Discovery of oxathiapiprolin, a new oomycete fungicide that targets an oxysterol binding protein.

Oxathiapiprolin is the first member of a new class of piperidinyl thiazole isoxazoline fungicides with exceptional activity against plant diseases caused by oomycete pathogens. It acts via inhibition of a novel fungal target-an oxysterol binding protein-resulting in excellent preventative, curative and residual efficacy against key diseases of grapes, potatoes and vegetables. Oxathiapiprolin is being developed globally as DuPont™ Zorvec™ disease control with first registration and sales anticipated in 2015. The discovery, synthesis, optimization and biological efficacy are presented.

Discovery and Optimization of Pyrazole Amides as Inhibitors of ELOVL1.

Accumulation of very long chain fatty acids (VLCFAs) due to defects in ATP binding cassette protein D1 (ABCD1) is thought to underlie the pathologies observed in adrenoleukodystrophy (ALD). Pursuing a substrate reduction approach based on the inhibition of elongation of very long chain fatty acid 1 enzyme (ELOVL1), we explored a series of thiazole amides that evolved into compound 27─a highly potent, central nervous system (CNS)-penetrant compound with favorable in vivo pharmacokinetics. Compound 27 selectively inhibits ELOVL1, reducing C26:0 VLCFA synthesis in ALD patient fibroblasts, lymphocytes, and microglia. In mouse models of ALD, compound 27 treatment reduced C26:0 VLCFA concentrations to near-wild-type levels in blood and up to 65% in the brain, a disease-relevant tissue. Preclinical safety findings in the skin, eye, and CNS precluded progression; the origin and relevance of these findings require further study. ELOVL1 inhibition is an effective approach for normalizing VLCFAs in models of ALD.

Structure of the ternary complex of phosphomevalonate kinase: the enzyme and its family.

The galacto-, homoserine-, mevalonate-, phosphomevalonate-kinase (GHMP) superfamily encompases a wide-range of protein function. Three members of the family (mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase) comprise the mevalonate pathway found in S. pneumoniae and other organisms. We have determined the 1.9 A crystal structure of phosphomevalonate kinase (PMK) from S. pneumoniae in complex with phosphomevalonate and AMPPNP.Mg(2+). Comparison of the apo and ternary PMK structures suggests that ligand binding reverses the side-chain orientations of two antiparallel lysines residues (100 and 101) with the result that Lys101 is switched into a position in which its ammonium ion is in direct contact with the beta,gamma-bridging atom of the nucleotide, where it is expected to stabilize both the ground and transition states of the reaction. Analysis of all available GHMP kinase ternary complex structures reveals that while their C(alpha)-scaffolds are highly conserved, their substrates bind in one of two conformations, which appear to be either reactive or nonreactive. The active site of PMK seems spacious enough to accommodate interconversion of the reactive and nonreactive conformers. A substantial fraction of the PMK active site is occupied by ordered water, which clusters near the charged regions of the substrate. Notably, a water pentamer that interacts extensively with the reactive groups of both substrates was discovered at the active site.

Crystal structure of the Streptococcus pneumoniae mevalonate kinase in complex with diphosphomevalonate.

Streptococcus pneumoniae, a ubiquitous gram-positive pathogen with an alarming, steadily evolving resistance to frontline antimicrobials, poses a severe global health threat both in the community and in the clinic. The recent discovery that diphosphomevalonate (DPM), an essential intermediate in the isoprenoid biosynthetic pathway, potently and allosterically inhibits S. pneumoniae mevalonate kinase (SpMK) without affecting the human isozyme established a new target and lead compound for antimicrobial design. Here we present the crystal structure of the first S. pneumoniae mevalonate kinase, at a resolution of 2.5 A and in complex with DPM.Mg(2+) in the active-site cleft. Structural comparison of SpMK with other members of the GHMP kinase family reveals that DPM functions as a partial bisubstrate analog (mevalonate linked to the pyrophosphoryl moiety of ATP) in that it elicits a ternary-complexlike form of the enzyme, except for localized disordering in a region that would otherwise interact with the missing portion of the nucleotide. Features of the SpMK-binding pockets are discussed in the context of established mechanistic findings and inherited human diseases linked to MK deficiency.

Mesoionic insecticides: a novel class of insecticides that modulate nicotinic acetylcholine receptors.

BACKGROUND: As the world population grows towards 9 billion by 2050, it is projected that food production will need to increase by 60%. A critical part of this growth includes the safe and effective use of insecticides to reduce the estimated 20-49% loss of global crop yields owing to pests. The development of new insecticides will help to sustain this protection and overcome insecticide resistance. RESULTS: A novel class of mesoionic compounds has been discovered, with exceptional insecticidal activity on a range of Hemiptera and Lepidoptera. These compounds bind to the orthosteric site of the nicotinic acetylcholine receptor and result in a highly potent inhibitory action at the receptor with minimal agonism. The synthesis, biological activity, optimization and mode of action will be discussed. CONCLUSION: Triflumezopyrim insect control will provide a powerful tool for control of hopper species in rice throughout Asia. Dicloromezotiaz can provide a useful control tool for lepidopteran pests, with an underexploited mode of action among these pests. © 2016 Society of Chemical Industry.

The trifunctional sulfate-activating complex (SAC) of Mycobacterium tuberculosis.

