Structural and Chemical Biology of the Interaction of Cyclooxygenase with Substrates and Non-Steroidal Anti-Inflammatory Drugs

Abstract

Cyclooxgenases are key enzymes of lipid signaling. They carry out the first step in the production of prostaglandins, important mediators of inflammation, pain, cardiovascular disease, and cancer, and they are the molecular targets for nonsteroidal anti-inflammatory drugs, which are among the oldest and most chemically diverse set of drugs known. Homodimeric proteins that behave as allosterically modulated, functional heterodimers, the cyclooxygenases exhibit complex kinetic behavior, requiring peroxide-dependent activation and undergoing suicide inactivation. Due to their important physiological and pathophysiological roles and keen interest on the part of the pharmaceutical industry, the cyclooxygenases have been the focus of a vast array of structural studies, leading to the publication of over 80 crystal structures of the enzymes in complex with substrates or inhibitors supported by a wealth of functional data generated by site-directed mutation experiments. In this review, we explore the chemical biology of the cyclooxygenases through the lens of this wealth of structural and functional information. We identify key structural features of the cyclooxygenases, break down their active site into regional binding pockets to facilitate comparisons between structures, and explore similarities and differences in the binding modes of the wide variety of ligands (both substrates and inhibitors) that have been characterized in complex with the enzymes. Throughout, we correlate structure with function whenever possible. Finally, we summarize what can and cannot be learned from the currently available structural data and discuss the critical intriguing questions that remain despite the wealth of information that has been amassed in this field.

Figure 1

Prostaglandin (PG) biosynthetic pathway. Bis-dioxygenation and cyclization of arachidonic acid at the cyclooxygenase active site of COX-1 or COX-2 yields PGG₂. Reduction of the 15-hydroperoxyl group of PGG₂ at the peroxidase active site of COX-1 or COX-2 yields PGH₂. PGH₂ serves as a substrate for five different synthases, producing four signaling PG products (PGE₂, PGI₂, PGF_2α, and PGD₂) or thromboxane A₂ (TXA₂). PGH₂ is chemically unstable under physiological conditions, and in the absence of the synthase enzymes, it is hydrolyzed to a mixture of PGE₂ and PGD₂.

Figure 2

Proposed mechanism of the cyclooxygenase reaction. Activation of the COX enzymes occurs through oxidation of the heme prosthetic group during reduction of a peroxide substrate at the peroxidase active site. Transfer of an electron from Tyr-385 in the cyclooxygenase active site to the heme then generates the catalytic tyrosyl radical. Step 1: the tyrosyl radical abstracts the 13-(pro)-S-hydrogen atom (indicated by an asterisk) from AA. Resonance places the unpaired electron at carbon-13, carbon-11, or carbon-15. Addition of oxygen at the carbon-11 or carbon-15 radical, followed by enzymatic or nonenzymatic reduction, results in the 11(R,S)-HETE or 15(R,S)-HETE minor products (indicated in blue), respectively. Step 2: antarafacial oxygen addition occurs at carbon-11. Step 3: the peroxyl group then attacks carbon-9, forming the endoperoxide ring and placing the unpaired electron at carbon-8. Step 4: bond formation between carbon-8 and carbon-12 generates the five-membered ring of PGG₂ and places the unpaired electron on carbon-13. Resonance enables migration of the unpaired electron to carbon-15. Step 5: attack of oxygen at carbon-15 follows, generating a peroxyl radical at that position. Step 6: transfer of a hydrogen atom from Tyr-385 reduces the peroxyl radical to a hydroperoxide, yielding PGG₂, and regenerates the tyrosyl radical for a new round of catalysis. Step 7: reduction of PGG₂ at the peroxidase active site yields PGH₂. Alternatively, attack of oxygen at carbon-13 rather than carbon-15 in step 5 followed by enzymatic or nonenzymatic reduction leads to a PGH₂ analog with the hydroxyl group at carbon-13 (shown in blue). Note that the overall mechanism requires a peroxidase turnover to produce the catalytic tyrosyl radical, but once this has occurred, the cyclooxygenase reaction is self-perpetuating.

