Cell-Impermeable Inhibitors Confirm That Intracellular Human Transglutaminase 2 Is Responsible for the Transglutaminase-Associated Cancer Phenotype
Abstract
:1. Introduction
2. Results and Discussion
2.1. Design
2.2. Synthesis
2.3. Kinetics
2.4. Isozyme Selectivity
2.5. Pharmacokinetic Properties
2.6. Fluorescent Labelling
2.7. Evaluation of Cancer Cell Proliferation and Migration
2.8. Pull-Down of TG2 from E. coli Lysate
3. Materials and Methods
3.1. Chemical Synthesis
3.2. Molecular Docking of Compounds 16, 18, and 29 into TG2 Active Site
3.3. rhTG2 Inhibition Assay
3.4. TGase Isozyme Selectivity
3.5. Fluorescent Labelling of Purified rhTG2
3.6. SH-SY5Y Fluorescent Microscopy
3.7. Cell Proliferation Assay
3.8. Real-Time Cell Migration Assay
3.9. HaCaT and SCC-13 Cell Staining
3.10. Pull-Down of rhTG2 from E. coli Lysate
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Compound | R | KI (µM) | kinact (min−1) | kinact/KI (×103 M−1 min−1) |
---|---|---|---|---|
17 * | 1.0 ± 0.3 | 0.39 ± 0.10 | 409 ± 22 | |
18 (NCEG2) * | 4.1 ± 2.1 | 2.33 ± 1.12 | 563 ± 13 | |
22 | 1.3 ± 1.1 | 0.24 ± 0.07 | 188 ± 167 | |
23 | n.d. | n.d. | 1186 ± 91 ** | |
29 (NCEG-RHB) | n.d. | n.d. | 223 ± 3 ** | |
31 * | 0.9 ± 0.5 | 0.46 ± 0.26 | 497 ± 33 |
Compound | 17 | 18 (NCEG2) |
---|---|---|
cLog P * | 0.94 | −1.19 |
Log D | <−2.73 | <−2.63 |
−Log Pe | >9.02 | >8.79 |
Papp(A-B) (10−6, cm/s) | 0.31 | 0.47 |
Papp(B-A) (10−6, cm/s) | 0.42 | 0.56 |
Efflux Ratio | 1.39 | 1.18 |
Papp(A-B) (10−6, cm/s) + Pgp Inh. | 0.33 | 0.57 |
Papp(B-A) (10−6, cm/s) + Pgp Inh. | 0.35 | 0.36 |
Efflux Ratio + Pgp Inh. | 1.04 | 0.66 |
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Gates, E.W.J.; Calvert, N.D.; Cundy, N.J.; Brugnoli, F.; Navals, P.; Kirby, A.; Bianchi, N.; Adhikary, G.; Shuhendler, A.J.; Eckert, R.L.; et al. Cell-Impermeable Inhibitors Confirm That Intracellular Human Transglutaminase 2 Is Responsible for the Transglutaminase-Associated Cancer Phenotype. Int. J. Mol. Sci. 2023, 24, 12546. https://doi.org/10.3390/ijms241612546
Gates EWJ, Calvert ND, Cundy NJ, Brugnoli F, Navals P, Kirby A, Bianchi N, Adhikary G, Shuhendler AJ, Eckert RL, et al. Cell-Impermeable Inhibitors Confirm That Intracellular Human Transglutaminase 2 Is Responsible for the Transglutaminase-Associated Cancer Phenotype. International Journal of Molecular Sciences. 2023; 24(16):12546. https://doi.org/10.3390/ijms241612546
Chicago/Turabian StyleGates, Eric W. J., Nicholas D. Calvert, Nicholas J. Cundy, Federica Brugnoli, Pauline Navals, Alexia Kirby, Nicoletta Bianchi, Gautam Adhikary, Adam J. Shuhendler, Richard L. Eckert, and et al. 2023. "Cell-Impermeable Inhibitors Confirm That Intracellular Human Transglutaminase 2 Is Responsible for the Transglutaminase-Associated Cancer Phenotype" International Journal of Molecular Sciences 24, no. 16: 12546. https://doi.org/10.3390/ijms241612546