Current methods of drug screening in human blood focus on the immediate products of the affected pathway and mostly rely on approaches that lack sensitivity and the capacity for multiplex analysis. We have developed a sensitive and selective method based on ultra-performance liquid chromatography–tandem mass spectrometry to scan the effect of drugs on the bioactive eicosanoid lipidome in vitro and ex vivo. Using small sample sizes, we can reproducibly measure a broad spectrum of eicosanoids in human blood and capture drug-induced substrate rediversion and unexpected shifts in product formation. Microsomal prostaglandin E synthase-1 (mPGES-1) is an antiinflammatory drug target alternative to COX-1/-2. Contrasting effects of targeting mPGES-1 versus COX-1/-2, due to differential substrate shifts across the lipidome, were observed and can be used to rationalize and evaluate drug combinations. Finally, the in vitro results were extrapolated to ex vivo studies by administration of the COX-2 inhibitor, celecoxib, to volunteers, illustrating how this approach can be used to integrate preclinical and clinical studies during drug development.
Liudmila L. Mazaleuskaya, John A. Lawson, Xuanwen Li, Gregory Grant, Clementina Mesaros, Tilo Grosser, Ian A. Blair, Emanuela Ricciotti, Garret A. FitzGerald
Antiinflammatory drug targets from the COX and 5-lipoxygenase pathways.
Arachidonic acid (AA) is metabolized by 3 major enzymatic cascades: COX (COX-1/-2), lipoxygenase (5-LOX, 12-LOX, 15-LOX), and cytochrome P450 (CYP). The 2 COX isoforms, COX-1 and COX-2, oxidize AA to the hydroperoxy-endoperoxide PGG2 and then reduce PGG2 to the unstable intermediate hydroxy-endoperoxide PGH2. PGH2 is transformed to eicosanoids PGI2, TxA2, PGE2, PGD2, and PGF2α by the tissue-specific isomerases prostaglandin I synthase (PGIS), thromboxane synthase (TxS), prostaglandin E synthases (cPGES, mPGES-1, mPGES-2), prostaglandin D synthases (L/H-PGDS), and prostaglandin F synthase (PGFS), respectively. Each eicosanoid acts at the corresponding receptor distributed in various tissues. The 5-LOX enzyme, together with the accessory protein 5-LOX–activating protein (FLAP), catalyzes the conversion of AA to 5-hydroperoxyeicosatetraenoic acid (5-HpETE) and then to the unstable intermediate leukotriene A4 (LTA4). 5-HpETE can be reduced by glutathione peroxidases into 5-HETE alcohol. The LTA4 hydrolase (LTA4H) metabolizes LTA4 to leukotriene B4 (LTB4), which acts at BLT1 and BLT2 receptors on target cells. Alternatively, LTA4 can be conjugated with glutathione (GSH) by LTC4 synthase (LTC4S) to yield cysteinyl leukotrienes LTC4, LTD4, and LTE4, which act at CysLT receptors. Enzyme inhibitors and receptor antagonists are shown in green, while drug targets are depicted in red. cPGES, cytosolic PGE synthase; mPGES-1 and mPGES-2, microsomal PGE synthase-1 and -2, respectively; L/H-PGDS, hematopoietic and lipocalin-type PGD synthases; IP, prostacyclin receptor; TP, thromboxane receptor; EP, E prostanoid receptor; DP, D prostanoid receptor; FP, F prostanoid receptor; EET, epoxyeicosatrienoic acid.