Functional investigations of thromboxane synthase (CYP5A1) in lipid bilayers of nanodiscs.

CYP5A1 is a membrane-associated cytochrome P450 that metabolizes the cyclooxygenase product prostaglandin (PGH2 ) into thromboxane A2 (TXA2 ), a potent inducer of vasoconstriction and platelet aggregation. Although CYP5A1 is an ER-bound protein, the role of membranes in modulating the thermodynamics and kinetics of substrate binding to this protein has not been elucidated. In this work, we incorporated thromboxane synthase into lipid bilayers of nanodiscs for functional studies. We measured the redox potential of CYP5A1 in nanodiscs and showed that the redox potential is within a similar range of other drug-metabolizing P450 enzymes in membranes. Further, we showed that binding of substrate to CYP5A1 can induce conformational changes in the protein that block small-molecule ligand egress by measuring the kinetics of cyanide binding to CYP5A1 as a function of substrate concentration. Notably, we observed that sensitivity to cyanide binding was different for two substrate analogues, U44069 and U46619, thus indicating that they bind differently to the active site of CYP5A1. We also characterized the effects of the different lipids on CYP5A1 catalytic activity by using nanodiscs to create unary, binary, and ternary lipid systems. CYP5A1 activity increased dramatically in the presence of charged lipids POPS and POPE, as compared to the unary POPC system. These results suggest the importance of lipid composition on modulating the activity of CYP5A1 to increase thromboxane formation.

Different behavior of artemisinin and tetraoxane in the oxidative degradation of phospholipid.

The reaction of trioxane and tetraoxane endoperoxides with unsaturated phospholipid 1 in the presence of Fe(II) was investigated in the absence of oxygen by means of tandem ESI-MS analysis. The spectral analyses for the reaction mixtures showed that artemisinin 2 with a trioxane structure gave no peak except that for the remaining intact phospholipid 1 (m/z 758.9), indicating that there was no structural change to 1. On other hand, the reaction mixture of 1 with tetraoxanes 3 and 4 afforded a number of new peaks (m/z 620-850) that were presumably assigned to oxidative degradation products originating from phospholipid 1. Since this reaction was completely inhibited by the addition of a phenolic antioxidant, the process was considered to involve some free radical species. The newly discovered marked differences in reactivity between the trioxane and the tetraoxanes possibly reflects their different anti-malarial mechanisms, and this reactivity may contribute to the classification of a number of anti-malarial endoperoxides whose mode of action is based on phospholipid oxidation.

Solution structure of a common substrate mimetic of cyclooxygenase-downstream synthases bound to an engineered thromboxane A2 synthase using a high-resolution NMR technique.

Understanding the docking mechanism of the common substrate, prostaglandin H(2) (PGH(2)), into the active sites of different cyclooxygenase(COX)-downstream synthases is a key step toward uncovering the molecular basis of the isomerization of PGH(2) to different prostanoids. A high-resolution NMR spectroscopy was used to determine the conformational changes and solution 3D structure of U44069, a PGH(2) analogue, bound to one of the COX-downstream synthases-an engineered thromboxane A(2) synthase (TXAS). The dynamic binding was clearly observed by (1)D NMR titration. The detailed conformational change and 3D structure of U44069 bound to the TXAS were demonstrated by 2D (1)H NMR experiments using transferred NOEs. Through the assignments for the 2D (1)H NMR spectra, TOCSY, DQF-COSY, NOESY, and the structural calculations based on the NOE constraints, they demonstrated that the widely open conformation with a triangle shape of the free U44069 changed to a compact structure with an oval shape when bound to the TXAS. The putative substrate-binding pocket of the TXAS model fits the conformation of the TXAS-bound U44069 appropriately, but could not fit the free form of U44069. It was the first to provide structural information for the dynamic docking of the PGH(2) mimic of the TXAS in solution, and to imply that PGH(2) undergoes conformational changes when bound to different COX-downstream synthases, which may play important roles in the isomerization of PGH(2) to different prostanoids. The NMR technique can be used as a powerful tool to determine the conformations of PGH(2) bound to other COX-downstream synthases.

Purification and characterization of recombinant human prostacyclin synthase.

