Michael Kurz

Antagonism of Pseudomonas cepacia against phytopathogenic fungi

Two strains ofPseudomonas cepacia, RJ3 and ATCC 52796, have been identified as potential antagonists of fungal plant pathogens. We have compared the antagnonistic activity of these two strains against various fungal pathogens. Although both strains displayed high levels of antagonism, ATCC 52796 was slightly more antagonistic than RJ3. The antagonist from RJ3 has been identified as the antifungal compound pyrrolnitrin after purification by HPLC and characterization by UV, IR, NMR, and mass spectroscopy. Both strains also antagonized the fungi by production of volatile compound(s), which have not yet been identified. Both strains are similar with respect to in vitro antagonism, mechanism of antagonism, and sensitivity to antibiotics.

Evaluation of Canines for Accelerant Detection at Fire Scenes

In recent years, canines have been successfully used in fire investigations to detect accelerant residues. We set out to determine the lower limits at which canines could reliably detect potential accelerants. Measured amounts ranging from 10 to as little as 0.01 microL of gasoline, kerosene, and isopars were applied to preselected spots along a continuous sample path (25 to 40 feet long) made out of burned and unburned wood or nylon carpeting strips at the testing site. Two canines were led past this sample path at least three times and positive alerts and negative responses were recorded. Both dogs were generally able to alert on spots containing 0.01 microL or more of all three accelerants, at or beyond the purge and trap recovery and gas chromatographic detection method employed. The canines did alert occasionally on background, especially that containing traces of styrene residues, either purposely added in specific amounts or formed upon partial pyrolysis of carpeting material. The dogs alerted on sites containing 0.1 to 1.0 microL of freshly applied gasoline or kerosene placed at actual heavily damaged fire scenes, but were less successful on samples containing smaller amounts.

Biosynthetic Studies of Telomycin Reveal New Lipopeptides with Enhanced Activity.

Telomycin (TEM) is a cyclic depsipeptide antibiotic active against Gram-positive bacteria. In this study, five new natural telomycin analogues produced by Streptomyces canus ATCC 12646 were identified. To understand the biosynthetic machinery of telomycin and to generate more analogues by pathway engineering, the TEM biosynthesis gene cluster has been characterized from S. canus ATCC 12646: it spans approximately 80.5 kb and consists of 34 genes encoding fatty acid ligase, nonribosomal peptide synthetases (NRPSs), regulators, transporters, and tailoring enzymes. The gene cluster was heterologously expressed in Streptomyces albus J1074 setting the stage for convenient biosynthetic engineering, mutasynthesis, and production optimization. Moreover, in-frame deletions of one hydroxylase and two P450 monooxygenase genes resulted in the production of novel telomycin derivatives, revealing these genes to be responsible for the specific modification by hydroxylation of three amino acids found in the TEM backbone. Surprisingly, natural lipopeptide telomycin precursors were identified when characterizing an unusual precursor deacylation mechanism during telomycin maturation. By in vivo gene inactivation and in vitro biochemical characterization of the recombinant enzyme Tem25, the maturation process was shown to involve the cleavage of previously unknown telomycin precursor-lipopeptides, to yield 6-methylheptanoic acid and telomycins. These lipopeptides were isolated from an inactivation mutant of tem25 encoding a (de)acylase, structurally elucidated, and then shown to be deacylated by recombinant Tem25. The TEM precursor and several semisynthetic lipopeptide TEM derivatives showed rapid bactericidal killing and were active against several multidrug-resistant (MDR) Gram-positive pathogens, opening the path to future chemical optimization of telomycin for pharmaceutical application.

Effect of Background Interference on Accelerant Detection by Canines

Additional studies were performed with respect to examining the lower limits at which canines can reliably detect products commonly used as accelerants and distinguish them from pyrolysis products or background hydrocarbons. As part of a testing exercise performed in conjunction with a national conference of the Canine Accelerant Detection Association (CADA), 34 canines were subjected to a series of tests, some of them were a recertification proficiency. In one of the tests, the dogs were nearly unanimously successful in locating one can (out of five) containing 50% evaporated gasoline at the 5 microL level on a burnt carpet matrix, and pinpointing the 6-in. square sector on a piece of plain carpeting where the same amount of gasoline (5 microL) was applied. However, only half were able to detect a second doped sample containing a lesser amount (0.05, 0.1, or 0.2 microL) of gasoline, and registered a number of alerts on samples containing only burnt carpeting material. The dogs were also tested on measured amounts (2 or 5 microL) of a variety of other light, medium, and heavy petroleum products applied to a variety of substances containing significant pyrolysis products. As a group, the canines were much less successful in pinpointing these products than they were with gasoline at this same level, and again registered a number of alerts on cans containing only pyrolysis products. The significant number of alerts by canines on samples not containing gasoline or other products points out the importance of obtaining laboratory confirmation on samples on which dogs alert, and on keeping accurate field and training records of canines to establish their credibility.