Figure 3

Domain structure of the cyclooxygenases. The top view shows the dimeric protein (COX-1) as observed from the side (i.e., parallel to the plane of the membrane). Each of the bottom views follows rotation of the top view by 90°. To the left, the protein is viewed looking down on the enzyme from above. To the right, the protein is viewed looking up from below. In all cases, the epidermal growth factor domain is gold, the membrane-binding domain is green, the catalytic domain is cyan, heme is sienna, and the flurbiprofen ligand is red. The membrane-binding domain inserts into the top leaflet of the underlying membrane bilayer. From PDB 1CQE.

Figure 4

Structure of the cyclooxygenase active site. Two wall-eyed stereo views of the COX-1 monomer (A and B) and a close-up view of the cyclooxygenase active site (C) are shown as observed from the side (i.e., parallel to the plane of the membrane). In all cases, Co³⁺-protoporphyrin IX (an inactive heme analog) is dark brown, and AA is red mesh. The side chains of the constriction residues (Arg-120, Tyr-355, and Glu-524) are displayed and labeled, as are the catalytic residue (Tyr-385) and the target of aspirin-mediated inactivation (Ser-530), which are located at the bend of the L-shaped channel. In A and B, His-388, the proximal heme ligand, and His-207, which serves as the distal heme ligand through a coordinating water molecule, are visible. Helices 2 (residues 195–207, light blue), 8 (residues 378–385, medium blue), 6 (residues 324–354, light green), and 17 (residues 519–535, dark green), which surround the active site, along with helices 5 (residues 295–320, dark purple), 11/12 (residues 444–459, medium purple), and 16 (residues 503–510, light purple) form a bundle that is conserved among a number of peroxidases, with helices 2, 5, 6, 8, and 11/12 involved in binding the heme prosthetic group. From PDB 1DIY.

Figure 5

(A) Wall-eyed stereo view of AA bound in the cyclooxygenase active site of COX-1 and the side chains that make up the proximal binding pocket (green), the central binding pocket (magenta), and the distal binding pocket (cyan). This view is similar to that used to depict most structures of fatty acids in complex with COX-1 or COX-2 throughout the review. (B) Wall-eyed stereo view of an overlay of the structures of four fatty acids in the active site of COX-1. Fatty acids and amino acid side chains are colored from lightest to darkest in the order of LA, DHLA, EPA, and AA. Notable is the minimal movement of active site residues to accommodate the structural differences among the various fatty acids. Monoscopic views of the individual structures are provided in Figure S1. From PDB 1DIY (A and B) and 1IGZ, 1FE2, and 1IGX (B only).

Figure 6

Wall-eyed stereo view of the proximal AA binding pocket as observed from the side (i.e., parallel to the plane of the membrane) (A) or looking downward toward the membrane from above (B). AA is colored by element, and its surface is shown as a mesh. Side chains of the residues comprising the pocket are displayed, and their surface is shown in solid tan. From PDB 1DIY.

Figure 7

Wall-eyed stereo view of the central AA binding pocket as observed from the side (i.e., parallel to the plane of the membrane) (A) or looking along the axis of the pocket (B). AA is colored by element, and its surface is shown as a mesh. Side chains of the residues comprising the pocket are displayed, and their surface is shown in solid tan. From PDB 1DIY.

Figure 8

Wall-eyed stereo view of AA bound to the active site of COX-1. (A) A pocket formed by Phe-381, Leu-384, Trp-387, Phe-518, and Met-522 provides space for formation of the endoperoxide ring. (B) Tyr-348, Phe-381, Typ-385, and Ser-530 surround carbon-15 of AA (green) dictating the orientation of oxygen addition at this site. AA is colored by element (with the exception of carbon-15 in B), and its surface is shown as a mesh. Side chains of the residues indicated are displayed, and their surface is shown in solid tan. From PDB 1DIY.

Figure 9

Wall-eyed stereo view of the distal AA binding pocket as observed from the side (i.e., parallel to the plane of the membrane) (A) or looking along the axis of the pocket (B). AA is colored by element, and its surface is shown as a mesh. Side chains of the residues comprising the pocket are displayed, and their surface is shown in solid tan. From PDB 1DIY.

Figure 10

(A) Wall-eyed stereo view of the structure of AA (nonproductive conformation) bound in the cyclooxygenase active site of COX-2 and the side chains that make up the proximal binding pocket (green), the central binding pocket (magenta), and the distal binding pocket (cyan). Note that AA is observed in two slightly different conformations. (B) Wall-eyed stereo view of an overlay of the structures of the nonproductive (dark gray) and productive (light gray) conformations of AA bound in the cyclooxygenase active site. Residues in the surrounding binding pockets are colored similarly to those in (A) with the lighter and darker colors corresponding to the productive and nonproductive conformations, respectively. Also shown is the marked difference in the position of Leu-531 between the productive conformation (gold) and the nonproductive conformation (sienna). From PDB 3HS5.

