Targeted degradation of p53-R175H hotspot mutant with an aptamer-based PROTAC for precise medicine
image: Molecular dynamics simulations calculate the structure of p53m and its binding interfaces with p53-R175H and CRBN (a), and PAGE electrophoresis confirms the successful connection between p53m-DA and CRBNL(b). Microscale thermophoresis measures the affinity of dp53m in p53-WT and p53-R175H (c). Streptavidin pull-down assays show that excess dp53m competitively binds p53-R175H (d). Fluorescence complementation experiments indicate that dp53m, CRBN, and p53-R175H form a ternary complex (e). AlphaLISA (f) and NanoBRET (g) experiments demonstrate that dp53m and CRBN can form a ternary complex with p53-R175H, but not p53-WT. Immunoblotting (h) and quantitative analysis (i) show that dp53m degrades p53-R175H in H1299 cells, with no effect on p53-WT. Immunoblotting (j) and quantitative analysis (k) show the impact of dp53m treatment on H1299 cells over specified time intervals. qPCR analysis shows the level of p53 mRNA in H1299-R175H cells 24 hours after transfection with dp53m (l). PAGE electrophoresis indicates the serum stability of dp53m (m, n).
Credit: ©Science China Press
This study was led by Songbo Xie & Diansheng Zhong from Tianjin Medical University General Hospital and Sijin Wu from Xi'an Jiaotong-Liverpool University. They reported an RNA aptamer-based PROTAC degrader capable of targeting and degrading the p53-R175H mutant previously. However, the serum instability limits its clinical translation. In this study, they developed the second-generation of p53-R175H degrader which demonstrated significant antitumor efficacy in p53-R175H-driven cancer cells both in vitro and in vivo, without toxicity.
Through an iterative molecular docking-guided post-SELEX approach, a high-performance DNA aptamer with improved affinity and excellent serum stability was identified. Subsequently, using this resulting aptamer as a warhead, a selective PROTAC, named dp53m, was developed, which degraded p53-R175H mutant while sparing wildtype p53 in a ubiquitin-proteasome-dependent mechanism.
Both in vitro and in vivo experiments demonstrated that dp53m had significant anti-tumor activity against cancer cells harboring the p53-R175H mutation. Additionally, dp53m significantly increased the sensitivity of p53-R175H-driven cancer cells to cisplatin.
This second-generation of p53-R175H degrader displays therapeutic potential, either alone or in combination with standard-of-care chemotherapeutic agents, for patients harboring the p53-R175H mutation.
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An engineered DNA aptamer-based PROTAC for precise therapy of p53-R175H hotspot mutant-driven cancer