Prostaglandin H synthase (PGHS), a key enzyme in prostanoid biosynthesis, exists as two isoforms. PGHS-1 is considered a basal enzyme; PGHS-2 is associated with inflammation and cell proliferation. A number of highly selective inhibitors for PGHS-2 cyclooxygenase activity are known. Inhibition by these agents involves an initial reversible binding, followed by a time-dependent transition to a much higher affinity enzyme-inhibitor complex, making these agents potent and poorly reversible PGHS-2 inhibitors. To investigate the PGHS-2 structural features that influence the time-dependent action of the selective inhibitors, we have constructed a three-dimensional model of human PGHS-2 by homologous modeling. Examination of the PGHS-2 model identified Val 1 catalyzes the first committed step in prostanoid biosynthesis, the bis-dioxygenation of arachidonic acid to form prostaglandin G 2 (1). Two isoforms of PGHS are known, with PGHS-1 generally ascribed housekeeping roles, whereas the strong induction of PGHS-2 by cytokines is believed to be a key part in inflammatory processes (2). Many cyclooxygenase inhibitors have been discovered; the most potent include agents, such as indomethacin, which trigger a time-dependent change in the protein once bound in the cyclooxygenase active site, thus achieving essentially irreversible inhibition without covalent modification of protein or agent (3-5). More recently, a set of time-dependent cyclooxygenase inhibitors with very high selectivity for PGHS-2 has been identified (6 -8). Little is known about the nature of the structural change(s) underlying noncovalent time-dependent cyclooxygenase inhibition of either isoform or about the protein structural features that lead to the remarkable specificity of the PGHS-2 inhibitors.We have constructed a three-dimensional model for human PGHS-2 based on the crystal structure of ovine PGHS-1 (9) and identified Val 509 in PGHS-2 as one of the few residues in the cyclooxygenase active site that is not conserved in PGHS-1. Recombinant human PGHS-2 was expressed with four Val 509 mutations to assess their effects on cyclooxygenase activity and on inhibition by agents specific for PGHS-2. Several of the Val 509 mutations led to a loss of the characteristic time-dependent action of the agents without a large perturbation of substrate or inhibitor binding. The results point to a role for Val 509 in the time-dependent structural transition, which makes these agents such potent and selective inhibitors of human PGHS-2 cyclooxygenase activity. EXPERIMENTAL PROCEDURESMaterials-Heme, dimethyl sulfoxide, and D-tryptophan were from Sigma; Tween 20 was from Pierce; arachidonate was from NuChek Preps, Inc.;[1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]arachidonate (55 mCi/mmol) was from Amersham Corp. Nimesulide was from Cayman Chemical Co. DuP697, SC58125, and NS398 were generous gifts from Drs. Chakk Ramesha (Roche Pharmaceuticals) and Paul J. Marshall (CIBA Pharmaceuticals).Homologous Modeling-The structural model for human PGHS-2 was built from th...

Prostacyclin (PGI2), a vascular protector with vasodilation and antithrombotic properties, is synthesized by coupling reactions of cyclooxygenase (COX, the first enzyme) with PGI2 synthase (PGIS, the second enzyme) using arachidonic acid (AA) as an initial substrate. The first COX product, prostaglandin H2 (PGH2) is also a command substrate for other prostanoid enzymes that produce distinct eicosanoids, such as thromboxane A2 (TXA2). The actions of TXA2 to cause vasoconstriction and platelet aggregation oppose the vasodilatory and anti-aggregatory effects of PGI2. Specifically upregulating PGI2 biosynthesis is an ideal model for the prevention and treatment of the TXA2-mediated thrombosis involved in strokes and myocardial infarctions. Here, we report that a single protein was constructed by linking COX-2 and PGIS together to form a new fusion enzyme through a transmembrane domain with 10 or 22 residues. The engineered protein expressed in HEK293 and COS-7 cells was able to continually convert AA to prostaglandin (PG) G2 (catalytic step 1), PGH2 (catalytic step 2), and PGI2 (catalytic step 3). The studies first demonstrate that a single protein with three catalytic functions could directly synthesize PGI2 from AA with a Km of approximately 3.2 microM. Specific upregulation of PGI2 biosynthesis through expression of the engineered single protein in the cells has shown strong activity in inhibiting platelet aggregation induced by AA in vitro, which creates a great potential for the fusion enzyme to be used as one of the new therapeutic interventions for strokes and heart attacks. The studies have also provided a model linking COX with its downstream enzymes to specifically regulate biosynthesis of eicosanoids which have potent biological functions.