Prostacyclin synthase (PGIS), which catalyzes the conversion of prostaglandin (PG) H(2) to prostacyclin (PGI(2)), is a member of the cytochrome P-450 (P450) superfamily, CYP8A1. To study the enzymatic and protein characteristics of human PGIS, the enzyme was overexpressed in Spodoptera frugiperda 21 (Sf21) cells using the baculovirus expression system. PGIS was expressed in the microsomes of the infected Sf21 cells after culture in 5 microg/ml hematin-supplemented medium for 72 h. The holoenzyme was isolated from the solubilized microsomal fraction by calcium phosphate gel absorption and purified to homogeneity by DEAE-Sepharose and hydroxyapatite column chromatography. The K(m) and V(max) values of the purified human PGIS for PGH(2) were 30 microM and 15 micromol/min/mg of protein at 24 degrees C, respectively. The optical absorption and EPR spectra of the enzyme revealed the characteristics of a low-spin form of P450 in the oxidized state. The carbon monoxide-reduced difference spectrum, however, exhibited a peak at 418 nm rather than 450 nm. The addition of a PGH(2) analogue, U46619, to the enzyme produced an oxygen-ligand type of the difference spectrum with maximum absorption at 407 nm and minimum absorption at 430 nm. Treatment with another PGH(2) analogue, U44069, produced a peak at 387 nm and a trough at 432 nm in the spectrum (Type I), while treatment with tranylcypromine, a PGIS inhibitor, produced a peak at 434 nm and a trough at 412 nm (Type II). A Cys441His mutant of the enzyme possessed no heme-binding ability or enzyme activity. Thus, we succeeded in obtaining a sufficient amount of the purified recombinant human PGIS from infected insect cells for spectral analyses that has high specific activity and the characteristics of a P450, indicating substrate specificity.

Resonance Raman investigation of the interaction of thromboxane synthase with substrate analogues.

Thromboxane synthase is a hemethiolate enzyme that catalyzes the isomerization of prostaglandin H2 to thromboxane A2. We report the first resonance Raman (RR) spectra of recombinant human thromboxane synthase (TXAS) in both the presence and the absence of substrate analogues U44069 and U46619. The resting enzyme and its U44069 complex are found to have a 6-coordinate, low spin (6c/ls) heme, in agreement with earlier experiments. The U46619-bound enzyme is detected as a 6c/ls heme too, which is in contradiction with a previous conclusion based on absorption difference spectroscopy. Two new vibrations at 368 and 424 cm(-1) are observed upon binding of the substrate analogues in the heme pocket and are assigned to the second propionate and vinyl bending modes, respectively. We interpret the changes in these vibrational modes as the disruption of the protein environment and the hydrogen-bonding network of one of the propionate groups when the substrate analogues enter the heme pocket. We use carbocyclic thromboxane A2 (CTA2) to convert the TXAS heme cofactor to its 5-coordinate, high spin (5c/hs) form, as is confirmed by optical and RR spectroscopy. In this 5c/hs state of the enzyme, the Fe-S stretching frequency is determined at 350 cm(-1) with excitation at 356.4 nm. This assignment is supported by comparison to the spectrum of resting enzyme excited at 356.4 nm and by exciting at different wavelengths. Implications of our findings for substrate binding and the catalytic mechanism of TXAS will be discussed.

Identification of the substrate interaction site in the N-terminal membrane anchor segment of thromboxane A2 synthase by determination of its substrate analog conformational changes using high resolution NMR technique.

The present studies describe an investigation for the interaction of N-terminal membrane anchor domain of thromboxane A(2) synthase (TXAS) with its substrate analog in a membrane-bound environment using the two-dimensional NMR technique. TXAS and prostaglandin I(2) synthase (PGIS), respectively, convert the same substrate, prostaglandin H(2) (PGH(2)), to thromboxane A(2) and prostaglandin I(2), which have opposite biological functions. Our topology studies have indicated that the N-terminal region of TXAS has a longer N-terminal endoplasmic reticulum (ER) membrane anchor region compared with the same segment proposed for PGIS. The differences in their interaction with the ER membrane may have an important impact to facilitate their common substrate, PGH(2), across the membrane into their active sites from the luminal to the cytoplasmic side of the ER. To test this hypothesis, we first investigated the interaction of the TXAS N-terminal membrane anchor domain with its substrate analog. A synthetic peptide corresponding to the N-terminal membrane anchor domain (residues 1-35) of TXAS, which adopted a stable helical structure and exhibited a membrane anchor function in the membrane-bound environment, was used to interact with a stable PGH(2) analog,. High resolution two-dimensional NMR experiments, NOESY and TOCSY, were performed to solve the solution structures of in a membrane-mimicking environment using dodecylphosphocholine micelles. Different conformations were clearly observed in the presence and absence of the TXAS N-terminal membrane anchor domain. Through combination of the two-dimensional NMR experiments, completed (1)H NMR assignments of were obtained, and the data were used to construct three-dimensional structures of in H(2)O and dodecylphosphocholine micelles, showing the detailed conformation change upon the interaction with the membrane anchor domain. The observation supported the presence of a substrate interaction site in the N-terminal region. The combination of the structural information of and was able to simulate a solution structure of the unstable TXAS and PGIS substrate, PGH(2).

A proposed common spatial pharmacophore and the corresponding active conformations of some TxA2 receptor antagonists.

