Evaluating metal ion salts as acid hydrolase mimics: metal-assisted hydrolysis of phospholipids at lysosomal pH

Abstract

Niemann-Pick disease and drug-induced phospholipidosis are lysosomal storage disorders in which there is an excessive accumulation of sphingomyelin in cellular lysosomes. Here we have explored the possibility of developing metal-based therapeutic agents to reverse phospholipid build-up through phosphate ester bond hydrolysis at lysosomal pH (~4.8). Towards this end, we have utilized a malachite green/molybdate-based colorimetric assay to quantitate the inorganic phosphate released upon the hydrolysis of sphingomyelin by twelve d- and f-block metal ion salts. In reactions conducted at 60 °C, the yields produced by the cerium(IV) complex Ce(NH(4))(2)(NO(3))(6) were superior. An Amplex(®) Red-based colorimetric assay and mass spectrometry were then employed to detect choline. The data consistently showed that Ce(IV) hydrolyzed sphingomyelin more efficiently at lysosomal pH: i.e., yields of choline and phosphate were 54 ± 4 and 22 ± 5 % at pH ~ 4.8, compared to 8 ± 1 and 5 ± 2 % at pH ~ 7.2. Hydrolysis at 60 °C could be significantly increased by converting sphingomyelin vesicles to mixed lipid vesicles and mixed micelles of Triton X-100. We then utilized cerium(IV) to cleave sphingomyelin at 37 °C (no Triton X-100). Although choline and phosphate levels were relatively low, hydrolysis continued to be considerably more efficient at lysosomal pH. A side by side comparison to phosphatidylcholine was then made. While the yields of choline and phosphate produced by phosphatylcholine were higher, the ratio of pH ~ 4.8 hydrolysis to pH ~ 7.2 hydrolysis was usually more favorable for sphingomyelin (37 and 60 °C).

Tuning Cerium(IV)‐Assisted Hydrolysis of Phosphatidylcholine Liposomes under Mildly Acidic and Neutral Conditions

“…Our best result for phosphatidylcholine liposomes was obtained in the presence of Ce IV and bis–tris propane (BTP). Compared to our first‐generation experiments,5d, 7c the yield of PC hydrolysis at ∼pH 4.8 and the ratio of pH 4.8 hydrolysis to pH 7.2 hydrolysis were significantly increased, and visible Ce IV precipitation was cleared from hydrolysis reactions. When the single‐chain nonionic surfactant Triton X‐100 was used to convert the liposomes to mixed micelles, cleavage of phosphatidylcholine occurred in near‐quantitative yields (37 °C and ∼pH 4.8).…”

“…Similar to our first generation, published experiments,5d, 7c the cleavage reactions contained 2 m M of PC in liposome form and 10 m M Ce IV (NH 4 ) 2 (NO 3 ) 6 and were run at ∼pH 4.8 and 7.2 for a period of 20 h. Twenty millimolar concentrations of each of the six ligands were added, and the reaction temperature was raised from 37 to 60 °C. The production of inorganic phosphate by Ce IV ‐assisted hydrolysis of phosphate ester bonds (Scheme ) was measured by using a commercially available malachite green/molybdate‐based colorimetric assay kit.…”

“…The reaction time (20 h) and remaining reagent concentrations (2 m M PC, 10 m M Ce(NH 4 ) 2 (NO 3 ) 6 ) were carried over from the preceding experiment. The inorganic phosphate liberated by Ce IV was quantitated with a malachite green/molybdate reagent, and a choline‐specific colorimetric assay based on Amplex Red dye5d, 7c was utilized in the event that the lower reaction temperature limited hydrolysis to the choline side of phosphorous ( 3 ; Scheme ). As desired, treatment of PC with Ce IV in the presence of BTP produced minimal amounts of phospholipid cleavage under near‐neutral conditions (Figure 2).…”

“…With the goal of designing a lysosomal phospholipase mimic, we treated liposomes of L ‐α‐phosphatidylcholine and sphingomyelin (PC and SM, respectively; 1 in Scheme ) with twelve metal ions that are known to hydrolyze activated and unactivated amide and/or phosphate ester bonds in peptides, nucleic acids, and other compounds (Ce IV , Zr IV , Hf IV , Co II , Cu II , Eu III , La III , Ni II , Pd II , Y III , Yb III , and Zn II ) 5d. 7c In the event that a metal ion cleaved both of the phosphate ester bonds of the phospholipid, free inorganic phosphate ( 2 ) would be released (Scheme ). In these first generation experiments, only Ce IV , Zr IV , Hf IV , Eu III , La III , Pd II , and Yb III were capable of hydrolyzing SM and/or PC.…”

“…Hydrolysis yields at ∼pH 4.8 and 7.2 were 18±3 % and 10±2 %, respectively 5d. 7c More efficient suppression of cleavage under neutral conditions would be critical for protecting cells from hydrolytic damage in the event of any leakage of a metal‐based therapeutic agent from the lysosome into the cytoplasm. Moreover, the hydrolysis reactions contained undesired Ce IV precipitation, and the yields of phosphate diester cleavage at 37 °C and ∼pH 4.8 were low.…”