Novel heteroaryl replacements of aromatic 3-tetrafluoroethoxy substituents in trifluoro-3-(tertiaryamino)-2-propanols as potent inhibitors of cholesteryl ester transfer protein.

A series of novel N,N-disubstituted trifluoro-3-amino-2-propanols has been prepared as potent inhibitors of cholesteryl ester transfer protein (CETP). Modifying the aromatic 3-tetrafluoroethoxy group in the lead molecule 1a with various heteroaryl moieties produced new 2-furyl analogues 2a,b with submicromolar potency in vitro.

Stereospecific inhibition of CETP by chiral N,N-disubstituted trifluoro-3-amino-2-propanols.

Chiral N,N-disubstituted trifluoro-3-amino-2-propanols represent a recently discovered class of compounds that inhibit the neutral lipid transfer activity of cholesteryl ester transfer protein (CETP). These compounds all contain a single chiral center that is essential for inhibitory activity. (R,S)SC-744, which is composed of a mixture of the two enantiomers, inhibits CETP-mediated transfer of [(3)H]cholesteryl ester ([(3)H]CE) from HDL donor particles to LDL acceptor particles with an IC(50) = 200 nM when assayed using a reconstituted system in buffer and with an IC(50) = 6 microM when assayed in plasma. Upon isolation of the enantiomers, it was found that the (R,+) enantiomer, SC-795, was about 10-fold more potent than the mixture, and that the (S,-) enantiomer, SC-794, did not have significant inhibitory activity (IC(50) > 0.8 microM). All of the activity of the (S,-)SC-794 enantiomer could be accounted for by contamination of this sample with a residual 2% of the highly potent (R,+) enantiomer, SC-795. The IC(50) of (R,+)SC-795, 20 nM, approached the concentration of CETP (8 nM) in the buffer assay. These chiral N,N-disubstituted trifluoro-3-amino-2-propanols were found to associate with both LDL and HDL, but did not disrupt overall lipoprotein structure. They did not affect the on or off rates of CETP binding to HDL disk particles. Inhibition was highly specific since the activities of phospholipid transfer protein and lecithin cholesterol acyl transferase were not affected. Competition experiments showed that the more potent enantiomer (R)SC-795 prevented cholesteryl ester binding to CETP, and direct binding experiments demonstrated that this inhibitor bound to CETP with high affinity and specificity. It is estimated, based on the relative concentrations of inhibitor and lipid in the transfer assay, that (R)SC-795 binds approximately 5000-fold more efficiently to CETP than the natural ligand, cholesteryl ester. We conclude that these chiral N,N-disubstituted trifluoro-3-amino-2-propanol compounds do not affect lipoprotein structure or CETP-lipoprotein recognition, but inhibit lipid transfer by binding to CETP reversibly and stereospecifically at a site that competes with neutral lipid binding.

Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast L-lactate dehydrogenase).

Flavocytochrome b(2) from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction. The crystal structure of the native yeast enzyme has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863] as well as that of the sulfite adduct of the recombinant enzyme produced in Escherichia coli [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]; several key active site residues were identified. In the sulfite adduct crystal structure, Arg289 adopts two alternative conformations. In one of them, its side chain is stacked against that of Arg376, which interacts with the substrate; in the second orientation, the R289 side chain points toward the active site. This residue has now been mutated to lysine and the mutant enzyme, R289K-b(2), characterized kinetically. Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotope effect) indicate the mutation affects k(cat) by a factor of about 10 and k(cat)/K(M) by up to nearly 10(2). Pre-steady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirely limited by flavin reduction. Inhibition studies on R289K-b(2) with a range of compounds show a general rise in K(i) values relative to that of wild-type enzyme, in line with the elevation of the K(M) for L-lactate in R289K-b(2); they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a loss of the inhibition by excess substrate. Altogether, the kinetic studies indicate that the mutation has altered the first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both in Michaelis complex and transition-state stabilization, as well as in ligand binding to the active site when the flavin is in the semiquinone state. In addition, it appears that the mutation has not affected electron transfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step, namely, transfer from flavin semiquinone to heme b(2). Finally, the X-ray crystal structure of R289K-b(2), with sulfite bound at the active site, has been determined to 2.75 A resolution. The lysine side chain at position 289 is well-defined and in an orientation that corresponds approximately to one of the alternative conformations observed in the structure of the recombinant enzyme-sulfite complex [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]. Comparisons between the R289K-b(2) and wild-type structures allow the kinetic results to be interpreted in a structural context.

