Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than non-malignant cells due to genetic alterations and abnormal growth and as a result, maintenance of the anti-oxidant glutathione (GSH) is essential for their survival and proliferation1–3. Under elevated ROS conditions endogenous l-Cysteine (l-Cys) production is insufficient for GSH synthesis, necessitating l-Cys uptake, predominantly in its disulfide form l-Cystine (CSSC) via the xCT(−) transporter. Here we show that administration of an engineered, pharmacologically optimized, human Cyst(e)inase enzyme mediates sustained depletion of the extracellular l-Cys and CSSC pool in mice and non-human primates, selectively causes cell cycle arrest and death (PI and Annexin-V staining) in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS, yet results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53−/− mice that develop disease resembling human chronic lymphocytic leukemia. The observation that enzyme-mediated depletion of the serum l-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods suggests that Cyst(e)inase represents a safe and effective therapeutic modality for inactivating anti-oxidant cellular responses in a wide range of malignancies4,5.

Cyclooxygenases (COX) play an important role in lipid signaling by oxygenating arachidonic acid to endoperoxide precursors of prostaglandins and thromboxane. Two cyclooxygenases exist which differ in tissue distribution and regulation but otherwise carry out identical chemical functions. The neutral arachidonate derivative, 2-arachidonylglycerol (2-AG), is one of two described endocannabinoids and appears to be a ligand for both the central (CB1) and peripheral (CB2) cannabinoid receptors. Here we report that 2-AG is a substrate for COX-2 and that it is metabolized as effectively as arachidonic acid. COX-2-mediated 2-AG oxygenation provides the novel lipid, prostaglandin H 2 glycerol ester (PGH 2 -G), in vitro and in cultured macrophages. PGH 2 -G produced by macrophages is a substrate for cellular PGD synthase, affording PGD 2 -G. Pharmacological studies reveal that macrophage production of PGD 2 -G from endogenous sources of 2-AG is calcium-dependent and mediated by diacylglycerol lipase and COX-2. These results identify a distinct function for COX-2 in endocannabinoid metabolism and in the generation of a new family of prostaglandins derived from diacylglycerol and 2-AG. Cyclooxygenase (COX 1; prostaglandin endoperoxide synthase, EC 1.14.99.1) catalyzes the bis-dioxygenation of arachidonic acid, generating prostaglandin (PG) H 2 , the precursor to a diverse family of lipid mediators including PGs, thromboxane, and prostacyclin (1). The discovery of a second COX isoform, COX-2, has provided important insights into the molecular basis of inflammation, hyperalgesia, and cancer and has established a novel pharmacological target for their treatment (2-4). The major functional differences between COX-1 and COX-2 are believed to be related to their differential regulation and tissue distribution (5). COX-1 is a constitutive enzyme, whereas COX-2 is inducible and highly regulated by a range of agonists (6, 7). COX-1 activity accounts for PG and thromboxane production in gastric mucosa, kidney, and platelets (3). COX-2 activity is primarily responsible for PG biosynthesis in the central nervous system and inflammatory cells (8 -10). These observations suggest that COX-1 and COX-2 serve different physiological and pathophysiological functions.The possibility that COX-2 has distinct biochemical functions has not been explored extensively. There are conserved structural differences between the active sites of COX-1 and COX-2 which have been exploited in the development of selective COX-2 inhibitors. It is possible that these or other structural differences may have evolved to support separate biochemical functions for the two COX isoforms. The first indication of a separate biochemical function for COX-2 was provided by the observation that it selectively oxygenates the neutral ethanolamide derivative of arachidonic acid, anandamide (11). Anandamide and 2-arachidonylglycerol (2-AG) are endogenous ligands for the cannabinoid receptors that bind ⌬ 9 -tetrahydrocannabinol and mediate its pharmacological effects (12). The ca...

Prostaglandins were discovered in human semen in 1930, but their low concentrations and instability precluded identification for nearly 30 years (for a brief historical review, see Ref. 1). Once they were identified, it was clear they arose from polyunsaturated fatty acids by a complex series of reactions involving oxygenation, cyclization, and the generation of five chiral centers from an achiral substrate. The mechanism of prostaglandin biosynthesis was outlined in 1967 by Hamberg and Samuelsson (2), and the basic tenets have been confirmed in subsequent studies. The key step in their proposed mechanism was the formation of bicyclic peroxides (endoperoxides) as the initial products of polyunsaturated fatty acid oxygenation (Fig. 1). The term cyclooxygenase (COX) 1, 2 was coined to describe the enzyme that carried out this complex chemical transformation, and its role was confirmed by the isolation of prostaglandin endoperoxides in 1973 (3, 4).In addition to catalyzing a fascinating metabolic transformation, COX is an enormously important pharmacological target. Vane reported in 1971 (5) that non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin formation and demonstrated that their relative inhibitory potency in vitro correlates to their antiinflammatory activity in vivo. This not only explained the beneficial activity of NSAIDs but also their side effects such as gastrointestinal toxicity and bleeding because prostaglandins and related molecules (i.e. thromboxane) are involved in a very broad range of physiological and pathophysiological responses. The importance of these molecules as autocrine and paracrine mediators has been confirmed recently by the phenotypes of mice bearing targeted deletions in COX genes or prostaglandin receptor genes.The discovery of a second gene (COX-2) coding for cyclooxygenase and the demonstration that its protein product is distributed differently from the originally discovered enzyme (COX-1) raised the possibility that some of the beneficial effects of NSAIDs may be separable from their side effects by development of isoform-selective inhibitors (6 -9). This hypothesis has been dramatically validated by the demonstration that selective COX-2 inhibitors are anti-inflammatory and analgesic but lack the gastric toxicity associated with all currently available NSAIDs (10, 11). Cyclooxygenase CatalysisSubstantial evidence supports the hypothesis that COX oxygenates arachidonic acid by a free radical mechanism (Fig. 1). Thus, COX appears to have co-opted the process that gives rise to isoprostanes to generate prostaglandins. The major differences between COX-catalyzed and spontaneous oxidation of arachidonic acid are the increased rate and high degree of stereochemical control of the enzymatic reaction (1 of 64 possible isomers predominates). Thus, the overall role of COX is rather simple: stereospecifically remove the 13-pro-S-hydrogen and control the stereochemistry of oxygenation. How does it do this?Oxidizing Agent-A protein tyrosyl radical appears to be the oxidiz...

