Wynona M. Johnson

Structure-based design of inhibitors of the rice blast fungal enzyme trihydroxynaphthalene reductase

Rice Blast Disease, caused by the fungus Pyricularia oryzae, is one of the most important diseases of rice. Several enzymes in the melanin biosynthetic pathway have proven to be valuable targets for development of rice blast fungicides. In particular, inhibitors of trihydroxynaphthalene reductase (3HNR), which catalyzes the conversion of trihydroxynaphthalene to vermelone, have yielded commercially useful rice fungicides. The X-ray structure of 3HNR has been published recently, presenting an opportunity to use this information in the de novo design of novel 3HNR inhibitors that may exhibit useful rice blast activity. We used the LeapFrog program to develop a docking model for interaction of ligands with the active site of THNR. The final model gave a good correlation between calculated binding energy and log Ki and was used to design novel ligands and score compounds for synthesis. Using this as a tool, we synthesized inhibitors in the nanomolar range and also developed several inhibitors that did not conform to the properties of the THNR active site. Leapfrog was able to locate a previously unrecognized binding pocket that could accommodate these otherwise anomalous regions of structure.

Binding inhibitors of the bacterial sliding clamp by design

The bacterial replisome is a target for the development of new antibiotics to combat drug resistant strains. The β(2) sliding clamp is an essential component of the replicative machinery, providing a platform for recruitment and function of other replisomal components and ensuring polymerase processivity during DNA replication and repair. A single binding region of the clamp is utilized by its binding partners, which all contain conserved binding motifs. The C-terminal Leu and Phe residues of these motifs are integral to the binding interaction. We acquired three-dimensional structural information on the binding site in β(2) by a study of the binding of modified peptides. Development of a three-dimensional pharmacophore based on the C-terminal dipeptide of the motif enabled identification of compounds that on further development inhibited α-β(2) interaction at low micromolar concentrations. We report the crystal structure of the complex containing one of these inhibitors, a biphenyl oxime, bound to β(2), as a starting point for further inhibitor design.

Ecdysone Receptors of Pest Insects–Molecular Cloning, Characterisation, and a Ligand Binding Domain-Based Fluorescence Polarization Screen

EcR- and USP-encoding cDNAs of four pest insects (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were cloned from high- quality lambda cDNA libraries and sequenced. Cognate EcR-USP cDNA pairs were shown to express functional ecdysone receptors in transfected cells. The amino acid sequences of the EcR ligand binding domains (LBDs) were employed in conjunc- tion with those known for other arthropods to construct a phylogenetic tree. Affinity tagged EcR-USP LBD heterodimers were co-expressed efficiently in insect cells using a baculovirus vector. The recombinant EcR and USP DE/F segments from each species associated spontaneously to form heterodimers that bound ecdysteroids with high affinity. An E/F segment pair (constructed only for H. armigera) also asso- ciated spontaneously to form a functional heterodimer, but one with ligand binding affinities several times lower than its DE/F counterpart. A fluorescein-inokosterone conjugate was synthesized and used to develop a novel ligand binding assay based on fluorescence polarization. This assay can be used in place of the classical ( 3 H)-ponasterone A binding assay, and is ideally suited to high-throughput screen- ing. The ligand binding data obtained in vitro using recombinant LBD heterodim- ers reflect the ability of agonists to induce ecdysone receptor controlled transgene expression in recombinant mammalian cells; in vitro binding data can also reflect the potency of ligands to act as insecticides.

Dihydro-1,2,4-triazin-6(1H)-ones. IV. Chemical Modification of 3-Phenyl-4,5-dihydro-1,2,4-triazin-6(1H)-ones

