Discovering and harnessing oxidative enzymes for chemoenzymatic synthesis and diversification of anticancer camptothecin analogues

The study addresses how to efficiently and selectively functionalize the camptothecin (CPT) scaffold to produce clinically relevant anticancer derivatives with improved properties. Although CPT exhibits potent anti-tumor activity through inhibition of DNA topoisomerase I, its clinical utility is limited by hydrophobicity and adverse side effects. Semi-synthetic derivatives such as topotecan and irinotecan are widely used but rely on harsh, labor-intensive chemical processes and limited natural availability of hydroxylated intermediates (e.g., 10- and 11-hydroxycamptothecin). The rarity of CPT and its oxidized derivatives in plant sources and the challenges in selective C–H functionalization create bottlenecks for diversification and scale-up. Enzymatic oxidation via cytochrome P450s offers regio- and stereoselective transformations under mild conditions. The purpose of this work is to discover CPT-oxidizing enzymes from C. acuminata and demonstrate their use in a chemoenzymatic pipeline to efficiently generate valuable CPT analogues, including approved drugs.

Background highlights include: CPT, vinblastine, and paclitaxel as prominent plant-derived anticancer agents; CPT’s role as a lead for semi-synthesis of topotecan and irinotecan. Conventional synthetic routes to hydroxylated CPT derivatives (e.g., 10HCPT) involve multi-step procedures with toxic reagents (Pd/C, Pb(OAc)2) and challenging C–H functionalization steps, complicating purity and scalability. Prior studies established cytochrome P450 enzymes as key oxidative biocatalysts in plant specialized metabolism, and MIAs rely on P450-mediated transformations. However, specific CPT 10- and 11-hydroxylases from C. acuminata had not been identified, and enzymatic routes to 11-hydroxylated CPTs—reported to have favorable therapeutic indices—remained underexplored.

- Metabolite profiling: Targeted metabolomics across C. acuminata tissues (young leaves, stems, fruits, bark) to correlate CPT and hydroxy-CPT (HCPT) distributions. Observed CPT accumulation in young leaves, with HCPTs primarily in stems, fruits, and bark. - Candidate gene discovery: Leveraged C. acuminata transcriptome and genome datasets and performed self-organizing map analysis to identify cytochrome P450 monooxygenases (CYP450s) co-expressed with monoterpenoid indole alkaloid (MIA) biosynthetic genes and HCPT accumulation. Nine CYP450 candidates spanning multiple clades were shortlisted. - Heterologous expression and in vivo assays: Cloned candidate CYP450 coding sequences into a galactose-inducible dual expression vector (pESC-Leu2KD) co-expressing a cytochrome P450 reductase (CPR). Transformed yeast cultures (100 µL) were fed 10 µM CPT for 48 h. LC-MS extracted ion chromatograms were used to detect products. A strain expressing CPR with candidate C23236 converted CPT to a product matching 10-hydroxycamptothecin (10HCPT) by m/z (365.2) and retention time. Another candidate, C23229, produced a compound of m/z 365.2 with a distinct retention time, indicating a different hydroxylation regioisomer. - In vitro microsomal assays: Prepared microsomal fractions from yeast expressing pESC-Leu2KD:CPR/C23236 or CPR/C23229. In the presence of NADPH, CPT was oxidized to products of m/z 365.2 as confirmed by LC-MS. - Structural elucidation: Isolated ~5–8 mg of enzymatic products for NMR characterization (1H, 13C, and 1D-TOCSY). Assignments confirmed that C23236 catalyzes C-10 hydroxylation to 10HCPT and C23229 catalyzes C-11 hydroxylation to 11HCPT. Substrate CPT NMR was recorded for comparison. No other products were detected. - Substrate scope: Assayed the two enzymes against 18 alkaloids spanning multiple MIA scaffolds (e.g., caryophylline, ajmaline, heteroyohimbine, quinolines). Activity was restricted to the CPT scaffold. Tested CPT-derived substrates: both enzymes accepted 7-ethyl-10HCPT to yield SN-38 (7-ethyl-10HCPT) and an isomer (2′-ethyl-1HCPT), respectively. CPTII accepted 10HCPT to form 10,11-dihydroxyCPT (7% conversion). 11HCPT was not a substrate for CPTI. Both enzymes converted 9-amino-CPT to two oxidized products (putatively 9-amino-10HCPT and 9-amino-11HCPT) based on m/z 380, though NMR confirmation was not feasible due to limited material and low conversion (~9%). - Chemoenzymatic synthesis: Leveraged enzymatic products to perform downstream chemical derivatizations under mild conditions (e.g., pH ~7, 30 °C) to access approved drugs and new analogues, including topotecan and irinotecan, with LC-MS confirmation against standards (Fig. 3).

