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Radiotherapy activates picolinium prodrugs in tumours

Nature Chemistry (2024)Cite this article

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Abstract

Radiotherapy-induced prodrug activation provides an ideal solution to reduce the systemic toxicity of chemotherapy in cancer therapy, but the scope of the radiation-activated protecting groups is limited. Here we present that the well-established photoinduced electron transfer chemistry may pave the way for developing versatile radiation-removable protecting groups. Using a functional reporter assay, N-alkyl-4-picolinium (NAP) was identified as a caging group that efficiently responds to radiation by releasing a client molecule. When evaluated in a competition experiment, the NAP moiety is more efficient than other radiation-removable protecting groups discovered so far. Leveraging this property, we developed a NAP-derived carbamate linker that releases fluorophores and toxins on radiation, which we incorporated into antibody–drug conjugates (ADCs). These designed ADCs were active in living cells and tumour-bearing mice, highlighting the potential to use such a radiation-removable protecting group for the development of next-generation ADCs with improved stability and therapeutic effects.

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Fig. 1: Radiation-induced NAP moiety activation to release functional molecules.
Fig. 2: NAP group was identified as a masking group that can be readily removed by e−aq generated in the water radiolysis.
Fig. 3: A release efficiency competition assay between the NAP group and other radiation-removable protecting groups.
Fig. 4: Radiation-induced reduction releases the fluorescent molecule MeRho from the NAPC-MeRho in the living system in a dose-dependent manner.
Fig. 5: The application of radiotherapy-induced NAPC system-controlled reduction-to-release strategy to ADC.
Fig. 6: Radiotherapy-induced controlled release of NAPC-ADC triggers tumour regression in mice.

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All data supporting the findings of this study are included in the article and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank J. Li and M. Zhai at Peking University for 60Co Source. We thank the facility support from the Analytical Instrumentation Center of Peking University. This study was funded by the National Nature Science Foundation of China (grant no. 22225603), the Ministry of Science and Technology of the People’s Republic of China (grant no. 2021YFA1601400), the Beijing Municipal Natural Science Foundation (grant no. Z200018) and Changping Laboratory to Z.L.

Author information

Author notes

  1. These authors contributed equally: Qunfeng Fu, Zhi Gu, Siyong Shen.

Authors and Affiliations

  1. Changping Laboratory, Beijing, China

    Qunfeng Fu, Zhi Gu & Zhibo Liu

  2. Beijing National Laboratory for Molecular Sciences, Radiochemistry and Radiation Chemistry Key Laboratory of Fundamental Science, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Qunfeng Fu, Zhi Gu, Siyong Shen, Yifei Bai, Xianglin Wang, Mengxin Xu, Pengwei Sun, Junyi Chen, Dongxuan Li & Zhibo Liu

  3. Peking University–Tsinghua University Center for Life Sciences, Peking University, Beijing, China

    Zhibo Liu

  4. Key Laboratory of Carcinogenesis and Translational Research of Ministry of Education, NMPA Key Laboratory for Research and Evaluation of Radiopharmaceuticals, Department of Nuclear Medicine, Peking University Cancer Hospital and Institute, Beijing, China

    Zhibo Liu

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Contributions

Z.L. conceived the study. Q.F., assisted by Z.G., S.S., Y.B., X.W. and P.S., performed chemical analysis, material synthesis and characterization. Q.F., assisted by S.S., J.C., M.X., Y.B., X.W. and D.L., performed radiosynthesis, positron emission tomography–computed tomography imaging, biodistribution and data analysis. Q.F., assisted by Z.G. and S.S., performed the cell viability assay. Z.G. analysed the NMR spectra and theoretical calculations. Q.F., assisted by Z.G. and S.S., performed all other experiments. Q.F., Z.G., S.S. and Z.L. analysed the data. Z.L. wrote the paper with inputs from all authors. All authors discussed the results and commented on the paper.

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Correspondence to Zhibo Liu.

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Nature Chemistry thanks Benoit Paquette, Jeremy Quintana and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Mechanism study of radiation-induced functional molecule release from picolinium compound.

