Synthetic prodrug design enables biocatalytic activation in mice to elicit tumor growth suppression

Abstract

Considering the intrinsic toxicities of transition metals, their incorporation into drug therapies must operate at minimal amounts while ensuring adequate catalytic activity within complex biological systems. As a way to address this issue, this study investigates the design of synthetic prodrugs that are not only tuned to be harmless, but can be robustly transformed in vivo to reach therapeutically relevant levels. To accomplish this, retrosynthetic prodrug design highlights the potential of naphthylcombretastatin-based prodrugs, which form highly active cytostatic agents via sequential ring-closing metathesis and aromatization. Structural adjustments will also be done to improve aspects related to catalytic reactivity, intrinsic bioactivity, and hydrolytic stability. The developed prodrug therapy is found to possess excellent anticancer activities in cell-based assays. Furthermore, in vivo activation by intravenously administered glycosylated artificial metalloenzymes can also induce significant reduction of implanted tumor growth in mice.

Fig. 1. Directions in prodrug design.

a General categorization of prodrug types can be made via their activation pathway, which are either naturally- or externally-triggered. This study aims to develop a working example of a synthetic prodrug. b Retrosynthetic prodrug design is a concept that aims to design prodrugs that transform into bioactive molecules via a bond-forming reaction. The proposed synthetic prodrug design relies on a sequential RCM/aromatization reaction to construct phenyl-containing bioactive agents.

Fig. 2. Model substrates for various pharmacophore fragments.

All reactions were carried out under the standardized conditions: 4 mM of substrate was incubated with 1 mol% of ruthenium catalyst 1 in PBS/1,4-dioxane (9:1) for 2 h at 37 °C. TON values were determined from product yields obtained by HPLC analysis; where TON_Arom refers to final product and TON_RCM refers to the combination of cyclohexadien-1-ol intermediate/final product. TON_RCM for compound 11 was calculated based on starting material conversion. Reactions were performed in triplicate.

Fig. 3. ArM-based activity studies with naphthalene-based precursors.

a The anchoring of 1 into the hydrophobic binding pocket of albumin (Alb) leads to the creation of Alb-Ru, which is capable of performing ring-closing metathesis. b Reactivity studies focused on the RCM activity of precursor 12 under various substrate concentrations and solvents. ^a20 mol% of glutathione added. Reactions were performed in triplicate; TON values were determined from product yields obtained by HPLC analysis. Michaelis–Menten kinetic graphs c and parameters d for substrates (2, 12, 14, 15). Summarized values are the substrate affinity (K_M), turnover frequency (k_cat), and catalytic efficiency (k_cat/K_M). Also presented are the TON_Arom using 1 mM substrate concentrations, which were incubated with 1 mol% of Alb-Ru in PBS/1,4-dioxane (9:1) for 2 h at 37 °C. Reactions were performed in triplicate. Data in c are represented as mean values ± SD, n = 3 independent experiments.

Fig. 4. Design and development of naphthylcombretastatin-based prodrugs.

a Crystal structure of the dimer interface between tubulin subunits (PDB ID: 5LYJ) showing a bound combretastain A-4 (green), and a modeled conformation of analog 20 (blue). Molecular docking studies of prodrugs 16–19 were also conducted to predict their relative binding affinities to the colchicine binding site of β-tubulin (via average calculated binding energies). b Alb-Ru activities using biologically relevant concentrations of prodrugs 16–19. Product yield was determined by HPLC analysis. Reactions were performed in triplicate. c Incubation of prodrugs 16–18 in blood was performed to determine ester stability via the detection of their respective hydrolyzed side product. d Michaelis–Menten kinetic graphs and parameters for prodrugs 16–18. Summarized values are the substrate affinity (K_M), turnover frequency (k_cat), and catalytic efficiency (k_cat/K_M). Reactions were performed in triplicate. Data in (d) are represented as mean values ± SD, n = 3 independent experiments.

Fig. 5. In cellulo prodrug therapy against cancer cell growth.

a Summary of calculated GR₅₀ values for drug 20, prodrug 17 and prodrug/Alb-Ru mixtures against HeLa, A549, PC3 and MCF7 cell lines. GR₅₀ values represent concentrations that gives half maximal growth rate inhibition. b Representative dose response curves against HeLa cells showing the cytostatic activities of drug 20, prodrug 17, and mixtures of Alb-Ru (0.5 μM) with prodrug 17. c Schematic depiction of HeLa targeted activation of prodrug 17 into drug 20 using GArM-Ru. The inhibition of HeLa cell growth was investigated using varying mixtures of GArM-Ru and prodrug 17. In general, the prodrug (and drug) was kept at constant concentrations of 4 μM, while varying concentrations of GArM-Ru was used. Data in (b) and (c) are represented as mean values ± SD, n = 3 biologically independent samples.

Fig. 6. In vivo synthetic prodrug therapy against HeLa tumor growth in mice.

a To highlight the biocatalytic activity of the GArM-Ru complex, the objective was to apply prodrug therapy via intravenous administration to treat subcutaneous xenograft tumors in mice. b Reactivity studies of prodrug 17 with Alb-Ru in mixtures of 5:4:1 blood/PBS/dioxane. Product yield was determined by HPLC analysis. Reactions were performed in triplicate. c Measurements of tumor size (mm³) in mice over time; Tumors were initially implanted in mice and developed over 4 days before therapy. Dosages were applied in 5 injections spread out over 8 days. d Extraction and visual comparison of tumor tissues shows the extent of growth inhibition 20 days after the start of therapy (n = 5). e Comparison of measured tumor burden (via weight) 20 days after the start of therapy (n = 5). P values were determined using a two-tailed Student’s t-test. **P = 0.0067 (Vehicle), *P = 0.0274 (Prodrug 17), *P = 0.0243 (GArM-Ru); *P < 0.05, **P < 0.01, and ***P < 0.001. Data in c are represented as mean value ± SD, n = 5 biologically independent samples.