Structure-activity relationship studies on 1-[2-(4-Phenylphenoxy)ethyl]pyrrolidine (SC-22716), a potent inhibitor of leukotriene A(4) (LTA(4)) hydrolase.

Leukotriene B(4) (LTB(4)) is a pro-inflammatory mediator that has been implicated in the pathogenesis of a number of diseases including inflammatory bowel disease (IBD) and psoriasis. Since the action of LTA(4) hydrolase is the rate-limiting step for LTB(4) production, this enzyme represents an attractive pharmacological target for the suppression of LTB(4) production. From an in-house screening program, SC-22716 (1, 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine) was identified as a potent inhibitor of LTA(4) hydrolase. Structure-activity relationship (SAR) studies around this structural class resulted in the identification of a number of novel, potent inhibitors of LTA(4) hydrolase, several of which demonstrated good oral activity in a mouse ex vivo whole blood assay.

Inhibitory activity of unsaturated fatty acids and anacardic acids toward soluble tissue factor-factor VIIa complex.

Five compounds, which inhibited the amidolytic activity of soluble tissue factor/activated factor VII complex (sTF/VIIa), were isolated from two traditional Chinese medicinal plants commonly used in the treatment of cardiovascular and cerebrovascular diseases. The active compounds were found to be linolenic, linoleic, and oleic acids from roots of Salvia miltiorrhiza; and two anacardic acids, 6-(8'Z-pentadecenyl)- and 6-(10'Z-heptadecenyl)-salicylic acids, from leaves of Ginkgo biloba. The IC50 values were in the range 30-80 micromol/L. Palmitic acid, isolated from roots of Salvia miltiorrhiza, and 2-[(3',7',11',15'-tetramethyl)-2'E,6'E,10'E, 14'E-hexadecatetraenyl]-1,4-hydroquinone, isolated from the marine sponge Adocia viola, did not inhibit sTF/VIIa. Further expansion of the structure-activity relationship to include anacardic acids, 6-(8'Z,11'Z-heptadecadienyl)- and 6-(8'Z, 11'Z, 14'Z-heptadecatrienyl)-salicylic acids from leaves of Anacardium spondias, and other fatty acids demonstrated that at least one cis double bond was essential for inhibitory activity, and that fatty acids containing two or three cis double bonds were optimal. Evidence from preincubation studies implied that these fatty acids may exert their effect by binding to VIIa and consequently preventing binding of sTF to VIIa.

Molecular basis for interprotein complex-dependent effects on the redox properties of amicyanin.

