Ligand-Substitution Reactions of the Tellurium Compound AS-101 in

Oct 11, 2016 - Ligand-Substitution Reactions of the Tellurium Compound AS-101 in. Physiological Aqueous and Alcoholic Solutions. Alon Silberman,. †,...
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Ligand-Substitution Reactions of the Tellurium Compound AS-101 in Physiological Aqueous and Alcoholic Solutions Alon Silberman,†,‡,§ Michael Albeck,† Benjamin Sredni,‡ and Amnon Albeck*,†,⊥ †

Department of Chemistry and ‡C.A.I.R. Institute, The Safdiè Center for AIDS and Immunology Research, The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel S Supporting Information *

cytokine IL-10.10,11 Indeed, AS-101 was recently found to specifically interact with spatially vicinal thiol groups (i.e., cysteines in close spatial proximity) located on the extracellular domain of the VLA-4 integrin, a key player in the metastatic cascade.12,13 This redox-modulated inhibition affected the conformation of VLA-4 and resulted in its impaired physiological activity in vivo. On the basis of these observations, others and we have speculated that the active labile ligands that participate in the ligand-substitution reactions of AS-101 with cysteine thiol residues are the chlorine atoms, whereas the ethylene glycol (EG) moiety was considered to be inert in relation to substitution with proteinaceous cysteine thiols but necessary for the three-dimensional fitting into the active site (Figure 1B).14 However, this speculation has not been validated experimentally. Furthermore, the chemical mechanism by which AS-101 substitutes its ligands with its target cysteines has never been studied. TeIV compounds may substitute their labile ligands with nucleophiles to yield Te(Nu)4 products, which may further either interact with other nucleophiles or undergo hydrolysis under aqueous conditions.15 Thus, ligand-substitution reactions of TeIV compounds include substitution of alcohols or halides with alcohols.16−18 Currently, in all preclinical and clinical studies, AS-101 is administered in aqueous physiological solutions such as saline and phosphate-buffered saline (PBS) solutions or in different aqueous propylene glycol (PG) mixtures. Taking it all together, we hypothesized that upon its dissolution and prior to entering the body, AS-101 may undergo ligandsubstitution reactions with the nucleophile aqueous/alcoholic solutions in which it is administered. This led us to initiate a set of experiments designed to study the chemical behavior of AS-101 under relevant aqueous/alcoholic biological solutions, which might also shed some light on other mechanistically open questions. AS-101 is poorly dissolved in PBS or saline solutions, in which it is mostly administered, only 150−200 μg/mL. Consequently, high signal-to-noise spectra are difficult to obtain even with prolonged data accumulation and high-field magnets. Therefore, in order to study the effect of the aqueous surroundings on AS101’s structure, we dissolved it in DMSO-d6 and reacted it with increasing amounts of D2O while following the outcome by 1H, 13 C, and 125Te NMR, in parallel with mass spectrometry analyses. AS-101’s anion has a typical trigonal-bipyramidal structure,19 with EG as a bidentate equatorial ligand (Figure 1A), and a mass

ABSTRACT: Since its first crystallization, the aqueous structure of the tellurium-containing experimental drug AS-101 has never been studied. We show that, under the aqueous conditions in which it is administered, AS-101 is subjected to an immediate ligand-substitution reaction with water, yielding a stable hydrolyzed oxide anion product that is identified, for the first time, to be TeOCl3−. Studying the structure of AS-101 in propylene glycol (PG), an alcoholic solvent often used for the topical and oral administration of AS-101, revealed the same phenomenon of ligand-substitution reaction between the alcoholic ligands. Upon exposure to water, the PGsubstituted product is also hydrolyzed to the same tellurium(IV) oxide form, TeOCl3−.

