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Cellular association, intracellular distribution, and efflux of auranofin via sequential ligand exchange reactions
Abstract:
Auranofin (AF), an orally active, antiarthritic agent, modulates the functional activities of macrophages in vivo and in vitro. To better understand the molecular mechanism of action of auranofin with macrophages we have investigated its cellular association, intracellular distribution, and efflux with RAW 264.7 cells using auranofin radiolabeled within the triethylphosphine (Et3P) [3H], the gold [195Au] or the tetraacetylthioglucose (TATG)[14C] moieties of the molecule. Evaluation of the effects of auranofin on RAW 264.7 cells demonstrates that (1) cellular association of this compound was concentration, time and temperature dependent; (2) cellular association of AF was inhibited by N-ethylmaleimide but not by 2,4-dinitrophenol and NaF; (3) cellular association and uptake of Au and Et3P into cells was reduced when the drug was preincubated with increasing concentrations of fetal calf serum and albumin; (4) no tetraacetylthioglucose from the auranofin molecule became cell associated whereas the Au and Et3P moieties were internalized and distributed between the nuclear, cytosolic and membrane fractions of cells; and (5) efflux of Au and Et3P from RAW 264.7 cells was time and temperature dependent. Based on these data we propose a model, a sequential ligand exchange process, that describes the molecular interactions of auranofin and possibly other gold compounds with these cells.
Insights
Auranofin (AF) interacts with macrophages, with gold and triethylphosphine moieties entering cells. A sequential ligand exchange model explains these interactions, crucial for understanding AF's antiarthritic effects.
Area of Science:
- Pharmacology
- Cell Biology
- Immunology
Background:
- Auranofin (AF) is an orally active antiarthritic drug.
- AF modulates macrophage functional activities in vivo and in vitro.
Purpose of the Study:
- Investigate the molecular mechanism of auranofin's action on macrophages.
- Determine AF's cellular association, intracellular distribution, and efflux using RAW 264.7 cells.
Main Methods:
- Used radiolabeled auranofin ([3H]Et3P, [195Au], [14C]TATG) to track molecular components.
- Evaluated concentration, time, and temperature dependence of cellular association.
- Assessed effects of N-ethylmaleimide, 2,4-dinitrophenol, NaF, fetal calf serum, and albumin.
- Analyzed intracellular distribution and efflux of drug moieties.
Main Results:
- Cellular association of AF was concentration, time, and temperature dependent.
- N-ethylmaleimide inhibited AF association, but 2,4-dinitrophenol and NaF did not.
- Uptake of gold (Au) and triethylphosphine (Et3P) was reduced by serum proteins.
- Tetraacetylthioglucose (TATG) was not cell-associated; Au and Et3P were internalized and distributed intracellularly.
- Efflux of Au and Et3P was time and temperature dependent.
Conclusions:
- Auranofin's interaction with macrophages involves internalization of Au and Et3P moieties.
- A sequential ligand exchange process is proposed to model these molecular interactions.
- This mechanism is relevant for understanding auranofin and other gold compounds' effects on cells.