Cellular Fates of Manganese(II) Pentaazamacrocyclic Superoxide Dismutase (SOD) Mimetics: Fluorescently Labeled MnSOD Mimetics, X-ray Absorption Spectroscopy, and X-ray Fluorescence Microscopy Studies

Abstract

Manganese(II) pentaazamacrocyclic complexes (MnPAMs) can act as small-molecule mimics of manganese superoxide dismutase (MnSOD) with potential therapeutic application in conditions linked to oxidative stress. Previously, the in vitro mechanism of action has been determined, their activity has been demonstrated in cells, and some representatives of this class of MnSOD mimetics have entered clinical trials. However, MnPAM uptake, distribution, and metabolism in cells are largely unknown. Therefore, we have used X-ray fluorescence microscopy (XFM) and X-ray absorption spectroscopy (XAS) to study the cellular fate of a number of MnPAMs. We have also synthesized and characterized fluorescently labeled (pyrene and rhodamine) manganese(II) pyane [manganese(II) trans-2,13-dimethyl-3,6,9,12,18-pentaazabicyclo[12.3.1]octadeca-1(18),14,16-triene] derivatives and investigated their utility for cellular imaging of MnPAMs. Their SOD activity was determined via a direct stopped-flow technique. XFM experiments show that treatment with amine-based manganese(II) pyane type pentaazamacrocycles leads to a 10-100-fold increase in the overall cellular manganese levels compared to the physiological levels of manganese in control cells. In treated cells in general, manganese was distributed throughout the cell body, with a couple of notable exceptions. The lipophilicity of the MnPAMs, examined by partitioning in octanol-buffer system, was a good predictor of the relative cellular manganese levels. Analysis of the XAS data of treated cells revealed that some fraction of amine-based MnPAMs taken up by the cells remained intact, with the rest transformed into SOD-active manganese(II) phosphate. Higher phosphate binding constants, determined from the effect of the phosphate concentration on in vitro SOD activity, were associated with more extensive metabolism of the amine-based MnPAMs to manganese(II) phosphate. In contrast, the imine-based manganese(II) pydiene complex that is prone to hydrolysis was entirely decomposed after uptake and free manganese(II) was oxidized to a manganese(III) oxide type species, in cytosolic compartments, possibly mitochondria. Complex stability constants (determined for some of the MnPAMs) are less indicative of the cellular fate of the complexes than the corresponding phosphate binding constants.

Potential Therapeutic Applications of MnSODs and SOD‐Mimetics

“…The complex M40403i st he prototypical complex of the family.S uch an MnSOD mimic acts as ac atalyst to scavenge the superoxide radical. [75] Ap hase 2c linical trial gave positive results by enhancing the analgesic effects forp ost-operative pain relief from dentalo perations, when combining M40403 with morphine. These types of synthetic enzymesa lso have the advantage that they do not have ab ell-shaped curve,a s opposed to native SOD enzymes.…”

“…The clinicalt rials that involve M40403 have now been either suspended (trial:N CT00033956) or terminated (trial: NCT00101621). [75] Ap hase 2c linical trial gave positive results by enhancing the analgesic effects forp ost-operative pain relief from dentalo perations, when combining M40403 with morphine. Another trial was performed on ab unionectomy post-operative pain model using such ac ombination approach.…”

Inhibition of Copper Transport Induces Apoptosis in Triple-Negative Breast Cancer Cells and Suppresses Tumor Angiogenesis

“…Analysis was performed using MAPS software (30) as described elsewhere (31). The nuclear region of the cell was defined as the region of high P and Zn content.…”

Radiation-Mediated Tumor Growth Inhibition Is Significantly Enhanced with Redox-Active Compounds That Cycle with Ascorbate

“…and tumor growth. As a result of their low metal/ligand stabilities, the therapeutic effects of Mn salens and Mn pentaazamacrocycles, seen elsewhere (3,20,55), may be the consequence of the moderate SOD-like activity of released manganese.…”