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he compound ammonium [trichloro(dioxoethylene-O,O′) tellurate] (AS101; Figure 1A) is a potent immunomodu-

Figure 1. (A) Structure of AS-101. (B) Previous hypothesized binding structure of AS-101, substituting its chlorine ligands with proteinaceous cysteine thiols, while the EG moiety was considered to be inert.

latory compound that exerts a variety of protective therapeutic applications in various in vivo models,1,2 as well as in preclinical and clinical studies.3−5 Much of the biological activity of AS-101 has been attributed to the pivotal TeIV atom and its specific chemical interactions with cysteine thiol residues.6−9 On the basis of the unique TeIV−S chemical affinity, we previously demonstrated that AS-101 interacts by ligand substitution with 4 equiv of L-cysteine as a thiol model compound to form a stable NMR-detectable Te(Cys)4 complex. Furthermore, AS-101 inhibited the cysteine proteases papain and cathepsin B, by inactivating the catalytic cysteines.9 The proteolytic activity could be recovered by adding reducing agents, suggesting either the formation of a TeIV−S chemical bond or the oxidation of catalytic thiol to a disulfide bond. Importantly, phase I and II clinical trials with advanced cancer patients showed a clear antitumoral effect of AS-101, which was later attributed to its ability to inhibit integrins and the ensuing secretion of the © XXXX American Chemical Society

Received: September 4, 2016

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DOI: 10.1021/acs.inorgchem.6b02138 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

structure, TeOCl3−, which is in agreement with all of our NMR findings, was consistently found with a typical tellurium trichloride “fingerprint” spectrum (Figure S2 in the SI). We further studied the interaction between AS-101 and PG, an alcoholic solvent that is often used as a solubilizing agent for the topical and oral administration of drugs, including AS-101. Following dissolution of AS-101 in DMSO-d6, we added gradual increasing amounts of PG to the NMR test tube and followed the products by 125Te NMR. The addition of 2 equiv of PG resulted in the formation of a major product at 1691 ppm, in addition to a residual 1680 ppm chemical shift of AS-101 (Figure 3A). 13C

of 295 (with the most abundant isotope of tellurium; Figure S1 in the Supporting Information, SI). Because of a plane of symmetry, the covalently bound form of EG gives a single 13C chemical shift at 67.3 ppm (Figure 2A). Adding 10 equiv of D2O into the NMR

Figure 2. AS-101, which is hydrolyzed upon the addition of water. (A) 13 C NMR spectrum of AS-101 in DMSO-d6. (B) 13C NMR spectrum of AS-101 in DMSO-d6 with the addition of 10 equiv of D2O. (C) 13C NMR spectrum of AS-101 in DMSO-d6 with the addition of a total of 50 equiv of D2O. (D) 125Te NMR spectrum of AS-101 in DMSO-d6. (E) 125 Te NMR spectrum of AS-101 in DMSO-d6 with the addition of 20 equiv of D2O. (F) 125Te NMR spectrum of AS-101 in DMSO-d6 with the addition of a total of 170 equiv of D2O.

Figure 3. AS-101 with PG substituted for its EG. (A) 125Te NMR spectrum of AS-101 in DMSO-d6 with 2 equiv of PG. (B) 13C NMR spectrum of AS-101 in DMSO-d6 with 2 equiv of PG. (C) 125Te NMR spectrum of AS-101 in DMSO-d6 with 2 equiv of PG and 223 equiv of D2O. (D) 13C NMR spectrum of AS-101 in DMSO-d6 with 2 equiv of PG and 223 equiv of D2O.

tube results in an immediate partial hydrolysis of the EG moiety, detected at 62.5 ppm (the chemical shift of free EG in this solvent), in addition to the presence of the starting material AS101 (Figure 2B). Adding larger amounts of D2O led to a continuous gradual hydrolysis toward its completion upon the addition of 50 equiv of D2O (Figure 2C). In order to quantitatively support our results regarding the full hydrolysis of the EG moiety in a mixture of DMSO-d6/D2O (Figure 2), we dissolved AS-101 in PBS (150 μg/mL), added a quantitative amount of pyridine, and followed the outcome by 1 H NMR. As expected, EG was fully hydrolyzed in PBS with no traces of AS-101 left (not shown). From the perspective of the tellurium atom, hydrolysis of the EG moiety may result in a few possible TeIV structures. Tellurium compounds exhibit a wide range of chemical shifts, from δ ∼ 2600−3300 ppm in highly deshielded compounds to δ ∼ 0−700 ppm in much more shielded ones, a fact that makes them easy to distinguish.20 Specifically, TeIV molecules exhibit a chemical shift range of δ ∼ 1000−2000 ppm. Applying 125Te NMR in parallel with the 1H and 13C NMR experiments described above revealed the formation of a few hydrolyzed TeIV-based structures. Specifically, upon the addition of 10−20 equiv of D2O, a few hydrolyzed products, in addition to the 1680 ppm signal of AS101 as a starting material (Figure 2D), were detected at 1642, 1626, and 1518 ppm (Figure 2E). However, adding 50 equiv of D2O resulted in the detection of only one stable hydrolyzed product at 1580 ppm, which is attributed to a TeIV compound (Figure 2F). In order to identify the EG-hydrolyzed TeIV product with the 1580 ppm resonance, we analyzed the same sample by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Indeed, a correlated oxide

