Distinction of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition from cyclooxygenase-2 inhibition in cells using a novel, selective mPGES-1 inhibitor
Graphical abstract
Introduction
Pain associated with human diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA) is treated with analgesic drugs, including nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 (COX-2) inhibitors (COXibs). These broadly used drugs work by inhibiting PGE2 synthesis through inhibition of the activity of COX-2 [1]. Consistent with the causative role of PGE2 in the signs and symptoms of RA and OA, antagonistic molecules that prevent PGE2 from binding to its receptor have shown efficacy in animal models of arthritis [2]. Furthermore, efficacy in pre-clinical models of inflammation and hyperalgesia was demonstrated with neutralizing PGE2 antibodies [3]. Thus, it was reasonable to hypothesize that inhibition of microsomal prostaglandin E synthase-1 (mPGES-1), the enzyme that is responsible for PGE2 synthesis in inflammation-associated pathologies, may be as efficacious as NSAIDs and COXibs.
mPGES-1 is the terminal enzyme one-step downstream of COX-2 that synthesizes prostaglandin E2 (PGE2) from PGH2, the unstable peroxide intermediate derived from arachidonate catabolism. mPGES-1 expression, like COX-2 expression, is induced by various inflammatory stimuli in normal cells, and in cells and tissues derived from human patients and animal disease models [4]. These include for example, macrophages and synovial fibroblasts derived from RA patients (RASF), OA chondrocytes and several other cell types involved in inflammatory diseases and certain cancers [5], [6]. The link between mPGES-1 and the production of PGE2 in inflammatory conditions was established first by data derived from genetic manipulation of mPGES-1 encoding gene. Indeed, mPGES-1-deleted cells produced significantly lower levels of PGE2 in response to inflammatory stimuli [7], [8], and consistently, mPGES-1 knockout (KO) mice were less sensitive to inflammatory and neuropathic pain and refractory to the development of joint pathology in rodent arthritis models [9], [10], [11]. These mice had no issues with thrombogenesis or blood pressure and renal function when placed on normal salt diet [12], [13]. The findings that indomethacin-induced gastrointestinal (GI) tract lesions in animals can be alleviated by the administration of the prostacyclin agonist Beraprost [14], suggest that depletion of both PGE2 and 6-keto-PGF1α (PGF1α) may be required for the development of GI tract lesions. Consistent with this notion, mPGES-1 inhibition had no inhibitory effects on the synthesis of other prostanoids, including thromboxane B2 (TXB2) and PGF1α and did not cause GI tract lesions in animals [15].
The exciting data generated with mPGES-1 KO mice triggered drug discovery efforts for the identification of pharmacological agents to inhibit this enzyme. In the process, compounds that modulate mPGES-1 expression or function have been identified [15], [16], [17], [18]. Efficacy in animal models of inflammation has been shown with some of these tool compounds [15], [16], though clinical benefits of these mPGES-1 inhibitors have yet to be demonstrated. Here, we described PF-9184 as a novel mPGES-1 inhibitor. PF-9184 is a potent inhibitor of mPGES-1 function, not its expression. In inflammation relevant cell systems, PF-9184 inhibited PGE2 synthesis while sparing the synthesis of PGF1α, PGF2α and TXB2. Thus, this class of mPGES-1 inhibitors opens avenues for the development of novel inhibitors of PGE2 synthesis and the treatment of signs and symptoms of OA and RA.
Section snippets
Reagents
Nonidet P-40, BSA, E. coli-derived lipopolysaccharide (LPS), phosphatase inhibitor cocktail, stannous (tin) chloride (SnCl2), pyridoxine hydrochloride, NaCl, Tween-20, diethyldithiocarbamate acid, phenylmethylsulfonyl fluoride, complete protease inhibitor mixture tablet, EDTA and dithiothreitol were obtained from Sigma (St. Louis, MO). Anti-GAPDH antibodies and Immobilon-P membranes were obtained from Millipore (Billerica, MA). Dulbecco's modified essential media (DMEM), Dulbecco's
PF-9184 is a novel, potent and selective inhibitor of mPGES-1
PF-9184 (N-(3′,4′-dichlorobiphenyl-4-yl)-4-hydroxy-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide) (Fig. 1) was identified from structure activity relationship of an oxicam series. It is a novel, potent inhibitor of recombinant human (rh) mPGES-1 (IC50 = 16.5 ± 3.8 nM, n = 8), and had no effect against rhCOX-1 and rhCOX-2 with IC50 of 118 and 263 μM, respectively.
Inflammation induces mPGES-1 expression in vivo in rat air pouch: effects of PF-9184
To investigate mPGES-1 expression in vivo in inflammatory conditions, air pouches generated in rats by subcutaneous injection of sterile air
Discussion
We identified PF-9184 from an oxicam series as a potent and highly selective inhibitor of human mPGES-1. The selectivity of PF-9184 was further demonstrated in a variety of biological systems optimally set up for determining direct effects of the inhibitors on mPGES-1 function, not its expression. Indeed, we showed that in cell systems, PF-9184 potently blocked mPGES-1 ability to synthesize PGE2 from PGH2, and consequently, increased the levels of PGF1α and PGF2α via a process known as shunting
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Cited by (0)
- 1
Present address: Indications Discovery, Pfizer Inc., 700 Chesterfield Parkway, Chesterfield, MO 63017, USA.
- 2
Present address: Icagen, Inc., 4222 Emperor Blvd., Durham, NC 27703, USA.