Article Figures & Data
Figures
- Fig. 1.
Duocarmycin analogs ICT2700 and ICT2726 (shown in boxes, differences colored in red). The ICT2700 analog has been reported to undergo bioactivation by CYP1A1 and CYP2W1 via a specific hydroxylation (red dotted oval) on the chloromethyl indoline substructure (Travica et al., 2013). Spontaneous spirocyclization occurs producing a reactive cyclopropane moiety. DNA adenine bases can be alkylated with this reactive group leading to cytotoxic DNA adducts. The ICT2726 analog does not undergo bioactivation. Instead, this analog can potentially serve as a biomarker for CYP2W1 activity due to the generation of a unique nontoxic metabolite specific to CYP2W1 (Sheldrake et al., 2013).
- Fig. 2.
Spectral binding experiments of CYP1A1 and CYP2W1 with the seco-duocarmycin enantiomers of ICT2726. Representative difference binding spectra are shown of CYP1A1 (1A1) [(A) and (B)] and CYP2W1 (2W1) [(C) and (D)], with the respective ICT2726 enantiomer. Insets display the difference in peak-trough absorbance (average of two technical duplicates) versus compound concentration and are fit by nonlinear regression to a tight-binding equation. The fitted compound affinities (Kd values) are reported with ± S.E. (error bars).
- Fig. 3.
Spectral binding experiments of CYP1A1 and CYP2W1 with the seco-duocarmycin enantiomers of ICT2700. Difference binding spectra are shown of CYP1A1 (1A1) [(A) and (B)] and CYP2W1 (2W1) [(C) and (D)] binding the ICT2700 enantiomers. Insets display the difference in peak-trough absorbance (average of two technical duplicates) versus compound concentration and are fit by nonlinear regression to a tight-binding equation. The fitted compound affinities (Kd values) are reported with ± S.E. (error bars).
- Fig. 4.
LC-MS chromatograms of ICT compounds incubated with CYP1A1 (1A1) or CYP2W1 (2W1) for 30 minutes; M6 = spirocyclized product. (A) ICT2700 R/S CYP1A1 metabolism. (B) ICT2700 R/S CYP2W1 metabolism. (C) ICT2726 R/S CYP1A1 metabolism (D) ICT2726 R/S CYP2W1 metabolism. AU, absorbance units; ESI+, electrospray ionization in positive mode; MS, mass; M+H, singly protonated mass; RT, retention time.
- Fig. 5.
Co-crystallization structures of CYP1A1 (ribbons) bound to (S)-ICT2726 (A) and (S)-ICT2700 (B) (gray sticks). Electron density is shown as 2Fo – Fc composite omit map contoured at 1.0 σ (blue mesh) around the ligand and heme (black sticks with orange Fe sphere). Potential hydrogen bonds (black dashes), halogen bonds (yellow dashes), and ligand atoms in closest proximity to the heme iron (red dashes) are shown with their corresponding distances.
Tables
CYP1A1/(S)-ICT2700 (6UDL) CYP1A1/(S)-ICT2726 (6UDM) Data collection Space group P212121 P3121 Cell dimensions (Å) 65.37, 196.15, 237.22 241.28, 241.28, 125.30 Molecules/asymmetric unit 4 4 Resolution (Å)a 50.00–2.85 (2.90–2.85) 39.64–3.07 (3.14–3.07) Total reflectionsa 510,182 (17,794) 783,762 (35,174) Unique reflectionsa 72,081 (3,422) 77,258 (3,944) Redundancya 7.1 (5.2) 10.1 (8.9) Rpima 0.080 (0.942) 0.054 (0.631) <I/σ(I)> a 28.0 (2.1) 9.7 (1.6) CC1/2a 0.990 (0.322) 0.997 (0.518) Completeness (%)a 99.8 (97.3) 99.0 (85.6) Refinement Resolution (Å) 48.799–2.850 39.64–3.10 No. reflections 71,709 76,831 R/Rfree (%) 23.8 / 28.7 20.1 / 22.1 Ramachandran (%) favored/allowed/outliers 96.19 / 3.81/ 0.00 97.15 / 2.85 / 0.00 No. non-H atoms/B factors (Å2) Protein 15,015 / 68.60 15,026 / 80.62 Ligand 54 / 57.62 104 / 80.66 Heme 172 / 57.09 172 / 68.49 CHAPS —/— 42 / 100.77 Water 1 / 49.31 —/— RMSD bond (Å) 0.008 0.007 RMSD angle (°) 0.765 0.636 Coordinate error (Å) 0.37 0.37 CC1/2, Pearson correlation coefficient; <I/σ(I)>, average intensity over variation in intensity; R, measure of error between the observed intensities from the diffraction pattern and the predicted intensities calculated from the model; Rfree, measure of error between the observed diffraction pattern intensities from the diffraction pattern and the predicted intensities for a subset of data not used in model refinement; RMSD, root mean square deviation; Rpim, precision-indicating merging R factor
a Statistics for highest resolution shell shown in parentheses.
Data Supplement
- Supplemental Data -
Table S1. Crystallographic data for ICT2700 and ICT2726 small molecule structures.
Table S2. LC-MS gradient method.
Figure S1. Structures of ICT2700 and ICT2726 as determined by single molecule X-ray crystallography.
- Supplemental Data -
