The role of the Hint1 protein in the metabolism of phosphorothioate oligonucleotides drugs and prodrugs, and the release of H 2 S under cellular conditions

• Published in: Biochemical pharmacology 2019-05, Vol.163, p.250
• Main Authors: Krakowiak, Agnieszka, Piotrzkowska, Danuta, Kocoń-Rębowska, Beata, Kaczmarek, Renata, Maciaszek, Anna
• Format: Article
• Language: English
• Subjects: A549 Cells; Adenosine Monophosphate - metabolism; Guanosine Monophosphate - analogs & derivatives; Guanosine Monophosphate - metabolism; HeLa Cells; Humans; Hydrogen Sulfide - metabolism; Lysine - analogs & derivatives; Lysine - metabolism; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Phosphorothioate Oligonucleotides - metabolism; RNA Interference
• ISSN: 1873-2968
• DOI: 10.1016/j.bcp.2019.02.018

Phosphorothioate oligonucleotides (PS-oligos) containing sulfur atom attached in a nonbridging position to the phosphorus atom at one or more internucleotide bond(s) are often used in medicinal applications. Their hydrolysis in cellular media proceeds mainly from the 3'-end, resulting in the appearance of nucleoside 5'-O-phosphorothioates ((d)NMPS), whose further metabolism is poorly understood. We hypothesize that the enzyme responsible for (d)NMPS catabolism could be Hint1, an enzyme that belongs to the histidine triad (HIT) superfamily and is present in all organisms. We previously found that (d)NMPS were desulfurated in vitro to yield (d)NMP and H S in a Hint1-assisted reaction. Here, we demonstrate that AMPS/GMPS/dGMPS introduced into HeLa/A549 cells are intracellularly converted into AMP/GMP/dGMP and H S. The level of the released H S was relative to the concentration of the compounds used and the reaction time. Using RNAi technology, we have shown decreased levels of AMPS/GMPS desulfuration in HeLa/A549 cells with reduced Hint1 levels. Finally, after transfection of a short Rp-d(A A A) oligomer into HeLa cells, the release of H S was observed. These results suggest that the metabolic pathway of PS-oligos includes hydrolysis into (d)NMPS (by cellular nucleases) followed by Hint1-promoted conversion of the resulting (d)NMPS into (d)NMP accompanied by H S elimination. Our observations may be also important for possible medicinal applications of (d)NMPS because H S is a gasotransmitter involved in many physiological and pathological processes.