer cell line HUH-7 with homozygous Y220C mutation. Conclusion: The p53-Y220C mutant is an excellent paradigm for the development of mutant p53 rescue drugs via protein stabilization. Similar rescue strategies may be applicable to other cavity-creating p53 cancer mutations.

Keywords: CRISPR/Cas9 p53 knockout; cancer mutations; cancer therapy; molecular chaperones; p53; protein stabilization; structure-based drug design; tumor suppression.

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Conflict of interest statement

Financial & competing interests disclosure

This work was funded by Worldwide Cancer Research (grants 14-1002, 18-0043), Sussex University (RN Jones), German Research Foundation (DFG) grant JO 1473/1-1 (AC Joerger) and ERC advanced grant 268506 (AR Fersht). The authors are grateful for support by the SGC, a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through Ontario Genomics Institute, Innovative Medicines Initiative (EU/EFPIA) (ULTRA-DD grant no. 115766), Janssen, Merck & Co., Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and the Wellcome Trust.

No writing assistance was utilized in the production of this manuscript.

Figures

Figure 1.
Figure 1.. Structure-based design strategy.
(A) Molecular surface representation of the p53 mutant Y220C in complex with the carbazole-based molecule PK083 (Protein Data Bank ID: 2VUK), highlighting different subsites of the binding pocket. Subsite 2 is not occupied by the ligand. The side chain of Cys220 blocks access to subsite 3 in the complex with PK083. Two different orientations are shown: cross-section through the binding site (top) and top view (bottom). (B) Scheme showing the design strategy for improving the binding affinity of PK083 by targeting subsite 2. See text for further details.
Figure 2.
Figure 2.. Inhibition of protein aggregation.
The effect of Y220C mutant stabilizers on the rate of aggregation of the mutant protein was measured at 37°C by monitoring light scattering at 500 nm. (A) Concentration-dependence of aggregation inhibition by PK9318. (B) Aggregation of the Y220C mutant at 15 μM PK9318 (green), PK9327 (red) or PK9328 (orange), and without compound (black).
Figure 3.
Figure 3.. Structures of p53-Y220C with subsite 2-targeting carbazole derivatives.
(A) Structure of the Y220C mutant bound to PK9284 (Protein Data Bank [PDB] ID 6GGA). The protein is shown as a gray surface representation, with the sulfur of Cys220 highlighted in yellow. The ligand is shown as a stick model. (B) Structure of the Y220C-PK9318 complex (PDB ID 6GGB). A cross-section of the binding pocket is shown. (C) Superposition of the binding modes of PK083 (gray carbon atoms; PDB ID 2VUK), PK9318 (green carbon atoms; PDB ID 6GGB), PK9320 (pink carbon atoms; PDB ID 6GGC) and PK9324 (cyan carbon atoms; PDB ID 6GGD) upon binding to the Y220C mutant. (D) Structure of the Y220C-PK9320 complex (PDB ID 6GGC). Orientation and color codes are the same as in panel B. In all four panels, chain B of the asymmetric unit is shown.
Figure 4.
Figure 4.. Binding mode of methylated thiophenes in subsite 2.
(A) Crystal structure of the Y220C-PK9328 complex (Protein Data Bank [PDB] ID 6GGF). Cross-section of the binding pocket. The Y220C mutant protein is shown as a surface representation. Selected side chains and the ligand are shown as stick models. 2Fo–Fc electron density for the ligand is shown in blue at a contour level of 1.5 σ. (B) Structure of Y220C-PK9328 (green) superimposed onto Y220C-PK9318 (gray; PDB ID 6GGB). Shown are Cα traces and selected side chains plus ligands as stick models. (C) Superposition of the binding modes of PK9328 (green stick model) and PK9318 (magenta stick model) shown in a different orientation, highlighting the rotation of the thiophene moiety upon methylation. The protein chain of the Y220C-PK9328 complex is shown in gray, with selected residues in the binding pocket displayed as stick models. (D) Structure of Y220C-PK9327 (pink; PDB ID 6GGE) superimposed onto Y220C-PK9318 (gray). Shown are Cα traces and selected side chains plus ligands as stick models. In all four panels, chain B of the asymmetric unit is shown.
Figure 5.
Figure 5.. Second-generation carbazole-based binders reduce the viability of liver cancer cell line HUH-7.
p53-Y220C-dependent cell viability reduction was observed for PK9318 (A) and PK9328 (B) after 72 h. PK9329 did not show any p53-Y220C-dependent viability reduction (C), consistent with its inability to stabilize the mutant. Cell viability was measured in quadruplicate and normalized against the values of blank (viability = 1) and no cell (viability = 0) controls. Data were measured in triplicates and are shown as mean ± SEM (paired t-test to test for significance in differences in HUH-7 and HUH-7 p53 KO viability reduction. *p < 0.05; **p < 0.01; ***p < 0.001. KO: Knockout; SEM: Standard error of the mean.
Figure 6.
Figure 6.. Heatmap of mRNA fold changes in p53 signaling after treatment with 10 μM PK9318 for 9 h of HUH-7 and the isogenic HUH-7 p53 knockout cell line.
The qPCR array comprises 84 genes related to p53-mediated signal transduction, classified into subgroups. Changes in mRNA levels were calculated using the ΔΔCt method. A value of 1 indicates no change in relative transcript levels between control- and PK9318-treated samples (values between 0.66 and 1.5 are shown in white). Increased mRNA levels are shown in green, starting from 1.5 (light green) to 4 (dark green), and decreased mRNA levels are shown in red, ranging from 0.66 (light red) to 0 (dark red). For ADGRB1 in HUH-7 p53 KO cells and TP73, WT1 in HUH-7 cells, no reliable ΔΔCt values could be obtained (shown as NA). Especially p53-target genes that are involved in apoptotic signaling (e.g., PUMA [BBC3], BTG2, ESR1, EGFR) and cell cycle modulation (GML, MDM2, SESN2) were selectively upregulated in HUH-7 (p53-Y220C) cells after PK9318 treatment, indicating Y220C-dependent induction of apoptosis and cell-cycle arrest in this cell line.
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References

    1. Joerger AC, Fersht AR. The p53 pathway: origins, inactivation in cancer, and emerging therapeutic approaches. Annu. Rev. Biochem. 85, 375–404 (2016). - PubMed

    2. •• Comprehensive review of p53 drug discovery and structural biology as well as of the evolutionary history of p53 family proteins.

    1. Vousden KH, Prives C. Blinded by the light: the growing complexity of p53. Cell 137, 413–431 (2009). - PubMed
    1. Bieging KT, Mello SS, Attardi LD. Unravelling mechanisms of p53-mediated tumour suppression. Nat. Rev. Cancer 14, 359–370 (2014). - PMC - PubMed
    1. Khoo KH, Verma CS, Lane DP. Drugging the p53 pathway: understanding the route to clinical efficacy. Nat. Rev. Drug Discov. 13, 217–236 (2014). - PubMed

    2. •• Excellent review of different p53 drug discovery strategies.

    1. Bouaoun L, Sonkin D, Ardin M. et al. TP53 variations in human cancers: new lessons from the IARC TP53 database and genomics data. Hum. Mutat. 37, 865–876 (2016). - PubMed

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