Four pharmacophore recognition sites have been proposed for active thromboxane A2 (TxA2) antagonists. We have sought to define the corresponding spatial pharmacophore for these four sites by performing conformational analysis and molecular superposition studies on five known antagonists: SQ 29,548, SQ 28,668, SQ 27,427, BM 13.505, and a Merck Frosst compound. The strategy was to identify a low intramolecular-energy conformer state for each antagonist for which the relative locations and orientations of the corresponding recognition site groups were in common when all five antagonists were superimposed. The conformations used in the successful molecular superpositions were then postulated to be the active conformations of each antagonist. Since SQ 29,548 is the most potent of the five antagonists, it was considered the reference structure in the molecular superposition. A unique spatial pharmacophore was identified and may be a useful template in designing a new TxA2 antagonists.

FTIR spectral study of intramolecular hydrogen bonding in thromboxane A2 receptor agonist (U-46619), prostaglandin (PG)E2, PGD2, PGF2 alpha, prostacyclin receptor agonist (carbacyclin), and their related compounds in dilute CCl4 solution: structure-activity relationships.

FTIR spectra measurements and full optimization curve analysis of their spectra were done to obtain parameters of the OH and C = O stretching vibration bands for intramolecular hydrogen bondings in thromboxane (TX)A2 receptor partial agonist (CTA2), prostaglandin (PG)E2, PGD2, PGF2 alpha, prostacyclin (PGI2) receptor agonist (carbacyclin), and their related compounds in dilute CCl4 solutions. For CTA2, PGE2, PGD2, and PGF2 alpha, cyclic intramolecular hydrogen bonds involving a 15-membered ring similar to that observed for the TXA2 receptor agonist (U-46619) were found between a carboxyl group of the alpha-side chain and a 15-hydroxyl group of the omega-side chain. The arrangement of these side chains was P-shaped, and the percentage of the intramolecular hydrogen-bonded molecules with the 15-membered ring in CCl4 solution showed a high value of ca. 80% for these compounds. In addition, it was found that the cyclic intramolecular hydrogen bonds involving the 13-, 12-, and 12-membered rings in PGE2, PGD2, and PGF2 alpha, respectively, are formed between the carboxyl group of the alpha-side chain and the 11-, 9-, and 9-hydroxyl groups of a cyclopentane ring, respectively, although the percentages of the intramolecular hydrogen-bonded molecules with these membered rings are very small. It was also found that the hydrogen bond is more easily formed in the order of the 11-, 9-, and 15-hydroxyl groups. For carbacyclin, the cyclic intramolecular hydrogen bond involving the 13-membered ring was found between the carboxyl group of the alpha-side chain and the 11-hydroxyl group. The percentage of the intramolecular hydrogen-bonded molecules showed the value of 58% for carbacyclin. On the basis of information on the side-chain conformations in CCl4, we examined the structure-activity relationships for U-46619 in place of TXA2, PGE2, PGD2, PGF2 alpha, and carbacyclin in place of PGI2.

PgH2 analogs as potential antiplatelet derivatives.

Previous observations implicating PgH2 as a direct activator of platelets suggested that derivatives of U46619, a well-characterized TxA2 receptor agonist having structural homology with PgH2, might possess antiplatelet activity. The present work describes the synthesis of [1S-(1 alpha,2 beta,3 alpha,4 alpha)]-3-[(tetrahydropyranyloxy)methyl]- 2-[2-[(triphenylmethyl)oxy]ethyl]-5-oxabicyclo[2.2.1]heptane (14) a potentially useful intermediate for the synthesis of various epoxymethano derivatives. The latter was converted to [1S-(1 alpha,2 beta (Z),3 alpha,4 alpha)]-7-[3-[[2- [(phenylamino)carbonyl]-hydrazino]methyl]-5-oxabicylo[2.2.1]hept-2 - yl]-5-heptenoic acid (23), an epoxymethano derivative of PgH2 containing a hydrazide lower side chain as previously used in the TxA2 antagonist, SQ 29,548. The intermediate 14 was also converted to [1S-(1 alpha,2 beta (Z),3 alpha,4 alpha)]-7- [3-[(hexylamino)methyl]-5-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (25) which contained a simple aza side chain as used in earlier antagonists. Derivatives 23 and 25 appeared to be specific antagonists of the human platelet TxA2 receptor as evidenced by their inhibition of U46619 (1.5 microM) induced aggregation of human platelet rich plasma (IC50 = 22 and 7 microM, respectively), while having little effect on ADP (2 microM) induced aggregation at much higher concentrations. In addition, one of these derivatives, the bicycloamine 25, was shown to compete for [3H]U46619 binding to washed human platelets with an IC50 value of 25 microM, supporting the notion that these derivatives were acting at the thromboxane receptor. However, the potency of these derivatives was less than for previously reported TxA2 antagonists, suggesting that simple linear combinations of functionality from molecules active at the human platelet thromboxane receptor will be of limited predictive value.