Distinction of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition from cyclooxygenase-2 inhibition in cells using a novel, selective mPGES-1 inhibitor

Abstract

Inflammation-induced microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme that synthesizes prostaglandin E₂ (PGE₂) downstream of cyclooxygenase-2 (COX-2). The efficacy of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in the treatment of the signs and symptoms of osteoarthritis, rheumatoid arthritis and inflammatory pain, largely attributed to the inhibition of PGE₂ synthesis, provides a rationale for exploring mPGES-1 inhibition as a potential novel therapy for these diseases. Toward this aim, we identified PF-9184 as a novel mPGES-1 inhibitor. PF-9184 potently inhibited recombinant human (rh) mPGES-1 (IC₅₀ = 16.5 ± 3.8 nM), and had no effect against rhCOX-1 and rhCOX-2 (>6500-fold selectivity). In inflammation and clinically relevant biological systems, mPGES-1 expression, like COX-2 expression was induced in cell context- and time-dependent manner, consistent with the kinetics of PGE₂ synthesis. In rationally designed cell systems ideal for determining direct effects of the inhibitors on mPGES-1 function, but not its expression, PF-9184 inhibited PGE₂ synthesis (IC₅₀ in the range of 0.5–5 μM in serum-free cell and human whole blood cultures, respectively) while sparing the synthesis of 6-keto-PGF_1α (PGF_1α) and PGF_2α. In contrast, as expected, the selective COX-2 inhibitor, SC-236, inhibited PGE₂, PGF_1α and PGF_2α synthesis. This profile of mPGES-1 inhibition, distinct from COX-2 inhibition in cells, validates mPGES-1 as an attractive target for therapeutic intervention.

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-PGF_1α (PGF_1α) 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 PGF_1α 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 PGF_1α, PGF_2α 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.