Abstract
Apoptosis plays a pivotal role in the etiology or pathogenesis of numerous medical disorders, and thus, targeting of apoptotic cells may substantially advance patient care. In our quest for novel low-molecular-weight probes for apoptosis, we focused on the uncommon amino acid γ-carboxyglutamic acid (Gla), which plays a vital role in the binding of clotting factors to negatively charged phospholipid surfaces. Based on the alkyl-malonic acid motif of Gla, we have developed and now present ML-10 (2-(5-fluoro-pentyl)-2-methyl-malonic acid, MW=206 Da), the prototypical member of a novel family of small-molecule detectors of apoptosis. ML-10 was found to perform selective uptake and accumulation in apoptotic cells, while being excluded from either viable or necrotic cells. ML-10 uptake correlates with the apoptotic hallmarks of caspase activation, Annexin-V binding and disruption of mitochondrial membrane potential. The malonate moiety was found to be crucial for ML-10 function in apoptosis detection. ML-10 responds to a unique complex of features of the cell in early apoptosis, comprising irreversible loss of membrane potential, permanent acidification of cell membrane and cytoplasm, and preservation of membrane integrity. ML-10 is therefore the most compact apoptosis probe known to date. Due to its fluorine atom, ML-10 is amenable to radio-labeling with the 18F isotope, towards its potential future use for clinical positron emission tomography imaging of apoptosis.
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Abbreviations
- ML-10:
-
(2-(5-fluoro-pentyl)-2-methyl-malonic acid)
- Gla:
-
(γ-carboxyglutamic acid)
- Anti-Fas:
-
(Anti-Fas antibody)
- PMD:
-
(plasma membrane depolarization)
- pHint:
-
(intracellular pH)
- pHext:
-
(extracellular pH)
- PET:
-
(positron emission tomography)
- PS:
-
(phosphatidylserine)
- TMRE:
-
(tetramethylrhodamine-ethyl ester)
- LDH:
-
(lactate dehydrogenase)
- DDC:
-
(didansyl-L-cystine)
- HBS:
-
(Hepes buffered saline solution)
- HBSNa:
-
(HBS with 140 mM Na+)
- HBSK:
-
(HBS with 140 mM K+)
- zVAD:
-
(Z-Val-Ala-Asp-fluoromethyl ketone)
- [K+]ext:
-
(extracellular potassium concentration)
- PI:
-
(propidium iodide)
- TB:
-
(trypan blue)
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Acknowledgements
We would like to thank Julia Dick and Mirit Argov from Aposense Ltd., for technical assistance. ML-10 is an investigational agent, developed by Aposense Ltd. (previously NeuroSurvival Technologies (NST) Ltd.).
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Cohen, A., Shirvan, A., Levin, G. et al. From the Gla domain to a novel small-molecule detector of apoptosis. Cell Res 19, 625–637 (2009). https://doi.org/10.1038/cr.2009.17
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DOI: https://doi.org/10.1038/cr.2009.17
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