Design of Novel Exendin-Based Dual Glucagon-like Peptide 1 (GLP-1)/Glucagon Receptor Agonists

Dual activation of the glucagon-like peptide 1 (GLP-1) and glucagon receptor has the potential to lead to a novel therapy principle for the treatment of diabesity. Here, we report a series of novel peptides with dual activity on these receptors that were discovered by rational design. On the basis of sequence analysis and structure-based design, structural elements of glucagon were engineered into the selective GLP-1 receptor agonist exendin-4, resulting in hybrid peptides with potent dual GLP-1/glucagon receptor activity. Detailed structure-activity relationship data are shown. Further modifications with unnatural and modified amino acids resulted in novel metabolically stable peptides that demonstrated a significant dose-dependent decrease in blood glucose in chronic studies in diabetic db/db mice and reduced body weight in diet-induced obese (DIO) mice. Structural analysis by NMR spectroscopy confirmed that the peptides maintain an exendin-4-like structure with its characteristic tryptophan-cage fold motif that is responsible for favorable chemical and physical stability.

Production of the Bengamide Class of Marine Natural Products in Myxobacteria: Biosynthesis and Structure–Activity Relationships

The bengamides, sponge-derived natural products that have been characterized as inhibitors of methionine aminopeptidases (MetAPs), have been intensively investigated as anticancer compounds. We embarked on a multidisciplinary project to supply bengamides by fermentation of the terrestrial myxobacterium M. virescens, decipher their biosynthesis, and optimize their properties as drug leads. The characterization of the biosynthetic pathway revealed that bacterial resistance to bengamides is conferred by Leu 154 of the myxobacterial MetAP protein, and enabled transfer of the entire gene cluster into the more suitable production host M. xanthus DK1622. A combination of semisynthesis of microbially derived bengamides and total synthesis resulted in an optimized derivative that combined high cellular potency in the nanomolar range with high metabolic stability, which translated to an improved half-life in mice and antitumor efficacy in a melanoma mouse model.

Novel Small Molecule Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa) from Natural Product Anabaenopeptin

Anabaenopeptins isolated from cyanobacteria were identified as inhibitors of carboxypeptidase TAFIa. Cocrystal structures of these macrocyclic natural product inhibitors in a modified porcine carboxypeptidase B revealed their binding mode and provided the basis for the rational design of small molecule inhibitors with a previously unknown central urea motif. Optimization based on these design concepts allowed for a rapid evaluation of the SAR and delivered potent small molecule inhibitors of TAFIa with a promising overall profile.

Nonclassical CH−π Supramolecular Interactions in Artemisinic Acid Favor a Single Conformation, Yielding High Diastereoselectivity in the Reduction with Diazene

The high diastereoselectivity of the hydrogenation of artemisinate by diazene to form dihydroartemisinate (diastereoselective ratio, dr, 97:3) necessary for efficient production of artemisin has been rationalized by state-of-the-art DFT calculations and identification of the noncovalent interactions by coupled ELF/NCI analysis. Remarkably, a single conformer of artemisinate is responsible for the high diastereoselectivity of the reaction. NMR studies confirm the preference for a single conformation that is found to be identical to that predicted by the calculations. The calculations and ELF/NCI analyses show that the hydrogenation of the exocyclic activated C═C double bond has a low energy barrier and that the lowest transition state and the preferred conformation of free artemisinate develop the same network of weak noncovalent interactions between the electron donor groups (oxygen and exocyclic C═C double bond) and CH bonds of the cis-decalene group of the artemisinate, which rationalize the high diastereoselectivity unusual for a strongly exothermic reaction.

Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)

Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1’ binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site.

Nitromethylation of aromatic hydrocarbons with nitromethane–manganese(III) acetate

Treatment of aromatic hydrocarbons with nitromethane and manganese(III) acetate produces aryl nitromethanes by way of homolytic aromatic substitution involving the ·CH2NO2 radical.