Disulfide-Linked Dinitroxides for Monitoring Cellular Thiol Redox Status through Electron Paramagnetic Resonance Spectroscopy

Abstract

Intracellular thiol-disulfide redox balance is crucial to cell health, and may be a key determinant of a cancer's response to chemotherapy and radiation therapy. The ability to assess intracellular thiol-disulfide balance may thus be useful not only in predicting responsiveness of cancers to therapy, but in assessing predisposition to disease. Assays of thiols in biology have relied on colorimetry or fluorimetry, both of which require UV-visible photons, which do not penetrate the body. Low-frequency electron paramagnetic resonance imaging (EPRI) is an emerging magnetic imaging technique that uses radio waves, which penetrate the body well. Therefore, in combination with tailored imaging agents, EPRI affords the opportunity to image physiology within the body. In this study, we have prepared water-soluble and membrane-permeant disulfide-linked dinitroxides, at natural isotopic abundance, and with D,(15)N-substitution. Thiols such as glutathione cleave the disulfides, with simple bimolecular kinetics, to yield the monomeric nitroxide species, with distinctive changes in the EPR spectrum. Using the D,(15)N-substituted disulfide-dinitroxide and EPR spectroscopy, we have obtained quantitative estimates of accessible intracellular thiol in cultured human lymphocytes. Our estimates are in good agreement with published measurements. This suggests that in vivo EPRI of thiol-disulfide balance is feasible. Finally, we discuss the constraints on the design of probe molecules that would be useful for in vivo EPRI of thiol redox status.

Imaging thiol redox status in murine tumors in vivo with rapid-scan electron paramagnetic resonance

“…PxSSPx and PxSH equilibrate between extracellular and intracellular compartments rapidly relative to the time scale of the experiment 4 ; therefore, assuming that cellular thiol concentration, [RSH], remains constant, the time course of cleavage can be analyzed by a simple kinetic model incorporating two processes: 1) cleavage of PxSSPx by intracellular thiols, which decreases PxSSPx and increases PxSH; and 2) clearance of PxSSPx and PxSH from the detection volume of the resonator. Process 1 is described by Eqs.…”

“…Spectroscopic results can be interpreted using the rate constant for cleavage of PxSSPx by RSH at 37 °C, estimated from in vitro measurements 4 . The bimolecular rate constant is 0.747 M −1 s −1 at pH 7.2 and 37 °C which, in combination with our observed cleavage kinetics yields an estimate of [RSH] 2.7 to 4.8 mM in untreated control tumors and [RSH] 1.5 to 2.1 mM in BSO-treated tumors.…”

“…We have recently synthesized a water-soluble, disulfide-linked dinitroxide (hereafter “disulfide-dinitroxide”) in both the normoisotopic and 2 H, 15 N-substituted forms 4 . In this report, the term “PxSSPx” designates the 2 H, 15 N-substituted disulfide-dinitroxide.…”

“…In this report, the term “PxSSPx” designates the 2 H, 15 N-substituted disulfide-dinitroxide. The disulfide-dinitroxides and the corresponding monomeric forms (generated through cleavage by thiols) have been characterized by EPR spectroscopy at X-band, L-band, and at 250 MHz 4,17 . Importantly, the rate of disulfide cleavage in living cells is markedly decreased after treatment with L -buthionine sulfoximine (BSO, a specific inhibitor of GSH biosynthesis), which suggests that the disulfide-dinitroxides could be useful as probes of thiol redox status in vivo.…”

Designing Molecular Probes To Prolong Intracellular Retention: Application to Nitroxide Spin Probes

“…For example, spin probes can be tailored for desired pharmacokinetics or designed to report specific physiological information (e.g., oxygen tension, pH, redox status). 2–10 …”