Oral Presentation Royal Australian Chemical Institute National Congress 2026

Bioorthogonal prodrug activation for targeted delivery of combretastatin A-4 prodrugs. (136722)

Julia Constantino Camilli 1 , Sushant Aryal 1 , Guntas S Ahluwalia 1 , Thomasin K Brind 1 , Jessica M Fairhall 1 , Parry Guilford 2 , Sarah Hook 1 , Allan B Gamble 1
  1. School of Pharmacy, University of Otago, Dunedin, New Zealand
  2. Department of Biochemistry, University of Otago, Dunedin, New Zealand

Bioorthogonal reactions are fast and precise reactions, between two chemical functional groups, that happen within a biological system without interfering with natural biochemical processes1, 2. One of the applications of bioorthogonal reactions is the ‘on demand’ activation of prodrugs3, 4. Tumor-targeted bioorthogonal prodrug activation is an emerging area aimed at selective delivery of cytotoxic agents, which are often limited by severe off-target toxicity. Bioorthogonal cytotoxic prodrugs can be designed to selectively activate in pre-targeted cancer cells, reducing side effects caused by off-target cytotoxicity in healthy tissues, and overcoming the main challenge faced by traditional chemotherapy.

In this study, an azide-linked bioorthogonal click-to-release prodrug system was developed. The anti-EGFR monoclonal antibody (cetuximab) was rationally modified by conjugation with a bioorthogonal strained trans-cyclooctene (d-TCO) group, enabling precise on-target activation and selective release of the cytotoxic agent Combrestatain A-4 (CA-4) in the tumor environment. Activation of the azide‑linked CA-4 prodrugs was achieved via a click-and-release [1,3]-dipolar cycloaddition and self-immolation of the prodrug linker.

Several azide-linked prodrugs containing Combrestatain A-4 (CA-4) as their cytotoxic payload were designed and synthesized. Prodrug activation and stability were investigated using in-house HPLC analysis. Based on these results, the lead prodrugs were assessed for in vitro cytotoxicity in the colorectal cancer cell line HCT116. Then, the prodrugs were used in cell studies using cetuximab-d-TCO conjugates. The efficacy of the EGFR targeting was assessed in vitro using the HCT116 EGFR knock-out cell line. HPLC activation and stability studies were also conducted in mice serum to support the translation of this strategy to in vivo studies. Based on the in vitro assays, two lead prodrugs were selected to be used in preliminary in vivo evaluation. This work highlights the potential of tumour-targeted bioorthogonal prodrug activation as a selective and safer approach for delivering cytotoxic agents to cancer cells.

 

 

 

 

 

 

  1. 1. Sletten EM, Bertozzi CR. From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions. Acc Chem Res. 2011;44(9):666-76.
  2. 2. Jewett JC, Bertozzi CR. Cu-free click cycloaddition reactions in chemical biology. Chem Soc Rev. 2010;39(4):1272-9.
  3. 3. Matikonda SS, Orsi DL, Staudacher V, Jenkins IA, Fiedler F, Chen J, et al. Bioorthogonal prodrug activation driven by a strain-promoted 1,3-dipolar cycloaddition. Chem Sci. 2015;6(2):1212-8.
  4. 4. Fairhall JM, Camilli JC, Gibson BH, Hook S, Gamble AB. EGFR-targeted prodrug activation using bioorthogonal alkene-azide click-and-release chemistry. Bioorg Med Chem. 2021;46:116361