The quinoprotein methylamine dehydrogenase (MADH), type I copper protein amicyanin, and cytochrome c-551i form a complex within which interprotein electron transfer occurs. It was known that complex formation significantly lowered the oxidation-reduction midpoint potential (Em) value of amicyanin, which facilitated an otherwise thermodynamically unfavorable electron transfer to cytochrome c-551i. Structural, mutagenesis, and potentiometric studies have elucidated the basis for this complex-dependent change in redox properties. Positively charged amino acid residues on the surface of amicyanin are known to stabilize complex formation with MADH and influence the ionic strength dependence of complex formation via electrostatic interactions. Altering the charges of these residues by site-directed mutagenesis had no effect on the Em value of amicyanin, ruling out charge neutralization as the basis for the complex-dependent changes in redox properties. The Em value of free amicyanin varies with pH and exhibits a pKa value for the reduced form of 7.5. The crystal structure of reduced amicyanin at pH 4.4 reveals that His95, which serves as a ligand for Cu2+, has rotated by 180 degrees about the Cbeta-Cgamma bond relative to its position in oxidized amicyanin and is no longer in the copper coordination sphere. At pH 7.7, the crystal structure of reduced amicyanin contains an approximately equal distribution of two active-site conformers. One is very similar to the structure of reduced amicyanin at pH 4.4, and the other is very similar to the structure of oxidized amicyanin at pH 4.8. Potentiometric analysis of amicyanin in complex with MADH indicates that its Em value is not pH-dependent from pH 6.5 to 8.5, and exhibits an Em value similar to that of free amicyanin at high pH. The structure of reduced amicyanin at pH 4.4, with His95 protonated and "flipped", was modeled into the structure of the complex of oxidized amicyanin with MADH. This showed that in the complex, the redox-linked pH-dependent rotation of His95 is hindered because it would cause an overlap of van der Waals' radii with residues of MADH. These results demonstrate that protein-protein interactions profoundly affect the redox properties of this type I copper protein by restricting a pH-dependent, redox-linked conformational change of one of the copper ligands.

Refined crystal structure of methylamine dehydrogenase from Paracoccus denitrificans at 1.75 A resolution.

The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans has been refined at 1.75 A resolution utilizing the DNA-based protein sequence. The final model incorporates 8034 atoms per molecule, including 552 molecules of solvent, and gives an R-factor of 0.163. The molecule is an H2L2 hetero-tetramer containing a non-crystallographic 2-fold axis of symmetry. The 373-residue H subunit is folded into seven repeats of a four-stranded antiparallel beta-sheet motif, arranged in a propeller-like pattern about a pseudo-7-fold rotational axis of symmetry. Each L subunit contains 131 residues folded in a tight structure composed of five beta-strands in two sheets and crosslinked by six disulfide bonds. In addition there is an intrasubunit covalent linkage between two tryptophan side-chains that form the unique redox center, tryptophan tryptophylquinone (TTQ). The active site contains the O-6 carbonyl of TTQ, the side-chains of Asp32L Asp76L, Tyr119L and Thr122L, and two solvent molecules. A potential "gate" (Phe55H) separates the closed active-site cavity from a channel containing a group of highly ordered water molecules to bulk solvent. Phe55H and Tyr119L, and a number of neighboring oxygen atoms, may also provide a binding site for monovalent cations that are known to affect the reactivity and spectral properties of TTQ as well as the oxidative half reaction. The overall reaction has been dissected into a number of discrete steps that may require participation by several individual amino acid residues in the active site acting as general acids and bases.

Four new clerodane diterpenes from the leaves of Casearia guianensis which inhibit the interaction of leukocyte function antigen 1 with intercellular adhesion molecule 1.

Four new clerodane diterpenes, casearinols A and B (1 and 2) and casearinones A and B (3 and 4), were isolated from the leaves of Casearia guianensis. These immunomodulatory compounds have been structurally elucidated primarily on the basis of 2D NMR analysis and spectral data comparison with known compounds. These compounds inhibited the binding of T-cell leukocyte function antigen 1 to intercellular adhesion molecule 1.

Isolation and structure of leukotriene-A4 hydrolase inhibitor: 8(S)-amino-2(R)-methyl-7-oxononanoic acid produced by Streptomyces diastaticus.

The novel amino acid 8(S)-amino-2(R)-methyl-7-oxononanoic acid (1) was isolated from the soil-borne microorganism Streptomyces diastaticus during our screening for inhibitors of leukotriene-A4 hydrolase (LTA4H), a requisite enzyme in the biosynthesis of the potent inflammatory mediator leukotriene-B4 (LTB4). The structure of 1 was determined by detailed spectroscopic analyses and is related to 7-keto-8-aminopelargonic acid (2), a biosynthetic precursor of biotin. The relative potency of 1 (LTA4H IC50 = 0.6 microM) warranted further biological studies.

