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From the Gla Domain to a Novel Small-Molecule Detector of Apoptosis

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From the Gla Domain to a Novel Small-Molecule Detector of Apoptosis Cell Research (2009) 19:625-637. © 2009 IBCB, SIBS, CAS All rights reserved 1001-0602/09 $ 30.00 npg ORIGINAL ARTICLE www.nature.com/cr From the Gla domain to a novel small-molecule detector of apoptosis Avi Cohen1, *, Anat Shirvan1, *, Galit Levin1, Hagit Grimberg1, Ayelet Reshef1, Ilan Ziv1 1Aposense Ltd, 5-7 Ha’Odem St, Kiryat Matalon, PO Box 7119, Petach Tikva, Israel Apoptosis plays a pivotal role in the etiology or pathogenesis of numerous medical disorders, and thus, target- ing 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 pro- totypical 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. Keywords: molecular imaging, apoptosis, Gla domain, plasma membrane potential, cellular acidification Cell Research (2009) 19:625-637. doi: 10.1038/cr.2009.17; published online 17 February 2009 Introduction monitoring of the course of disease, or early assessment of efficacy of treatment. Positron emission tomography There is considerable evidence indicating an impor- (PET) has emerged as the leading modality for molecular tant role for apoptosis in the etiology or pathogenesis of imaging, enabling sensitive and quantitative assessment disease. Molecular detection of this fundamental ‘cell of cellular processes. Current approaches for in vivo de- suicide’ process may therefore be very useful in clini- tection of apoptosis are mostly based on large proteins cal practice, assisting in diagnosis or staging of disease, or peptides, e.g., Annexin-V [1], C2A domain of synap- totagmin-I [2], or caspase substrates [3, 4], which have limitations such as low clearance rate, suboptimal bio- *These two authors contributed equally to this work. distribution or potential immunogenicity. No PET tracer Correspondence: Avi Cohen for apoptosis is currently available for clinical use. Our Tel: +972-3-9215717; Fax: +972-3-9215714 E-mail: [email protected] objective was to address this need by developing novel, Abbreviations: ML-10 (2-(5-fluoro-pentyl)-2-methyl-malonic acid); Gla low molecular weight apoptosis-detectors, potentially (γ-carboxyglutamic acid); Anti-Fas (Anti-Fas antibody); PMD (plasma suitable to serve as PET tracers. membrane depolarization); pHint (intracellular pH); pHext (extracellular pH); Annexin-V and the C2A domain of synaptotagmin-I PET (positron emission tomography); PS (phosphatidylserine); TMRE (tetramethylrhodamine-ethyl ester); LDH (lactate dehydrogenase); DDC are used as apoptosis-probes due to their ability to bind (didansyl-L-cystine); HBS (Hepes buffered saline solution); HBSNa (HBS to anionic phospholipid surfaces, in relation with the with 140 mM Na+); HBSK (HBS with 140 mM K+); zVAD (Z-Val-Ala- apoptosis-related externalization of phosphatidylserine + Asp-fluoromethyl ketone); [K ]ext (extracellular potassium concentration); (PS). Another class of proteins that bind to anionic mem- PI (propidium iodide); TB (trypan blue) Received 16 July 2008; revised 7 October 2008; accepted 7 November branes is the γ-carboxyglutamic-acid (Gla)-domain pro- 2008; published online 17 February 2009 teins, including the clotting factors II, VII, IX, and X, the npg ML-10 – a novel apoptosis probe 626 anticoagulant proteins C and S [5], and the anti-apoptotic therefore comprised the following steps: (1) attachment protein gas6 [6]. However, these proteins or their related of an alkyl chain to render the molecule amphipathic for domains have not yet been explored as potential apop- membrane interaction; (2) optimization of the alkyl-chain tosis probes. For all these proteins, membrane binding length to five carbon atoms, based on pharmacokinetic is mediated by a domain rich in the uncommon amino considerations; (3) attachment of a methyl group at the acid Gla, synthesized post-translationally in the liver by α-position to block metabolism in vivo; and (4) attach- γ-carboxylation of glutamate [7]. The functional impor- ment of a fluorine atom, towards potential future radio- tance of this carboxylation is demonstrated by the ef- labeling with the 18F radio-isotope for PET. These modi- fect of oral anticoagulant drugs such as warfarin, which fications were guided, in