NMR analysis of the same NMR tube revealed a new PGsubstituted product, in addition to AS-101, suggesting a rapid ligand-substitution reaction between PG and EG, as was also evidenced by the detection of a free hydrolyzed EG (Figure 3B). In order to support the 13C NMR findings, we applied MALDITOF analysis. Indeed, a new PG-substituted anion was detected at m/z 309, in addition to AS-101’s anion structure detected at m/z 295 (Figure S3 in the SI). While topically or orally administered, AS-101 is dissolved in different PG/H2O mixtures. Thus, we examined whether the PGsubstituted product (Figures 3A and S3 in the SI) might also be subjected to hydrolysis upon exposure to aqueous conditions. Thus, adding 20 equiv of D2O into the same NMR test tube (containing the TeIV−PG product) led to the formation of a few hydrolyzed products at 1652, 1623, and 1515 ppm in 125Te NMR (Figure S4 in the SI). Upon the addition of 200 equiv of D2O, only one TeIV hydrolyzed product was detected at 1580 ppm (Figure 3C). Intriguingly, the latter is identical with the chemical shift of the hydrolyzed form of AS-101, which we identified to be TeOCl3− (see above and Figure S2 in the SI). Application of 13C NMR confirmed our observations regarding the full hydrolysis process because neither AS-101 (detected at 67.48 ppm; Figure 3B) nor the PG-substituted product (detected at 18.57, 72.77, and 74.52 ppm; Figure 3B) was detected (Figure 3D). Our accumulated results suggest that, upon dissolution of AS101 in aqueous mixtures, it immediately substitutes its bidentate EG ligand with water to yield the tellurium(IV) oxide based product TeOCl3−. Furthermore, dissolving AS-101 in PG results in the same phenomenon of the ligand-substitution reaction between the bidentate alcoholic ligands, followed by hydrolysis B

DOI: 10.1021/acs.inorgchem.6b02138 Inorg. Chem. XXXX, XXX, XXX−XXX

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to TeOCl3− upon exposure to an aqueous environment (Scheme 1). Our results suggest that whether intravenously administered

Communication

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b02138. 125 Te NMR and mass spectra (PDF)

Scheme 1. Suggested Model of Ligand-Substitution Reactions of AS-101 with Either Water (Hydrolysis) or PG, and Their Further Hydrolysis To Generate TeOCl3−, Prior to Entering the Body

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Addresses §

Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel. ⊥ The Julius Spokojny Bioorganic Chemistry Laboratory, Department of Chemistry, Bar-Ilan University, Ramat-Gan 5290002, Israel.

in PBS/saline solutions or orally/topically administered in aqueous PG solution as a dissolving agent, AS-101 only exists as its hydrolyzed form, TeOCl3−, and not in its crystal form as intact AS-101 (Scheme 1). These results contradict the previous hypothesis (Figure 1B) by which the EG moiety of AS-101 is important for the three-dimensional fitting into the biological active site because it hydrolyzes prior to any interaction with cysteine thiols. Interestingly, the trivalent arsenic compound phenylarsine oxide (PAO), an established biological inhibitor known for its high affinity toward thiols, also interacts with vicinal thiol groups via its oxide moiety, forming a cyclic S−AsIII(Ph)−S adduct (Scheme 2A).21−23 Thus, it is intriguing to suggest that

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We greatly appreciate the useful advice of Drs. Hugo Gottlieb and Keren Keinan-Adamsky from Bar-Ilan University and of Dr. Arye Tishbee from The Weizmann Institute of Science. This study was partially supported by the Raoul Wallenberg Chair for Immunological Chemistry and by The Dr. Tovi CometWalerstein Cancer Research endowment.

