William C. Stallings

Structural insights into the stereochemistry of the cyclooxygenase reaction.

Cyclooxygenases are bifunctional enzymes that catalyse the first committed step in the synthesis of prostaglandins, thromboxanes and other eicosanoids. The two known cyclooxygenases isoforms share a high degree of amino-acid sequence similarity, structural topology and an identical catalytic mechanism. Cyclooxygenase enzymes catalyse two sequential reactions in spatially distinct, but mechanistically coupled active sites. The initial cyclooxygenase reaction converts arachidonic acid (which is achiral) to prostaglandin G2 (which has five chiral centres). The subsequent peroxidase reaction reduces prostaglandin G2 to prostaglandin H2. Here we report the co-crystal structures of murine apo-cyclooxygenase-2 in complex with arachidonic acid and prostaglandin. These structures suggest the molecular basis for the stereospecificity of prostaglandin G2 synthesis.

Closing down on glyphosate inhibition—with a new structure for drug discovery

Among enzyme inhibitors used in agriculture, glyphosate ( N -phosphomethyl glycine) is remarkable. A nonselective herbicide discovered in 1970 by a group of scientists at Monsanto led by Dr. John Franz (1), glyphosate, since first commercialization under the trade name Roundup, has been used globally as a safe and effective means of weed control. The discovery of glyphosates herbicidal activity was not quite serendipity, but instead resulted from a synthetic strategy based on the hypothesis that the weak herbicidal activities of related compounds derived from the possibility of their similar metabolic fate (2). Nevertheless, the initial discovery by greenhouse screening has been followed by intensive biochemical studies that have now led to nearly complete understanding of glyphosates mode of action. In 1972, scientists at Monsanto led by Dr. E. Jaworski observed (3) that application of glyphosate resulted in the inhibition of aromatic amino acid biosynthesis in plants. In 1980, Professor N. Amrhein and coworkers (4) identified its target enzyme from the shikimate pathway (4): 5- enol pyruvoylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19). EPSPS is a key enzyme involved in aromatic amino acid biosynthesis (5). The enzyme catalyzes an unusual reaction, wherein the enolpyruvoyl group from phosphoenol pyruvate (PEP) is transferred to the 5-hydroxyl of shikimate-3-phosphate (S3P) to form the products 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate (Pi). The only other enzyme known to catalyze carboxyvinyl transfer by using PEP is UDP- N -acetylglucosamine enolpyruvyl transferase (MurA), which catalyzes the first committed step in the biosynthesis of the peptidoglycan layer of the bacterial cell. In the case of EPSPS, the reaction proceeds through a tetrahedral intermediate (Scheme S1) formed from S3P and PEP (6). Inhibition of EPSPS by glyphosate has been shown to proceed through the formation of an EPSPS-S3P-glyphosate ternary complex and the binding is ordered with glyphosate binding to the enzyme only …

Structure-based drug design of pyrazinone antithrombotics as selective inhibitors of the tissue factor VIIa complex

Structure-based drug design coupled with polymer-assisted solution-phase library synthesis was utilized to develop a series of pyrazinone inhibitors of the tissue factor/Factor VIIa complex. The crystal structure of a tri-peptide ketothiazole complexed with TF/VIIa was utilized in a docking experiment that identified a benzyl-substituted pyrazinone as a P(2) surrogate for the tri-peptide. A 5-step PASP library synthesis of these aryl-substituted pyrazinones was developed. The sequence allows for attachment of a variety of P(1) and P(3) moieties, which led to synthesis pyrazinone 23. Compound 23 exhibited 16 nM IC(50) against TF/VIIa with >6250x selectivity versus Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for pre-clinical intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a primate model of thrombosis.

Polymer-Assisted solution-Phase (PASP) parallel synthesis of an α-Ketothiazole library as tissue factor VIIa inhibitors

A solution-phase synthesis of an alpha-ketothiazole library of the general form D-Phe-L-AA-L-Arg-alpha-ketothiazole is described. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase purification protocols, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multi-step synthesis affords the desired alpha-ketothiazole products in excellent purities and yields. A variety of L-amino acid inputs were used to probe the S2 pocket of the tissue factor (TF) VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound 10e bound to the TF/VIIa complex was obtained that explains the observed selectivity. The alpha-ketothiazoles were found to be potent, reversible-covalent inhibitors of tissue factor VIIa, with some analogues demonstrating selectivity versus thrombin.