part, by our experience gained function by inhibiting γ-carboxylation, thus abolishing in the development of fluorescent apoptosis probes of the membrane binding and respective catalysis of the ApoSense family. coagulation cascade [8]. These lines of evidence point at the alkyl-malonate group of Gla as a potential critical Selective detection of apoptosis by ML-10 structural moiety, enabling binding to cells with anionic To test ML-10 as an apoptosis detector, we utilized a phospholipid surfaces. We therefore hypothesized that tritium-labeled derivative (3H-ML-10). Cultured Jurkat alkyl-malonate could serve as a potential building block cells were induced to undergo apoptosis by treatment for novel small-molecule probes for apoptosis. with anti-Fas antibody (Anti-Fas) [13]. As shown in Based on this hypothesis and on our previous experi- Figure 2A, control, untreated cells manifested low levels ence in developing fluorescent small-molecule apoptosis of 3H-ML-10 uptake. However, upon induction of apop- probes of the ApoSense family, such as didansyl-L- tosis, a nearly 10-fold increase in tracer uptake was ob- cystine (DDC) [9, 10, 11] and NST-732 [12], we now served. Interestingly, this uptake was not calcium-depen- present ML-10 (2-(5-fluoro-pentyl)-2-methyl-malonic dent, since there was no calcium in the medium. Uptake acid), as a rationally designed molecule, with an amphip- of 3H-ML-10 was totally blocked by concomitant treat- athic compact structure (MW=206 Da). We also describe ment with the pan-caspase inhibitor, zVAD. The increase the performance of this novel Gla-derived probe as a de- in 3H-ML-10 uptake was time-dependent (Figure 2B), tector of apoptosis, and its mechanism of action. respective of the progression of the apoptotic process, being minimal up to 30 min after initiation of exposure Results to the apoptotic trigger, reaching a maximum at 180 min after apoptosis induction, and maintaining a steady state Considerations in the design of ML-10 thereafter. By contrast, 3H-ML-10 uptake by the control ML-10 (Figure 1) was rationally designed, based on viable cells was minimal and did not increase up to 6 h the alkyl-malonate motif, with the modifications required post incubation. These data demonstrate 3H-ML-10 as an to support its potential future use as a detector of apop- apoptosis-sensitive probe, manifesting selective and cas- tosis in vivo with PET. The molecular design of ML-10 pase-dependent uptake into cells undergoing apoptosis. Similar results were obtained in utilizing a fluorescent ML-10 analog, comprising a dansyl group. Cells were co-stained with both ML-10-dansyl and propidium io- A HO OH B dide (PI), serving as a marker for membrane disruption. While no uptake of ML-10-dansyl or PI was observed O O by the control viable cells (Figure 2C), exposure to the apoptotic trigger caused numerous cells to manifest ML- 10-dansyl uptake (turquoise fluorescence), yet excluding PI (red fluorescence, Figure 2D). This indicates uptake of ML-10-dansyl upon induction of apoptosis, at a stage wherein membrane integrity is still preserved. This fea- ture was also observed with tritium-labeled 3H-ML-10, 3 F in which induction of apoptosis was associated with H- ML-10 uptake, yet with the exclusion of PI or trypan blue (TB) (Figure 2E). Importantly however, 3H-ML-10 Figure 1 (A) Structure of ML-10 (2-(5-fluoro-pentyl)-2-methyl- malonic-acid). (B) Three-dimensional model of ML-10 in its di- uptake occurred in parallel to Annexin-V binding (Figure anionic form. Carbon atoms are in green, oxygen atoms in red, 2E), indicating externalization of PS on the membrane 3 hydrogen atoms in white and the fluorine atom in light blue. surface. We then explored subcellular localization of H- Cell Research | Vol 19 No 5 | May 2009 Avi Cohen et al. npg 627 0.8 A B * 0.4 * Control 0.7 Anti-Fas 0.6 * 0.3 0.5 0.2 0.4 0.3 * 0.1 0.2 H-ML-10 uptake (%) H-ML-10 uptake (%) 3 3 0.1 0.0 0.0 Control Anti-Fas zVAD zVAD +Anti-Fas 0 30 60 90 120 150 180 210 240 270 Treatment Time (min) C D Control Anti-Fas E 14 F 70 3H-ML-10 12 60 Anx 10 PI 50 8 TB 40 6 30 4 20 Anti-Fas / Control 2 10 % of total ML-10 uptake 0 0 0 30 60 90 120 150 180 210 Nuclei Mitochondria Membrane Cytosol Time (min) Subcellular fractions Figure 2 3H-ML-10 as a selective marker for apoptosis.
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