Novel Nontoxic 5,9-Disubstituted SN38 Derivatives: Characterization of Their Pharmacological Properties and Interactions with DNA Oligomers
Abstract
:1. Introduction
2. Results and Discussion
2.1. Chemical and Stereochemical Stability of 1 and 2 in Water
2.2. Aggregation Studies of 1 and 2 in D2O Buffer
2.3. Binding of 1 or 2 with d(GCGATCGC)2, Characterized by DOSY
2.4. Mode of Binding of Diastereomers 1 and 2 to d(GCGATCGC)2 Based on 1H NMR Titration
2.5. Molecular Modeling
3. Materials and Methods
3.1. Chemical Substrates
3.2. HPLC Analysis
3.2.1. HPLC Separation and Purification
3.2.2. Compounds Purity
3.2.3. HPLC Analysis of Incubation Samples
3.3. Sample Preparation
3.4. NMR Experiments
- 1D 1H NMR spectra: spectral width 8000 Hz, 16–256 scans, 32 K complex points, acquisition time 2 s, relaxation delay 2 s.
- NOESY: spectral widths 5000 Hz in both dimensions, 1024 complex points in t2, 512 complex points in t1, 64 scans per increment, relaxation delay 1 s, and mixing time 200 ms.
- 1H-13C HSQC: spectral widths 6000 Hz in F2 and 21,600 Hz in F1, 1024 complex points in t2, 400 complex points in t1, 64 scans per increment, relaxation delay 1s, 1J(C,H) = 146 Hz.
- 1H-13C HMBC: spectral widths 6000 Hz in F2 and 23,880 Hz in F1, 1024 complex points in t2, 400 complex points in t1, 256 scans per increment, relaxation delay 1 s, nJ(C,H) = 5 or 8 Hz.
3.5. Calculating Self-Association Constants of 1 and 2
3.6. Calculating Binding Constants Based on 1H NMR Titration
3.7. Calculating Binding Constants from the Diffusion Coefficients
3.8. Molecular Dynamics Calculations
3.8.1. Calculating Binding Free Energies (Enthalpies) Using MM-PBSA and MM-GBSA Methods
3.8.2. PM7 Semi-Empirical Calculations
3.8.3. Cluster Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Δδ for 1 | ||||||||||||
c1 | Ring A | Ring B | Ring C | Ring D | Ring E | |||||||
H12 | H11 | H23 | H5 | H14 | H17a | H17b | H18 | H19 | ||||
0.005 | (8.15) 2 | (7.60) | (1.26) | (6.16) | (7.59) | (5.65) | (5.50) | (2.05) | (1.02) | |||
0.05 | 23.6 | 11.5 | −8.7 | 10.6 | 19.7 | 5.3 | 4.2 | 3.8 | 2.2 | |||
0.09 | 39.8 | 20.6 | −15.1 | 17.9 | 33.4 | 9.6 | 7.9 | 7.6 | 4.9 | |||
0.29 | 96.1 | 47.2 | −35.8 | 37.7 | 78.7 | 23.6 | 18.8 | 17.4 | 11.5 | |||
0.65 | 142.0 | 69.5 | −54.7 | 53.8 | 117.1 | 34.6 | 27.4 | 25.1 | 16.4 | |||
1.20 | 180.3 | 88.1 | −71.5 | 67.2 | 150.1 | 44.1 | 34.1 | 32.2 | 20.9 | |||
Ka Δδmax 3 | 1.61 367.0 | 1.74 174.6 | 1.34 −153.4 | 2.28 121.9 | 1.59 305.7 | 1.64 89.4 | 1.76 67.9 | 1.70 64.3 | 1.76 41.3 | |||
the average value of Ka | 1.71 ± 0.24 | |||||||||||
Δδ for 2 | ||||||||||||
c2 | Ring A | Ring B | Ring C | Ring D | Ring E | |||||||