Crystallization and preliminary X-ray characterization of a soybean seed lipoxygenase.

An isoenzyme of soybean (Glycine max L. Merrill cv. Provar) lipoxygenase (EC 1.13.11.12) has been crystallized using the vapor diffusion method. Crystals were grown from solutions of the protein (7 mg/ml) using 10 to 20% (w/v) polyethylene glycol 8000 in citrate/phosphate buffer (pH 5.7) containing 0.5% (w/v) n-octyl-beta-D-glucopyranoside. The crystals reached maximum dimensions of 0.3 mm x 0.2 mm x greater than 2 mm. The enzyme crystallized in space group C222(1) with unit cell dimensions a = 246 A, b = 193 A and c = 75 A. A calculated Vm value of 2.35 A3/dalton was obtained assuming two molecules per asymmetric unit. The density of the crystals was found to be 1.16 g/ml, which confirmed the presence of two molecules per asymmetric unit and indicated a solvent content of 47.5%.

STRUCTURE OF A THYMINE‐THYMINE ADDUCT FORMED BY MENADIONE PHOTOSENSITIZATION

Abstract— The structure of one of the dithymine adducts formed by near‐UV photosensitization of aqueous oxygenated thymine in the presence of 2‐methyl‐ 1, 4‐naphthoquinone is reported. The compound, 6′β‐hydroxy‐5′β‐1 ‐ [5‐methylpyrimidinyl‐ 2,4‐dione]‐5′‐6′‐dihydrothymine has one thymine ring (ring I) linked through the 1‐nitrogen atom to the C5′ atom of a second thymine, which has been hydroxylated af C6′ (ring II). Crystals are monoclinic, space group 12/c, and the structure was refined to Robs= 0.036 for 2072 unique reflections with intensities I greater than 2.33s̀(I). Ring I is planar, whereas ring II is not. Ring I and the OH group on ring II are cis to ring II. The planar ring I and the OH group are attached, respectively, in an equatorial and axial manner to ring II (which is in the sofa conformation). The planar rings I of close‐lying pairs of molecules stack parallel to each other. The structure is held together by a hydrogen‐bonding system consisting of the water molecules, the NH groups, two of the C=O groups and the OH group. The chemical formula and relative configurations at C5′ and C6 are established by this analysis.

The structure of a biological alkylating agent with antiumor properties: N-{2-[(chloroethyl)thio]ethyl}-10-methyl-9-anthracenemethylamine hydrochloride and of its selfalkylation product 10-methyl-9-[(4-thiomorpholino)methyl]anthracene

The structures of a biological alkylating agent, N{ 2-[ (2-chloroethyl)thio ]ethyl }- 10-methyl-9-anthracenemethylamine hydrochloride (I), with antitumor properties, and of its self-alkylation product, 10-methyl9-[(4-thiomorpholino)methyl]anthracene (II), have been determined. Crystal data for (I) are C20H23CINS +. Cl-, M r = 380.38, a = 22.748 (2), b = 13.007 (1), c = 6.3443 (8) A, V= 1877.2 (4) A 3, D C = 1.35 Mg m -3, space group P21212 ~, Z = 4. Crystal data for (II) are C20H2~NS, M r = 307.46, a = 35.816 (6), b = 6.8965 (9), c = 13.895 (2) A, fl= 105.89 (1) °, V= 3300.9 (8)/k s, Dc = 1.24 Mg m -3, space group P21/a, Z = 8 (2 molecules per asymmetric unit). The final R values were 0.050 for 1220 observed data for (I) and 0.047 for 4513 observed data for (II). In the alkylating agent (I) there is no internal NH... S hydrogen bond in the side chain, unlike the case of other ICR compounds which are acridine alkylating agents with intramolecular NH...N hydrogen bonds in their side chains. This lack of hydrogen bonding in (I) may facilitate the self-alkylation process. The flexibility of the anthracene ring system is shown by the variability in the angle between the planes of the two outer rings as observed in these and other structures. In both structures the anthracene ring systems of the molecules do not overlap in planes 3.4 A apart, but they do lie in parallel planes, approximately 3.5 A apart, with a remarkably similar motif for each.