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REFERENCES

(1) Sredni, B.; Caspi, R. R.; Klein, A.; Kalechman, Y.; Danziger, Y.; Benyaakov, M.; Tamari, T.; Shalit, F.; Albeck, M. A New Immunomodulating Compound (as-101) with Potential Therapeutic Application. Nature 1987, 330, 173−176. (2) Kalechman, Y.; Gafter, U.; Gal, R.; Rushkin, G.; Yan, D. H.; Albeck, M.; Sredni, B. Anti-IL-10 therapeutic strategy using the immunomodulator AS101 in protecting mice from sepsis-induced death: Dependence on timing of immunomodulating intervention. J. Immunol. 2002, 169, 384−392. (3) Strand, V. Biologic agents and innovative interventional approaches in the management of systemic lupus erythematosus. Curr. Opin. Rheumatol. 1999, 11, 330−340. (4) Kalich-Philosoph, L.; Roness, H.; Carmely, A.; Fishel-Bartal, M.; Ligumsky, H.; Paglin, S.; Wolf, I.; Kanety, H.; Sredni, B.; Meirow, D. Cyclophosphamide Triggers Follicle Activation and ″Burnout″; AS101 Prevents Follicle Loss and Preserves Fertility. Sci. Transl. Med. 2013, 5 (185), 185ra62. (5) Sredni, B. Immunomodulating tellurium compounds as anti-cancer agents. Semin. Cancer Biol. 2012, 22, 60−69. (6) Piovan, L.; Alves, M. F.; Juliano, L.; Bromme, D.; Cunha, R. L.; Andrade, L. H. Structure-activity relationships of hypervalent organochalcogenanes as inhibitors of cysteine cathepsins V and S. Bioorg. Med. Chem. 2011, 19, 2009−2014. (7) Caracelli, I.; Vega-Teijido, M.; Zukerman-Schpector, J.; Cezari, M. H. S.; Lopes, J. G. S.; Juliano, L.; Santos, P. S.; Comasseto, J. V.; Cunha, R. L. O. R.; Tiekink, E. R. T. A tellurium-based cathepsin B inhibitor: Molecular structure, modelling, molecular docking and biological evaluation. J. Mol. Struct. 2012, 1013, 11−18. (8) Sekhon, B. S. Metalloid compounds as drugs. Res. Pharm. Sci. 2013, 8, 145−158. (9) Albeck, A.; Weitman, H.; Sredni, B.; Albeck, M. Tellurium compounds: Selective inhibition of cysteine proteases and model reaction with thiols. Inorg. Chem. 1998, 37, 1704−1712. (10) Sredni, B.; Tichler, T.; Shani, A.; Catane, R.; Kaufman, B.; Strassmann, G.; Albeck, M.; Kalechman, Y. Predominance of TH1 response in tumor bearing mice and cancer patients treated with AS101. J. Natl. Cancer I 1996, 88, 1276−1284.

Scheme 2. (A) Formation of the Cyclic PAO Adduct and (B) the Corresponding Proposed Interaction of Hydrolyzed AS101 with Two Vicinal Thiol Groups

the oxide/hydrate moiety of AS-101’s hydrolysis product might be necessary for the ligand-substitution reactions with the proteinaceous cysteine thiols (Scheme 2B), much like the oxide moiety of PAO. Structural studies that aim to decipher the exact chemical mechanism of the interaction of AS-101 with in vitro and in vivo proteinaceous cysteine thiols are underway. In summary, our results show that the experimental drug AS101, which was previously established by us to bind proteinaceous vicinal thiols, exists only as its hydrolyzed form TeOCl3− when administered in vitro and in vivo. This fact indicates that the newly formed oxide moiety is necessary for AS-101’s biological activity. Therefore, from the perspective of future inorganic tellurium drug design, the tellurium oxide moiety should be considered and evaluated as a reactive group toward substitution with cysteine thiols. C