Figure 11

Wall-eyed stereo view of the structure of PGG₂/H₂ overlaid with that of AA (productive conformation) bound in the active site of COX-2. Side chains that make up the central binding pocket are shown (light and dark magenta), as are Leu-384 (gold and sienna), proximal binding pocket residues Arg-120 and Tyr-355 (light and dark green), and distal binding pocket residue Phe-381 (dark and light blue). PGG₂ (dark gray) and AA (light gray) are colored by heteroatom. In the case of residue side chains, the darker and lighter colors denote positions in the complexes containing PGG₂/H₂ and AA, respectively. The major shift in the position of Tyr-385 is readily apparent, as is the upward shift in the position of the carboxyl group of PGG₂ relative to that of AA. These displacements are enabled by the absence of heme and likely would not occur in the holoenzyme. From PDB 3HS5 (chain B) and 1DDX.

Figure 12

Wall-eyed stereo view of the interaction of the proximal inhibitor binding pocket with (S)-flurbiprofen as observed from the side (i.e., parallel to the plane of the membrane) (A) or looking upward from the membrane (B). (S)-Flurbiprofen is colored by element, and its surface is shown as a mesh. Side chains of the residues comprising the pocket are displayed, and their surface is shown in solid tan. From PDB 1EQH.

Figure 13

Wall-eyed stereo view of the interaction of the central inhibitor binding pocket with (S)-flurbiprofen as observed from the side (i.e., parallel to the plane of the membrane) (A) or looking upward from the membrane (B). (S)-Flurbiprofen is colored by element, and its surface is shown as a mesh. Side chains of the residues comprising the pocket are displayed, and their surface is shown in solid tan. From PDB 1EQH.

Figure 14

Wall-eyed stereo views of overlays of the structures of (R)- and (S)-flurbiprofen (A) and (R)- and (S)-naproxen (B), bound in the cyclooxygenase active site of COX-2 and the side chains that make up the proximal binding pocket (light/dark green) and the central binding pocket (pink/magenta). In each case, structures related to the (R)-enantiomer (tan) are shown in the lighter color and those related to the (S)-enantiomer (sienna) are shown in the darker color. From PDB 3PGH and 3RR3 (A) and PDB 3NT1 and 3Q7D (B).

Figure 15

Wall-eyed stereo view of the interaction of the proximal inhibitor binding pocket with indomethacin as observed from the side (i.e., parallel to the plane of the membrane) (A) or from above (B). Indomethacin is colored by element, and its surface is shown as a mesh. Side chains of the residues comprising the pocket are displayed, and their surface is shown in solid tan. From PDB 4COX.

Figure 16

Wall-eyed stereo view of the interaction of the central inhibitor binding pocket with indomethacin as observed from the side (i.e., parallel to the plane of the membrane) (A) or looking upward from the membrane (B). Indomethacin is colored by element, and its surface is shown as a mesh. Side chains of the residues comprising the pocket are displayed, and their surface is shown in solid tan. From PDB 4COX.

Figure 17

Wall-eyed stereo views of the structure of indomethacin-dansyl conjugate 1 (A) and indomethacin-dansyl conjugate 2 (B) bound in the cyclooxygenase active site of COX-2. The side chains that make up the proximal binding pocket (green), the central binding pocket (magenta), and membrane-binding domain residues that interact with the dansyl moiety (cyan) are shown. From PDB 6BL4 (A) and 6BL3 (B).

Figure 18

(A) Wall-eyed stereo view of the interaction of the oxicam binding pocket with isoxicam as observed from the side (i.e., parallel to the plane of the membrane), and comparison of the size of the oxicam binding pocket (B) with the comparable region in the structure of COX-2 complexed to naproxen (C). The difference in the position of Leu-531 is clearly visible, as is the marked difference in the size of the pockets between the two complexes. Isoxicam and naproxen are colored by element, and their surface is shown as a mesh. Side chains of the residues comprising the pocket are displayed, and their surface is shown in solid tan. From PDB 4M10 and 3NT1.