H12 | H11 | H24 | H23 | H51 | H28 | H28 | H14 | H17a | H17b | H18 | ||
0.003 | (8.15) 2 | (7.60) | (5.10) | (1.26) | (6.20) | (4.81) | (4.35) | (7.57) | (5.66) | (5.46) | (2.02) | |
0.05 | 2.5 | 1.9 | 1.0 | 0 | 2.5 | 2.1 | 1.2 | 2.3 | 1.6 | 1.6 | 0.5 | |
0.08 | 3.4 | 2.1 | 1.9 | 0 | 3.2 | 2.3 | 1.4 | 2.8 | 2.0 | 1.7 | 1.7 | |
0.26 | 8.3 | 4.5 | 3.6 | −2.8 | 8.1 | 4.2 | 2.7 | 7.3 | 3.3 | 3.2 | 2.7 | |
0.43 | 11.5 | 5.3 | 4.4 | −6.4 | 11.0 | 5.0 | 2.6 | 10.1 | 3.6 | 3.9 | 2.2 | |
0.77 | 19.4 | 8.9 | 7.5 | −8.4 | 18.6 | 8.6 | 4.9 | 17.6 | 7.1 | 8.0 | 5.4 | |
Ka Δδmax 3 | 0.40 98.0 | 0.91 27.7 | 0.68 26.9 | 0.20 −67.3 | 0.42 89.4 | 0.82 27.7 | 1.03 14.8 | 0.35 96.5 | 1.53 15.7 | 0.41 40.9 | 0.98 15.1 | |
the average value of Ka | 0.70 ± 0.38 |
1 | 2 | ||
---|---|---|---|
c [mm] | Di × 10−10 [m2s−1] | c [mm] | Di × 10−10 [m2s−1] |
1.5 | 1.92 ± 0.05 | 0.77 | 2.18 ± 0.05 |
0.29 | 2.13 ± 0.10 | 0.19 | 2.22 ± 0.10 |
0.23 | 2.13 ± 0.10 | 0.13 | 2.24 ± 0.10 |
Concentration [mm] | DOBS | DOBS-3 | MFcomp | Ka ± 0.3 | ||
---|---|---|---|---|---|---|
c | c3 | × 10−10 [m2 s−1] | × 10−10 [m2 s−1] | [mm−1] | ||
1 | 0.29 | 0.92 | 1.11 ± 0.05 | 0.86 ± 0.05 | 0.80 ± 0.05 | 5.9 |
0.45 | 0.92 | 1.14 ± 0.05 | 0.91 ± 0.05 | 0.81 ± 0.05 | 7.7 | |
1.89 | 1.26 | 1.34 ± 0.05 | 0.88 ± 0.05 | 0.55 ± 0.05 | 5.5 | |
2 | 0.29 | 0.28 | 1.83 ± 0.10 | 0.93 ± 0.10 | 0.29 ± 0.05 | 2.1 |
0.29 | 0.42 | 1.67 ± 0.10 | 0.92 ± 0.10 | 0.41 ± 0.05 | 2.4 | |
0.29 | 0.87 | 1.45 ± 0.10 | 0.92 ± 0.10 | 0.59 ± 0.05 | 2.0 | |
0.9 | 1.47 | 1.35 ± 0.10 | 0.84 ± 0.10 | 0.63 ± 0.05 | 1.8 |
Δδ for 1 | ||||||||||
c3 | Ring A | Ring B | Ring C | Ring D | Ring E | |||||
H12 | H11 | H22 | H5 | H28 | H14 | H17a | H17b | H18 | H19 | |
0 | (7.95) 2 | (7.51) | (3.36) | (6.10) | (4.76) | (7.44) | 5.60 | (5.46) | (2.01) | (0.99) |
0.02 | 3.9 | 6.6 | 10.1 | 11.2 | 5.0 | 2.6 | 4.3 | 4.3 | 3.6 | 2.4 |
0.06 | 6.2 | 13.5 | 19.3 | 28.3 | 12.1 | 4.7 | 8.7 | 10.9 | 8.2 | 5.5 |
0.12 | 13.2 | 26.8 | 31.3 | 56.5 | 23.0 | 9.8 | 17.5 | 21.6 | 16.2 | 10.7 |
0.25 | 23.4 | 42.1 | 49.3 | 89.2 | 36.9 | 18.3 | 30.7 | 35.1 | 26.8 | 18.1 |
0.56 | 46.2 | 69.2 | 80.7 | 143.5 | 60.4 | 35.4 | 51.2 | 57.2 | 44.1 | 30.1 |
0.87 | 59.0 | 82.7 | 93.2 | - | 71.1 | 44.2 | 60.0 | 67.6 | 52.9 | 35.6 |
Ka Δδmax 3 | 1.37 4 118.8 | 5.34 105.7 | 7.57 111.4 | 7.72 197.6 | 5.89 89.4 | 1.54 4 85.0 | 4.76 79.4 | 5.56 85.7 | 5.38 66.9 | 4.89 47.0 |
the average value of Ka | 5.00 ± 2.02 (5.89 ± 1.07) 4 | |||||||||
Δδ for 2 | ||||||||||
c4 | Ring A | Ring B | Ring C | Ring D | Ring E | |||||
H12 | H11 | H22 | H23 | H5 | H28 | H14 | H17a | H17b | H19 | |