(A) Proposed mechanism of the radiation-induced release reaction. The reaction yields radical intermediates and releases protected acetic acid by a one-electron reduction after 4-[(Acetyloxy)methyl]-1-methylpyridinium (compound 7’) irradiated with high-energy radiation. (B) 1H NMR spectrum of 7’ (up) and the radiation-irradiated product (bottom). A new single peak arose with a chemical shift of 2.10 ppm in D2O indicating the release of acetic acid. (C) UPLC chromatogram of a deoxygen solution containing 2 mM TEMPO and 1 mM 7’ with/without receiving a dose of 5,000 Gy γ-ray. The detector wavelength is 254 nm. (D) The mass spectral signal corresponding to the peak with a retention time of 2.26 minutes matches the molecular weight of the captured intermediate adduct. (E) UPLC chromatogram of 1 mM 7’ with/without received a dose of 5,000 Gy γ-ray. The detector wavelength is 254 nm. (F) The mass spectral signal corresponds to the peak with a retention time of 1.07 minutes, which matches the molecular weight of 7’. (G) The mass spectral signal corresponds to the peak with a retention time of 0.64 minutes, which matches the molecular weight of methylpicolinium.

Extended Data Fig. 2 Colony formation assay of 4T1-FAP cells treated with radiotherapy-induced NAPC-ADC activation.

4T1-FAP cells cultured in 6-well cell culture plates were treated as shown. (A) Photograph of colony formation in which colonies are stained with crystal violet. (B, C) Quantitative analysis of the number of colonies consisting of at least 50 cells under different treatments (n = 6, mean ± s.d.). The number of colonies in the group without ADC treatment and without X-ray irradiation in a normoxic environment was defined as 100%. Three independent experiments were performed and representative results are shown.

Source data

Extended Data Fig. 3 Pharmacokinetic studies of NAPC-ADC through positron emission tomography imaging and biodistribution.

(A) Representative positron emission tomography–computed tomography imaging of [89Zr]NAPC-ADC in BALB/c mice. Three independent experiments were performed and representative results are shown. (B) Time activity curves of [89Zr]NAPC-ADC in the blood (n = 3 independent experiments; 3 mice, mean ± s.d.). Mice received 14.8 MBq of [89Zr]NAPC-ADC (0.15 MBq/μg). Biodistribution of [89Zr]NAPC-ADC in both (C) BALB/c mice and (D) 4T1-FAP tumour-bearing BALB/c mice were performed at the indicated time points (n = 5 independent biological samples from 5 mice per group, mean ± s.d.). Mice received 0.74 MBq of [89Zr]NAPC-ADC (7.4 kBq/μg).

Source data

Extended Data Fig. 4 Blood biochemistry and complete blood panel analysis to evaluate the toxicology of NAPC-ADC in blood.

Healthy BALB/c mice were intravenously injected with PBS or NAPC-ADC (5 mg/kg) through the tail vein. Blood samples were collected at a series of time points: 1-, 3-, 7-, 14-, and 30-days post-injection (n = 6 independent biological samples, mean ± s.d.). The analysis reveals almost negligible difference in blood biochemical parameters and complete blood count, indicating the absence of any toxicological effects caused by NAPC-ADC on the blood system. (RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCHC, mean corpuscular hemoglobin concentration; WBC, white blood cells; NEUT, neutrophils; Lym, lymphocyte; Mon, monocyte; PLT, platelet; ALT, alanine transaminase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; TBIL, total bilirubin; TP, total protein; ALB, albumin; GLO, globulin; UREA, urea; CRE, creatinine; Ca, calcium; P, phosphate).

Source data

Extended Data Fig. 5 Synthetic routes of related compounds.

(A) Synthetic route of compounds 1 and 7. Compounds 2–6 and 8–9 shared the same synthesis route. (B) Synthetic route of NAPC-AMC. (C) Synthetic route of NAPC-MeRho. (D) Synthetic route of the payload of NAPC-ADC.

Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–17, NMR spectra and high-resolution mass spectra (Figs. 18–65).

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Fu, Q., Gu, Z., Shen, S. et al. Radiotherapy activates picolinium prodrugs in tumours. Nat. Chem. (2024). https://doi.org/10.1038/s41557-024-01501-4

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