Figure 19

(A) and (B) Wall-eyed stereo views of the structure of isoxicam overlaid with that of (S)-flurbiprofen bound in the COX-2 active site along with the residues that form the oxicam pocket. Isoxicam is shown in sienna, and the associated amino acid residues are in dark blue. (S)-Flurbiprofen is in tan, and the associated amino acids are in light blue. Note the large difference in the conformation of Leu-531 between the two structures. This rotation is necessary to provide access to the oxicam pocket. Note also the difference in the positions of Met-113, Val-116, and Leu-117 that results from the movement of helix D. From PDB 4M10 and 3PGH.

Figure 20

Wall-eyed stereo view of isoxicam bound in the cyclooxygenase active site of COX-2, and the hydrogen -bonded water molecules through which the inhibitor establishes polar contacts with the side chains of residues in the proximal binding pocket (A and C) and the central binding pocket (B and C). Side chains are colored in green (proximal binding pocket) or magenta (central binding pocket) with the exception of Arg-120, Tyr-355, Tyr-385, and Ser-530, which are colored by heteroatom on a sienna background. Isoxicam is colored by atom. The coordinated water molecule is shown as a red sphere. From PDB 4M10.

Figure 21

Wall-eyed stereo view of the interaction of the COX-2 side pocket with the phenylsulfonamide group of SC-558 as seen from two different views. SC-558 is colored by element, and its surface is shown as a mesh. Side chains of the residues comprising the pocket are displayed, and their surface is shown in solid tan. In (B), the phenylsulfonamide group of SC-558 is completely surrounded by the pocket, so that only the sulfonamide can be seen. From PDB 6COX.

Figure 22

(A) Wall-eyed stereo view of the structure of SC-558 bound in the cyclooxygenase active site of COX-2 highlighting residues involved in side pocket formation and interactions. Overlaid are the same residues as observed in the structure of (S)-flurbiprofen bound in the cyclooxygenase active site of COX-2. Highlighted are Phe-518, which packs against Val-434 (pink/magenta) to open the side pocket, residues 352–355 (tan/sienna), which move to enlarge the side pocket upon diarylheterocycle binding, Val-523 (light/dark green), which is Ile-523 in COX-1, and other side pocket residues (light/navy blue). In each case, the darker colors correspond to residues in the SC-558-COX-2 structure whereas the lighter colors correspond to the (S)-flurbiprofen-COX-2 structure. (B) Same as (A) except that the structure of the SC-558-COX-2 complex (dark colors) is overlaid with the corresponding residues from the structure of (S)-flurbiprofen bound in the cyclooxygenase active site of COX-1 (light colors). Note the three key residues at positions 434, 513, and 523 that are different between the two isoforms. In particular, Ile-523 in COX-1 encroaches on the side pocket, and Ile-434 in COX-1 prevents the movement of Phe-518 that provides access into the pocket. From PDB 6COX, 1EQH, and 3PGH.

Figure 23

(A) Wall-eyed stereo view of the structure of harmaline compound 3 bound in the cyclooxygenase active site of COX-2. The side chains that make up the proximal binding pocket (green), the central binding pocket (magenta), and the oxicam pocket (cyan) are shown. Note that Val-349 is part of both the proximal pocket and the oxicam pocket. It is colored in cyan. (B) Wall-eyed stereo view of an overlay of the structures of harmaline compound 3 and indomethacin bound in the active site of COX-2, including residues that make up the proximal (light/dark green) and central (pink/magenta) binding pockets. Indomethacin and compound 3 are shown in light and dark gray, respectively, and the lighter residue colors correspond to those in the indomethacin-COX-2 complex. Also shown is Leu-531 (yellow/sienna) which moves away from the constriction to accommodate the tricyclic harmaline nucleus. From PDB 63VR.

Figure 24

(A) and (B) Wall-eyed stereo views of the structure of (S)-flurbiprofen bound in the cyclooxygenase active site of a wild-type/R12Q COX-1 heterodimer and the side chains that make up the proximal binding pocket (green) and the central binding pocket (magenta). The structures of the two subunits are overlaid, with the wild-type subunit (containing flurbiprofen) shown in the lighter colors. Also shown are residues 121–129, which exist in two conformations in the R120Q subunit (sienna) depending on the presence or absence of bound flurbiprofen. Subunits containing inhibitor exhibit the same conformation as is observed in the wild-type subunit (tan), whereas those lacking inhibitor diverge between Ser-121 and Pro-125. From PDB 3N8W.