- Two C. acuminata CYP450 monooxygenases were discovered and functionally characterized: - C23236 (CPTI): camptothecin 10-hydroxylase producing 10HCPT. - C23229 (CPTII): camptothecin 11-hydroxylase producing 11HCPT. - LC-MS evidence: Enzymatic products showed m/z 365.2 with distinct retention times corresponding to 10- vs 11-hydroxylation; in vitro microsomal assays with NADPH confirmed oxidation. - NMR confirmation: 1H, 13C, and 1D-TOCSY NMR (5–8 mg purified products) established C-10 and C-11 hydroxylation sites, respectively; no additional byproducts detected. - Substrate specificity: Activity largely restricted to the CPT scaffold among 18 MIA representatives. - Derivatization capability: - Both enzymes accepted 7-ethyl-10HCPT, enabling formation of SN-38 and an isomeric product (2′-ethyl-1HCPT) via regioselective oxidation steps. - CPTII further hydroxylated 10HCPT to 10,11-dihydroxyCPT with low conversion (7%). - 11HCPT was not a substrate for CPTI. - Both enzymes oxidized 9-amino-CPT to two products (m/z 380), likely 9-amino-10HCPT and 9-amino-11HCPT; structural confirmation pending. - Chemoenzymatic applications: Enzymatic hydroxylations enabled concise synthesis of approved drugs topotecan (Hycamtin) and irinotecan (Camptosar), and access to 11-hydroxyl analogues, using mild conditions. - Performance metrics: High regioselectivity for C-10 and C-11 positions; conversion rates reported at 62%–67% for targeted hydroxylations under chemoenzymatic conditions. - Therapeutic relevance: 11HCPT and derivatives exhibit a greater therapeutic index and may mitigate interpatient variability and resistance compared with irinotecan, highlighting the value of enzymatic access to 11-hydroxylated series.

The identification of CPTI and CPTII as regioselective 10- and 11-hydroxylases provides biocatalysts that directly address the challenge of selective C–H oxidation on the CPT scaffold. These enzymes enable access to key hydroxylated intermediates under mild, aqueous conditions, circumventing harsh and toxic reagents required for traditional semi-synthetic routes. Their strict regioselectivity facilitates targeted derivatization at C-10 and C-11, streamlining synthesis of topotecan, irinotecan, and expanded libraries of CPT analogues. The enzymatic route to 11HCPT is particularly impactful, as 11-hydroxylated derivatives have been associated with improved therapeutic index and potential to overcome variability and resistance. The work advances sustainable manufacturing by integrating biocatalysis with chemical steps to deliver clinically relevant entities, demonstrating a practical, greener pipeline for anticancer agent diversification from a single natural product scaffold.

This study discovers and characterizes two C. acuminata CYP450 monooxygenases—CPTI (10-hydroxylase) and CPTII (11-hydroxylase)—that enable regioselective oxidation of camptothecin. By integrating these enzymes into a chemoenzymatic workflow, the authors achieved efficient access to approved drugs (topotecan, irinotecan) and valuable 11-hydroxylated analogues under mild, sustainable conditions. The findings illuminate camptothecin metabolism and provide practical tools to expand the chemical space of CPT-derived therapeutics. Future directions include enzyme engineering to broaden substrate scope and improve conversions (e.g., for dihydroxylation), optimization and scale-up of biocatalytic steps, and exploration of additional oxidative tailoring enzymes to further diversify CPT analogues.

- Substrate scope is largely restricted to the camptothecin scaffold among tested MIAs. - Low conversion for secondary hydroxylation: CPTII forms 10,11-dihydroxyCPT at only ~7% yield under tested conditions. - Limited material and low conversion (~9%) for 9-amino-CPT oxidations prevented NMR-based structural confirmation of products. - Some chemoenzymatic steps and overall conversions (62%–67%) may require optimization for industrial scale-up and cost-effectiveness.