X-ray structure of the cupredoxin amicyanin, from Paracoccus denitrificans, refined at 1.31 A resolution.

High-resolution X-ray diffraction data to d(min) = 1.31 A were collected on a Xuong-Hamlin area detector from crystals of the blue-copper protein amicyanin, isolated from P. denitrificans. With coordinates from the earlier 2.0 A structure determination as a starting point, simulated annealing and restrained positional and temperature factor refinements using the program X-PLOR resulted in a final R factor of 15.5%, based on 21 131 unique reflections in the range 8.0-1.3 A. Comparison of the 1.31 A structure with that at 2.0 A shows the same basic features. However, the high-resolution electron-density maps clearly reveal additional solvent molecules and significant discrete disorder in protein side chains and within the solvent structure. As a consequence of modelling side-chain disorder, several new hydrogen-bonding interactions were identified.

Enzymatic and electron transfer activities in crystalline protein complexes.

Enzymatic and electron transfer activities have been studied by polarized absorption spectroscopy in single crystals of both binary and ternary complexes of methylamine dehydrogenase (MADH) with its redox partners. Within the crystals, MADH oxidizes methylamine, and the electrons are passed from the reduced tryptophan tryptophylquinone (TTQ) cofactor to the copper of amicyanin and to the heme of cytochrome c551i via amicyanin. The equilibrium distribution of electrons among the cofactors, and the rate of heme reduction after reaction with substrate, are both dependent on pH. The presence of copper in the ternary complex is not absolutely required for electron transfer from TTQ to heme, but its presence greatly enhances the rate of electron flow to the heme.

Refinement and structural analysis of bovine cytochrome b5 at 1.5 A resolution.

The structure of bovine liver cytochrome b(5), a soluble 93-residue proteolytic fragment of a 16 kDa membrane-bound hemoprotein, initially solved at 2.0 A resolution, has been refined at 1.5 A using data collected on a diffractometer. Refinement to 2.0 A resolution used the Hendrickson-Konnert procedure PROLSQ and was then extended to 1.5 A resolution using the program PROFFT. Only residues 3-87 could be identified in the model and these residues together with 93 water molecules gave an agreement factor of R = 0.161 for data in the resolution range 1.5-5 A. The structure was finally refined using the program X-PLOR, which enabled alternate conformers to be modelled for several surface side chains. Residues 1 and 2 at the amino terminus of the protein and residue 88 near the carboxyl terminus could be identified from these electron-density maps. However the remaining disordered carboxy-terminal residues could not successfully be included in the model. A total of 117 solvent molecules were included in the final refinement to give R = 0.164 for the data between 1.5 and 10 A.

Structure of an electron transfer complex: methylamine dehydrogenase, amicyanin, and cytochrome c551i.

The crystal structure of a ternary protein complex has been determined at 2.4 angstrom resolution. The complex is composed of three electron transfer proteins from Paracoccus denitrificans, the quinoprotein methylamine dehydrogenase, the blue copper protein amicyanin, and the cytochrome c551i. The central region of the c551i is folded similarly to several small bacterial c-type cytochromes; there is a 45-residue extension at the amino terminus and a 25-residue extension at the carboxyl terminus. The methylamine dehydrogenase-amicyanin interface is largely hydrophobic, whereas the amicyanin-cytochrome interface is more polar, with several charged groups present on each surface. Analysis of the simplest electron transfer pathways between the redox partners points out the importance of other factors such as energetics in determining the electron transfer rates.

Crystal structure analysis and refinement at 2.15 A resolution of amicyanin, a type I blue copper protein, from Thiobacillus versutus.