The sulfate activation pathway is essential for the assimilation of sulfate and, in many bacteria, is comprised of three reactions: the synthesis of adenosine 5'-phosphosulfate (APS), the hydrolysis of GTP, and the 3'-phosphorylation of APS to produce 3'-phosphoadenosine 5'-phosphosulfate (PAPS), whose sulfuryl group is reduced or transferred to other metabolites. The entire sulfate activation pathway is organized into a single complex in Mycobacterium tuberculosis. Although present in many bacteria, these tripartite complexes have not been studied in detail. Initial rate characterization of the mycobacterial system reveals that it is poised for extremely efficient throughput: at saturating ATP, PAPS synthesis is 5800 times more efficient than APS synthesis. The APS kinase domain of the complex does not appear to form the covalent E.P intermediate observed in the closely related APS kinase from Escherichia coli. The stoichiometry of GTP hydrolysis and APS synthesis is 1:1, and the APS synthesis reaction is driven 1.1 x 10(6)-fold further during GTP hydrolysis; the system harnesses the full chemical potential of the hydrolysis reaction to the synthesis of APS. A key energy-coupling step in the mechanism is a ligand-induced isomerization that enhances the affinity of GTP and commits APS synthesis and GTP hydrolysis to the completion of the catalytic cycle. Ligand-induced increases in guanine nucleotide affinity observed in the mycobacterial system suggest that it too undergoes the energy-coupling isomerization.

Molecular functions of conserved aspects of the GHMP kinase family.

The sequences and three-dimensional structures of the galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase (GHMP) family were compared to identify highly conserved surface residues. The functions of these solvent-accessible residues were assessed by determining the effects of their substitution, via mutagenesis, on the initial-rate parameters of a representative member of the GHMP kinase family, phosphomevalonate kinase from Streptococcus pneumoniae. What emerges from this study is a profile of the conserved surface-linked functions of the family. Certain substitutions produce highly selective effects on the steady-state affinity of a particular substrate, while one residue, Asp150, appears to be a pure k(cat) effector. Substitutions elsewhere affect multiple initial-rate parameters with varying, and sometimes compensatory, patterns. An alpha-helix that repositions during catalysis was substituted along its length to assess how its different segments contribute to catalysis-the substrate-proximal edge of the helix affects ATP recognition and k(cat), while the distal edge affects recognition of both substrates without affecting turnover. GHMP kinase mutations at the conserved surface residues corresponding to Ser291 and Ala293 in phosphomevalonate kinase are linked to mevalonic acid deficiency, which can lead to early fatality, and galactokinase deficiency, which causes cataracts. Our results suggest that the molecular basis for this particular galactokinase deficiency is an increase in the K(m) for galactose.

Mouse folylpoly-gamma-glutamate synthetase isoforms respond differently to feedback inhibition by folylpolyglutamate cofactors.

Folylpoly-gamma-glutamate synthetase (FPGS) is the enzyme responsible for metabolic trapping of reduced folate cofactors in cells for use in nucleotide and amino acid biosynthesis. There are two isoforms of FPGS expressed in mouse tissues, one is expressed in differentiated tissue, principally liver and kidney, and the other in all rapidly proliferating cell types. The present study sought the functional difference that would explain the evolution of two mouse FPGS species. Recombinant cytosolic mouse isozymes were compared with respect to steady state kinetics, chain length of polyglutamate derivatives formed, and end-product inhibition by the major reduced folylpentaglutamate cofactors. Both isoforms were equally effective in catalyzing the addition of a mole of glutamic acid to reduced folate monoglutamate substrates. Each isoform was also capable of forming long chain polyglutamate derivatives of the model folate, 5,10-dideazatetrahydrofolate. In contrast, the FPGS isoform derived from rapidly proliferating tissue was much more sensitive to inhibition by (6R)-5,10-CH(2)-H(4)PteGlu(5) and (6S)-H(4)PteGlu(5) than the isoform expressed in differentiated tissues, as demonstrated by 13- and 6-fold lower inhibition constants (K(i)), respectively. Interestingly, each isozyme was equally sensitive to inhibition by (6R)-10-CHO-H(4)PteGlu(5). We drew the conclusion that the decreased sensitivity of the FPGS expressed in mouse liver and kidney to feedback inhibition by 5,10-CH(2)-H(4)PteGlu(5-6) and H(4)PteGlu(5-6) may have evolved to permit accumulation of a larger folate cofactor pool than that found within rapidly proliferating tissue.

Streptococcus pneumoniae isoprenoid biosynthesis is downregulated by diphosphomevalonate: an antimicrobial target.

The toll that Streptococcus pneumoniae exacts on the welfare of humanity is enormous. This organism claims the lives of approximately 3700 people daily, the majority of whom are children below the age of 5, and the situation could worsen due to the increasing incidence of pernicious, multiple-antibiotic-resistant strains. Here we report the discovery and characterization of a new allosteric site, shown to be absent in humans, that can be used to switch off an essential pathway in S. pneumoniae, the mevalonate pathway. Diphosphomevalonate (DPM), an intermediate in the pathway, binds with high affinity (K(d) = 530 nM) to mevalonate kinase, the first enzyme in the pathway, and inactivates it. Steady-state and equilibrium binding measurements reveal that DPM binding is noncompetitive versus substrates. DPM binds at an allosteric site, and inhibition cannot be overcome by an increasing substrate concentration. The DPM-binding site is a promising target for the development of new antimicrobial agents.