Thromboxane A(2) receptor (TP receptor), a prostanoid receptor, belongs to the G protein-coupled receptor family, composed of three intracellular loops and three extracellular loops connecting seven transmembrane helices. The highly conserved extracellular domains of the prostanoid receptors were found in the second extracellular loop (eLP(2)), which was proposed to be involved in ligand recognition. The 3D structure of the eLP(2) would help to further explain the ligand binding mechanism. Analysis of the human TP receptor model generated from molecular modeling based on bacteriorhodopsin crystallographic structure indicated that about 12-14 A separates the N- and C-termini of the extra- and intracellular loops. Synthetic loop peptides whose termini are constrained to this separation are presumably more likely to mimic the native loop structure than the corresponding loop region peptide with unrestricted ends. To test this new concept, a peptide corresponding to the eLP(2) (residues 173-193) of the TP receptor has been made with the N- and C-termini connected by a homocysteine disulfide bond. Through 2D nuclear magnetic resonance (NMR) experiments, complete (1)H NMR assignments, and structural construction, the overall 3D structure of the peptide was determined. The structure shows two beta-turns at residues 180 and 185. The distance between the N- and C-termini of the peptide shown in the NMR structure is 14.2 A, which matched the distance (14.5 A) between the two transmembrane helices connecting the eLP(2) in the TP receptor model. This suggests that the approach using the constrained loop peptides greatly increases the likelihood of solving the whole 3D structures of the extra- and the intracellular domains of the TP receptor. This approach may also be useful in structural studies of the extramembrane loops of other G protein-coupled receptors.

The pathogenesis of numerous cardiovascular, pulmonary, inflammatory, and thromboembolic diseases can be related to arachidonic acid (AA) metabolites. One of these bioactive metabolites of particular importance is thromboxane A(2) (TXA(2)). It is produced by the action of thromboxane synthase on the prostaglandin endoperoxide H(2)(PGH(2)), which results from the enzymatic degradation of AA by the cyclooxygenases. TXA(2) is a potent inducer of platelet aggregation, vasoconstriction and bronchoconstriction. It is involved in a series of major pathophysiological states such as asthma, myocardial ischemia, pulmonary hypertension, and thromboembolic disorders. Therefore, TXA(2) receptor antagonists, thromboxane synthase inhibitors and drugs combining both properties have been developed by several pharmaceutical companies since the early 1980s. Several compounds have been launched on the market and others are under clinical evaluation. Moreover, the recent literature reported the interest of thromboxane modulators, which combine another pharmacological activity such as, platelet activating factor antagonism, angiotensin II antagonism, or 5-lipoxygenase inhibition. In this review, we will propose a description of the recently described thromboxane modulators of major interest from both a pharmacological and a chemical point of view.

Tumor suppressor protein p53, our most critical defense against tumorigenesis, can be made powerless by mechanisms such as mutations and inhibitors. Fortilin, a 172-amino acid polypeptide with potent anti-apoptotic activity, is up-regulated in many human malignancies. However, the exact mechanism by which fortilin exerts its anti-apoptotic activity remains unknown. Here we present significant insight. Fortilin binds specifically to the sequence-specific DNA binding domain of p53. The interaction of fortilin with p53 blocks p53-induced transcriptional activation of Bax. In addition, fortilin, but not a double point mutant of fortilin lacking p53 binding, inhibits p53-dependent apoptosis. Furthermore, cells with wild-type p53 and fortilin, but not cells with wild-type p53 and the double point mutant of fortilin lacking p53 binding, fail to induce Bax gene and apoptosis, leading to the formation of large tumor in athymic mice. Our results suggest that fortilin is a novel p53-interacting molecule and p53 inhibitor and that it is a logical molecular target in cancer therapy.Tumor suppressor protein p53 keeps us free of cancer when it is functional. Mice lacking p53 (p53 Ϫ/Ϫ ) spontaneously develop numerous neoplasms within 6 months (1). Mutated p53 genes are seen in more than 50% of all human cancers, making them the most frequently observed genetic derangement in human cancer (2). At a molecular level, the ability of p53 to eliminate cancerous cells relies on its ability to induce apoptosis, through either the transcriptional activation of proapoptotic genes such as Noxa (3), PUMA 4 (4), and Bax (5) or the direct transcription-independent activation of Bax on mitochondria (6). Growing cancers manage to keep p53 in check either by mutating the p53 gene itself (7-9) or by expressing p53 inhibitors such as Mdm2 (9, 10). The function of fortilin, a ubiquitous, highly conserved, 172-amino acid polypeptide also known as "translationally controlled tumor protein," or TCTP, remained unknown (11,12). Investigation in our laboratory and others showed that fortilin possesses potent anti-apoptotic activity (13-15). Fortilin is overexpressed in human cancers (16,17), the depletion of which is associated with spontaneous death of cancerous cells (13, 18). Higher levels of fortilin are associated with more malignant cancer phenotypes (14). Although heterozygous fortilin-deficient mice (fortilin ϩ/Ϫ ) were normal in appearance and fertile, homozygous fortilin-deficient (fortilin Ϫ/Ϫ ) mice were embryonically lethal around 3.5 days postcoitus due to massive apoptosis, as reported by our laboratory and others (19 -21).The mechanism by which fortilin functions as an anti-apoptotic molecule has been under robust investigation. First, based on the fact that fortilin physically interacts with myeloid cell leukemia protein-1 (MCL1), an anti-apoptotic Bcl-2 family member, it was suggested that fortilin stabilizes and exerts its anti-apoptotic activity through MCL1 (22). However, fortilin is capable of protecting cells from apoptosis in the...