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Inorganic Chemistry (11) Sredni, B.; Albeck, M.; Tichler, T.; Shani, A.; Shapira, J.; Bruderman, I.; Catane, R.; Kaufman, B.; Kalechman, Y. Bone MarrowSparing and Prevention of Alopecia by As1o1 in Non-Small-Cell LungCancer Patients Treated with Carboplatin and Etoposide. J. Clin. Oncol. 1995, 13, 2342−2353. (12) Layani-Bazar, A.; Skornick, I.; Berrebi, A.; Pauker, M. H.; Noy, E.; Silberman, A.; Albeck, M.; Longo, D. L.; Kalechman, Y.; Sredni, B. Redox Modulation of Adjacent Thiols in VLA-4 by AS101 Converts Myeloid Leukemia Cells from a Drug-Resistant to Drug-Sensitive State. Cancer Res. 2014, 74, 3092−3103. (13) Rashidi, A.; Uy, G. L. Targeting the microenvironment in acute myeloid leukemia. Curr. Hematol Malig Rep 2015, 10, 126−131. (14) Ba, L. A.; Doring, M.; Jamier, V.; Jacob, C. Tellurium: an element with great biological potency and potential. Org. Biomol. Chem. 2010, 8, 4203−4216. (15) Patai, S.; Rapoport, Z. Organic Selenium and Tellurium Compounds; Wiley: New York, 1987; Vol. 2. (16) Denney, D. B.; Denney, D. Z.; Hammond, P. J.; Hsu, Y. F. Preparation and Nmr-Studies of Tetraalkoxyselenuranes and Tetraalkoxytelluranes. J. Am. Chem. Soc. 1981, 103, 2340−2347. (17) Gottlieb, H. E.; Hoz, S.; Elyashiv, I.; Albeck, M. Ligand exchange processes in tetraalkoxytelluranes. Inorg. Chem. 1994, 33, 808−811. (18) Cunha, R. L.; Urano, M. E.; Chagas, J. R.; Almeida, P. C.; Bincoletto, C.; Tersariol, I. L.; Comasseto, J. V. Tellurium-based cysteine protease inhibitors: evaluation of novel organotellurium(IV) compounds as inhibitors of human cathepsin B. Bioorg. Med. Chem. Lett. 2005, 15, 755−760. (19) Albeck, M.; Tamari, T.; Sredni, B. Synthesis and Properties of Ammonium Trichloro(Dioxyethylene-O,O′)Tellurate (as-101) - a New Immunomodulating Compound. Synthesis 1989, 1989, 635−636. (20) O’Brien, D. H.; Dereu, N.; Grigsby, R. A.; Irgolic, K. J.; Knapp, F. F. Tellurium-125 chemical shifts of symmetric and unsymmetric dialkyl ditellurides. Organometallics 1982, 1, 513−517. (21) Owen, T. S.; Xie, X. J.; Laraway, B.; Ngoje, G.; Wang, C.; Callahan, B. P. Active site targeting of hedgehog precursor protein with phenylarsine oxide. ChemBioChem 2015, 16, 55−58. (22) Manickam, N.; Ahmad, S. S.; Essex, D. W. Vicinal thiols are required for activation of the alphaIIbbeta3 platelet integrin. J. Thromb. Haemostasis 2011, 9, 1207−1215. (23) Oeste, C. L.; Diez-Dacal, B.; Bray, F.; Garcia de Lacoba, M.; de la Torre, B. G.; Andreu, D.; Ruiz-Sanchez, A. J.; Perez-Inestrosa, E.; Garcia-Dominguez, C. A.; Rojas, J. M.; Perez-Sala, D. The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways. PLoS One 2011, 6, e15866.

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DOI: 10.1021/acs.inorgchem.6b02138 Inorg. Chem. XXXX, XXX, XXX−XXX