0.00 | (8.16) 2 | (7.62) | (3.37) | (1.27) | (6.19) | (4.81) | (7.57) | (5.65) | (5.46) | (0.99) |
0.02 | 5.7 | 4.1 | 6.7 | 1.4 | 7.3 | 2.8 | 4.3 | 2.1 | 1.5 | 0 |
0.07 | 17.3 | 12.9 | 15.5 | 3.4 | 22.7 | 9.1 | 13.6 | 6.1 | 4.1 | −0.9 |
0.13 | 31.1 | 23.4 | 28.7 | 5.5 | 40.3 | 15.8 | 24.3 | 10.4 | 7.0 | −1.7 |
0.28 | 60.2 | 45.4 | 57.3 | 9.6 | 76.7 | 30.0 | 46.8 | 21.3 | 13.2 | −3.9 |
0.65 | - | 74.9 | 88.1 | 14.5 | - | 49.0 | 77.4 | - | 20.5 | −6.5 |
1.13 | 123.5 | - | 110.9 | 18.4 | 153.0 | 62.5 | 96.2 | - | - | −7.3 |
1.47 | 133.5 | - | 115.3 | 20.7 | 163.2 | 65.7 | 104.6 | - | - | −7.0 |
Ka Δδmax 3 | 3.11 167.5 | 3.52 119.9 | 3.93 138.0 | 3.86 23.8 | 3.64 199.3 | 3.40 89.4 | 3.09 130.4 | 1.66 5 81.2 | 6.48 5 28.0 | 4.88 −8.85 |
the average value of Ka | 3.76 ± 1.19 |
Δδ | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|
G1-H8 | G1-H1′ | G1-H5′ | C2-H6 | C2-H5 | A4-H8 | A4-H2 | A4-H1′ | T5-H6 | C8-H1′ | |
(7.98) 2 | (5.99) | (3.72) | (7.42) | (5.38) | (8.24) | (7.79) | (6.28) | (7.19) | (6.19) | |
1 | 60.2 | 36.4 | −28.8 | 20.3 | 28.3 | 11.4 | 10.5 | 9.9 | 10.3 | 31.9 |
2 | 11.3 | 26.4 | −16.3 | 5.0 | 9.6 | 1.4 | 1.4 | 3.0 | 1.9 | 3.5 |
No. | Structure | PM7 ENERGY [kcal/mol] | % Population | |
---|---|---|---|---|
Clust. Average | 10% Clust. Min. | |||
1 | −1174.25 ± 3.79 | −1181.15 | 20.39 | |
2 | −1179.37 ± 3.39 | −1185.18 | 17.14 | |
3 | −1177.11 ± 3.94 | −1184.58 | 14.25 |
No. | Structure | PM7 ENERGY [kcal/mol] | % Population | |
---|---|---|---|---|
Clust. Average | 10% Clust. Min. | |||
1 | −1178.76 ± 4.77 | −1186.83 | 20.19 | |
2 | −1177.52 ± 4.07 | −1183.82 | 16.28 |
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Bednarek, E.; Bocian, W.; Urbanowicz, M.; Sitkowski, J.; Naumczuk, B.; Kozerski, L. Novel Nontoxic 5,9-Disubstituted SN38 Derivatives: Characterization of Their Pharmacological Properties and Interactions with DNA Oligomers. Int. J. Mol. Sci. 2021, 22, 8190. https://doi.org/10.3390/ijms22158190
Bednarek E, Bocian W, Urbanowicz M, Sitkowski J, Naumczuk B, Kozerski L. Novel Nontoxic 5,9-Disubstituted SN38 Derivatives: Characterization of Their Pharmacological Properties and Interactions with DNA Oligomers. International Journal of Molecular Sciences. 2021; 22(15):8190. https://doi.org/10.3390/ijms22158190
Chicago/Turabian StyleBednarek, Elżbieta, Wojciech Bocian, Magdalena Urbanowicz, Jerzy Sitkowski, Beata Naumczuk, and Lech Kozerski. 2021. "Novel Nontoxic 5,9-Disubstituted SN38 Derivatives: Characterization of Their Pharmacological Properties and Interactions with DNA Oligomers" International Journal of Molecular Sciences 22, no. 15: 8190. https://doi.org/10.3390/ijms22158190