A variety of drugs inhibit the conversion of arachidonic acid to prostaglandin G 2 by the cyclooxygenase (COX) activity of prostaglandin endoperoxide synthases. Several modes of inhibitor binding in the COX active site have been described including ion pairing of carboxylic acid containing inhibitors with Arg-120 of COX-1 and COX-2 and insertion of arylsulfonamides and sulfones into the COX-2 side pocket. Recent crystallographic evidence suggests that Tyr-385 and Ser-530 chelate polar or negatively charged groups in arachidonic acid and aspirin. We tested the generality of this binding mode by analyzing the action of a series of COX inhibitors against site-directed mutants of COX-2 bearing changes in Arg-120, Tyr-355, Tyr-348, and Ser-530. Interestingly, diclofenac inhibition was unaffected by the mutation of Arg-120 to alanine but was dramatically attenuated by the S530A mutation. Determination of the crystal structure of a complex of diclofenac with murine COX-2 demonstrates that diclofenac binds to COX-2 in an inverted conformation with its carboxylate group hydrogen-bonded to Tyr-385 and Ser-530. This finding represents the first experimental demonstration that the carboxylate group of an acidic non-steroidal anti-inflammatory drug can bind to a COX enzyme in an orientation that precludes the formation of a salt bridge with Arg-120. Mutagenesis experiments suggest Ser-530 is also important in time-dependent inhibition by nimesulide and piroxicam.The cyclooxygenase (COX) 1 activity of prostaglandin endoperoxide synthase catalyzes the incorporation of two molecules of O 2 into arachidonic acid to yield the hydroperoxy endoperoxide, prostaglandin G 2 (PGG 2 ) (1, 2). PGG 2 diffuses from the cyclooxygenase active site and binds at the peroxidase active site where it is reduced to the hydroxy endoperoxide, PGH 2 , the precursor to prostaglandins, thromboxane, and prostacyclin (3). Two COX isoforms exist that differ in expression pattern, mode of regulation, and biological function (4). COX-1 is generally considered the homeostatic form of the enzyme as it is constitutively expressed in a number of tissues, whereas COX-2 is sensitive to induction in many tissues by a broad range of physiological and pathological stimuli (5). Inhibition of COX enzymes by non-steroidal anti-inflammatory drugs (NSAIDs) accounts for their anti-inflammatory and analgesic activities, as well as their gastrointestinal toxicity (6). Development of selective COX-2 inhibitors has reduced the gastrointestinal liability (7).Structural and functional analysis is providing an increasingly detailed picture of the molecular determinants of COXsubstrate and COX-inhibitor interactions (3). COX-1 and COX-2 have very similar structures characterized by a membrane-binding domain comprised of amphipathic helices forming the entrance to a long hydrophobic channel (8 -10). This channel leads deep inside the protein, and at its upper end comprises the cyclooxygenase active site. The cyclooxygenase active site is separated from the opening near the membran...

All nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the cyclooxygenase (COX) isozymes to different extents, which accounts for their anti-inflammatory and analgesic activities and their gastrointestinal side effects. We have exploited biochemical differences between the two COX enzymes to identify a strategy for converting carboxylate-containing NSAIDs into selective COX-2 inhibitors. Derivatization of the carboxylate moiety in moderately selective COX-1 inhibitors, such as 5,8,11,14-eicosatetraynoic acid (ETYA) and arylacetic and fenamic acid NSAIDs, exemplified by indomethacin and meclofenamic acid, respectively, generated potent and selective COX-2 inhibitors. In the indomethacin series, esters and primary and secondary amides are superior to tertiary amides as selective inhibitors. Only the amide derivatives of ETYA and meclofenamic acid inhibit COX-2; the esters are either inactive or nonselective. Inhibition kinetics reveal that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Site-directed mutagenesis of murine COX-2 indicates that the molecular basis for selectivity differs from the parent NSAIDs and from diarylheterocycles. Selectivity arises from novel interactions at the opening and at the apex of the substrate-binding site. Lead compounds in the present study are potent inhibitors of COX-2 activity in cultured inflammatory cells. Furthermore, indomethacin amides are orally active, nonulcerogenic, anti-inflammatory agents in an in vivo model of acute inflammation. Expansion of this approach can be envisioned for the modification of all carboxylic acid-containing NSAIDs into selective COX-2 inhibitors.