Reaction of 3-phenyl-4,5-dihydro-1,2,4-triazin-6(1H)-one (1) and its 5,5-dimethyl derivative (4) with phosphoruspentasulfide gave the corresponding 6-thiones (2) and (5); methylation of (2) gave the 6-methylsulfanyl-3-phenyl-4,5-dihydro-1,2,4-triazine (3), also obtained by reaction of 6-chloro-3-phenyl-4,5-dihydro-1,2,4-triazine (8) withsodium thiomethoxide. Reaction of (8) with morpholine afforded the 6-(morpholin-4¢-yl) derivative (9). Reaction of (1) with isopropyl isocyanate gave N-isopropyl-6-oxo-3-phenyl-1,4,5,6-tetrahydro-1,2,4-triazine-1-carb-oxamide (19a), and a Mannich reaction of (1) with morpholine and formaldehyde gave the 1-morpholinomethylene derivative (20a). Attempts to effect cycloaddition of 1-methyl-3-phenyl-1,2,4-triazin-6(1H)-one (12) with a benzonitrile oxide failed. Reaction of the thione (2) with 1,3-dipolarophiles gave unstable adducts.

Construction of the 1,2,3,4-tetrahydro-1,4,6,2-oxathiazaphosphorine ring system

The novel 1,2,3,4-tetrahydro-1,4,6,2-oxathiazaphosphorine ring system has been prepared by reaction of the sodium salt of diethyl mercaptomethylphosphonate with nitrile oxides, followed by cyclization of the resulting hydroximinoyl sulfide. The stability of the new ring system is investigated.

2‐Methyl‐3‐phenyl‐2,5‐dihydro‐1,2,4‐triazin‐6(1H)‐one Methanol Solvate and 4‐Methyl‐3‐phenyl‐4,5‐dihydro‐1,2,4‐triazin‐6(1H)‐one

Tautomeric detail in the two title triazinones has been accurately defined. 2-Methyl-3-phenyl-2,5-dihydro-1,2,4-triazin-6(1H)-one methanol solvate, C 10 H 11 N 3 O.CH 4 O, was found to adopt a zwitterionic form with a dihedral angle between the mean planes of the dihydrotriazinone ring (r.m.s. deviation 0.12 A) and the phenyl ring of 44.5(2)°. In the crystal, the molecules are linked by intermolecular hydrogen bonds between the protonated ring-N atom at position 4 and the carbonyl-O atom. 4-Methyl-3-phenyl-4,5-dihydro-1,2,4-triazin-6(1H)-one, C 10 H 11 N 3 O, adopts a non-ionic tautomeric form. The dihedral angle between the mean planes of the dihydrotriazinone ring (r.m.s. deviation 0.15 A) and the phenyl ring is 33.7 (1)°. The molecules in the crystal are linked into dimers by hydrogen bonds between the protonated ring-N atom at position 1 and the carbonyl-O atom.

Synthesis of some 5,6-dihydro-7H-1,4,2-oxathiazepin-7-ones and 5H-4,1,3-benzoxathiazepin-5-ones

The novel heterocyclic systems 5,6-dihydro-7H-1,4,2-oxathiazepin-7-one (1) and 5H-4,1,3-benzoxathiazepin-5-one (2) were prepared by the reaction of hydroximoyl chlorides (3) with mercapto-carboxylic acids, followed by cyclization with 1,3-dicyclohexylcarbodiimide (6) or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl (7)

NEW ARYL-POLYFLUOROCYCLOBUTANE INSECTICIDES. CHEMICAL AND BIOLOGICAL STUDIES

Abstract Insecticidally potent and toxicologically safe, aryltetrafluorocyclobutane esters were designed based on combined features of DDT and pyrethroid structures. For some of the esters the only remaining structural resemblance to the natural pyrethrin insecticides, based on chrysanthemic acid is the -COOCH2- of the ester linking group. Biochemical evidence indicates that mixed function oxidase (MFO) enzymes could play a major role not only in the degradation but also in the action of the compounds. This role is highlighted by the finding that the more active diastereoisomers of several of the insecticides are better substrates for the induced enzymes. This effect is contrary to the expected decrease in insecticidal activity for more rapidly degraded forms of a neuro-active agent. Studies of MFO inhibition in vitro and induction of MFO in the blowfly in vivo , indicate that a specific MFO enzyme cpmplex is located at an hydrophobic site accessible to the highly lipid soluble MFO inhibitors e.g. sesamex and that its active site is stereospecific.