The crystal structure of the type I blue copper protein amicyanin from Thiobacillus versutus has been determined by Patterson search techniques on the basis of the molecular model of amicyanin from Paracoccus denitrificans, and refined by energy-restrained least-squares methods. Amicyanin crystallizes in the trigonal space group P3(2) with unit cell dimensions of a = b = 87.40 A, c = 38.20 A. The asymmetric unit is composed of three independent molecules centred on the crystallographic 3(2) axes. The final R-value is 17.4% for 15,984 reflections to a resolution of 2.15 A. The polypeptide fold in amicyanin is based on the beta-sandwich structure commonly found in blue copper proteins. Nine beta strands are folded into two twisted beta-sheets that pack together with a filling of non-polar residues between them. The geometry of the copper site is similar to that of plastocyanin. There are four ligands, arranged approximately as a distorted tetrahedron, to the copper atom: His54, Cys93, His96 and Met99. One of the copper ligands, His96, is exposed to the surface and lies in the centre of a cluster of seven hydrophobic residues.

Isolation and structure of harzianum A: a new trichothecene from Trichoderma harzianum.

A new trichothecene, harzianum A [1], was isolated from the soil-borne fungus Trichoderma harzianum. The structure of 1 was determined by extensive spectral analyses including the nmr techniques of PS-COSY, HMQC, HMBC, and NOESY. Harzianum A [1] contains a (Z,E,E)-2,4,6-octatriendioic acid esterified on the 4 beta hydroxyl group of trichodermol and is structurally related to the trichoverroids. Harzianum A [1] showed no cytotoxicity against baby hamster kidney cells, no activity against Gram-negative and Gram-positive bacteria, but modest antifungal activity at 100 micrograms/ml.

Crystal structure analysis of amicyanin and apoamicyanin from Paracoccus denitrificans at 2.0 A and 1.8 A resolution.

The crystal structure of amicyanin, a cupredoxin isolated from Paracoccus denitrificans, has been determined by molecular replacement. The structure has been refined at 2.0 A resolution using energy-restrained least-squares procedures to a crystallographic residual of 15.7%. The copper-free protein, apoamicyanin, has also been refined to 1.8 A resolution with residual 15.5%. The protein is found to have a beta-sandwich topology with nine beta-strands forming two mixed beta-sheets. The secondary structure is very similar to that observed in the other classes of cupredoxins, such as plastocyanin and azurin. Amicyanin has approximately 20 residues at the N-terminus that have no equivalents in the other proteins; a portion of these residues forms the first beta-strand of the structure. The copper atom is located in a pocket between the beta-sheets and is found to have four coordinating ligands: two histidine nitrogens, one cysteine sulfur, and, at a longer distance, one methionine sulfur. The geometry of the copper coordination is very similar to that in the plant plastocyanins. Three of the four copper ligands are located in the loop between beta-strands eight and nine. This loop is shorter than that in the other cupredoxins, having only two residues each between the cysteine and histidine and the histidine and methionine ligands. The amicyanin and apoamicyanin structures are very similar; in particular, there is little difference in the positions of the coordinating ligands with or without copper. One of the copper ligands, a histidine, lies close to the protein surface and is surrounded on that surface by seven hydrophobic residues. This hydrophobic patch is thought to be important as an electron transfer site.

Crystal structure of an electron-transfer complex between methylamine dehydrogenase and amicyanin.

The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-A resolution using molecular replacement. The search model was MADH from Thiobacillus versutus. The amicyanin could be located in an averaged electron density difference map and the model improved by refinement and model building procedures. Nine beta-strands are observed within the amicyanin molecule. The copper atom is located between three antiparallel strands and is about 2.5 A below the protein surface. The major intermolecular interactions occur between amicyanin and the light subunit of MADH where the interface is largely hydrophobic. The copper atom of amicyanin and the redox cofactor of MADH are about 9.4 A apart. One of the copper ligands, His 95, lies between the two redox centers and may facilitate electron transfer between them.