Activity-based targeting of secretory phospholipase A2 enzymes: A fatty-acid-binding-protein assisted approach

Abstract

Syntheses and enzymological characterization of fluorogenic substrate probes targeting secretory phospholipase A2 (sPLA2) for detection and quantitative assays are presented. Three fluorogenic phosphatidylcholine analogs PC-1, PC-2, and PC-3 each containing the duo of 7-mercapto-4-methyl-coumarin fluorophore and 2,4-dinitroanaline quencher on either tail were synthesized from (R)-3-amino-1,2-propanediol and R-(−)-2,2-dimethyl-1,3-dioxolane-4-methanol. These small reporter groups are advantageous in preserving natural membrane integrity. Phosphocholine was incorporated into the sn-3 position of the glycerol backbone. Acyl amino group at the sn-1 position in PC-1 and PC-2 is meant to block sPLA1. The sn-1 and sn-2 positions of the glycerol backbone in PC-1 have a quencher terminated 12-carbon chain and fluorophore terminated 11-carbon chain respectively. PC-2 has a quencher terminated 3-carbon chain at the sn-2 and chain terminating fluorescent reporter at the sn-1 positions. PC-3 resembles PC-1 except for an ester instead of amide at the sn-1 position, because of which it is more similar to natural phospholipids than PC-1. It was designed to elucidate the effect of replacing the ester group with amide by comparing its hydrolysis rate with that of PC-1. Design principles apply to synthesis of other labeled phospholipids. Enzymological characterization using bee-venom sPLA2 was performed by a fatty-acid-binding-protein fluorescence assay and by pH-Stat method in which the amount of fatty acid released by hydrolysis is given by the amount of base required to maintain a constant pH of 8.0. Hydrolytic activity toward PC-1 and PC-3 were each about 238 ± 25 μmol/mg/min and 537 μmol/mg/min on unmodified phospholipid. Ester to amide change did not affect hydrolysis rates. Activity toward PC-2 was about 45-μmol/mg/min. PC-1 and PC-3 show potential for targeted real-time spectrophotometric assay of sPLA2.

Arginine 123 of apolipoprotein A-I is essential for lecithin:cholesterol acyltransferase activity

“…The effect of the apoA-I mutations on LCAT phospholipid hydrolysis was studied using a fluorometric assay kit for measuring phospholipase A2 activity of LCAT (STA-615; Cell Biolabs, Inc., San Diego, CA). The approach utilizes a fluorogenic dual-labeled POPC with fluorophores attached to the terminal ends of both acyl chains, similar to the fluorescence assays developed for measuring the phospholipase A2 activity of LCAT (41) and other enzymes (42,43). The fluorophores are in a quenched state when the phospholipid is uncleaved.…”

Insight on Mercapto-Coumarins: Synthesis and Reactivity

“…Hajdu and his co-workers reported the synthesis and enzymological characterization of three fluorogenic phosphatidylcholine analogs PC-1 ( 77 ), PC-2 ( 78 ), and PC-3 ( 79 ), targeting the detection and the quantitative assays of phospholipase A2 (sPLA2) [ 79 ]. Each demonstrated molecule contained a 7-mercapto-4-methyl coumarin fluorophore and 2,4-dinitroaniline quencher on both tails ( Scheme 27 ) [ 79 ]. The small size of these molecules helps to not disrupt the natural membrane.…”

Phosphorus-Based Probes as Molecular Tools for Proteome Studies: Recent Advances in Probe Development and Applications

“…Much work has been dedicated to the development of probes for phospholipase A, including a fluorogenic probe in which the ester in the sn-1 position was exchanged for a nonhydrolyzable ether or amide moiety. Potential permeability issues due to the ionic character of the phospholipid probe were addressed by the application of a prodrug strategy. , …”

“…The Hajdu group , designed fluorogenic probes 66 – 68 that target secretory phospholipase sPLA 2 . Analogs modified with amide or ester groups in the sn-1 position did not show a difference in hydrolysis rates.…”

“…Potential permeability issues due to the ionic character of the phospholipid probe were addressed by the application of a prodrug strategy. 109,111 The Hajdu group 110,112 designed fluorogenic probes 66−68 that target secretory phospholipase sPLA 2 . Analogs modified with amide or ester groups in the sn-1 position did not show a difference in hydrolysis rates.…”