Inhibition of antibiotic-resistant Staphylococcus aureus by the broad-spectrum dihydrofolate reductase inhibitor RAB1.

The bacterial burden on human health is quickly outweighing available therapeutics. Our long-term goal is the development of antimicrobials with the potential for broad-spectrum activity. We previously reported phthalazine-based inhibitors of dihydrofolate reductase (DHFR) with potent activity against Bacillus anthracis, a major component of Project BioShield. The most active molecule, named RAB1, performs well in vitro and, in a cocrystal structure, was found deep within the active site of B. anthracis DHFR. We have now examined the activity of RAB1 against a panel of bacteria relevant to human health and found broad-spectrum applicability, particularly with regard to gram-positive organisms. RAB1 was most effective against Staphylococcus aureus, including methicillin- and vancomycin-resistant (MRSA/VRSA) strains. We have determined the cocrystal structure of the wild-type and trimethoprim-resistant (Phe 98 Tyr) DHFR enzyme from S. aureus with RAB1, and we found that rotational freedom of the acryloyl linker region allows the phthalazine moiety to occupy two conformations. This freedom in placement also allows either enantiomer of RAB1 to bind to S. aureus, in contrast to the specificity of B. anthracis for the S-enantiomer. Additionally, one of the conformations of RAB1 defines a unique surface cavity that increases the strength of interaction with S. aureus. These observations provide insights into the binding capacity of S. aureus DHFR and highlight atypical features critical for future exploitation in drug development.

Identification and ecdysteroid antagonist activity of three oligostilbenes from the seeds of Carex pendula (Cyperaceae).

Methanolic extracts of seeds of several (Carex species were found to antagonise the action of 20-hydroxyecdysone in the Drosophila melanogaster microplate-based B(II) cell bioassay. Bioassay-guided HPLC analysis of seeds of Carex pendula (drooping sedge) provided one previously unknown tetrastilbene (cis-miyabenol A) and two known oligostilbenes (kobophenol B and cis-miyabenol C) as the biologically active compounds (EC50 values were 31, 37 and 19 microM, respectively, vs. 5 x 10(-8) M 20-hydroxyecdysone). The structures and relative stereochemistries of these compounds were deduced by comprehensive ID- and 2D-NMR experiments. These compounds are isolated from Carex pendula for the first time. In vitro experiments with dipteran and lepidopteran ecdysteroid receptor proteins demonstrate that the oligostilbenes are able to compete with radiolabelled ecdysteroid ([3H]ponasterone A) for occupancy of the ligand binding site. IC50/Ki values are similar to the EC50 values obtained in the B(II) bioassay.

Assessment of natural products in the Drosophila melanogaster B(II) cell bioassay for ecdysteroid agonist and antagonist activities.

Ecdysteroid agonist and antagonist activities can be detected and quantified with the Drosophila melanogaster B(II) cell bioassay. This bioassay is convenient, sensitive and robust. We report the assessment with this bioassay of the activities of a wide range of compounds representing a number of classes of natural products. Many compounds were inactive over a wide concentration range (10(-8) to 10(-4) or 10(-3) M) or cytotoxic at high concentrations. However, antagonisitic activity was associated with several classes of compounds: cucurbitacins and withanolides (extending previous findings) and phenylalkanoids and certain alkaloids (described for the first time). A withanolide (withaperuvin D) is identified which possesses agonistic activity. Brassinosteroids, which have been ascribed (ant)agonistic properties in the past, were not found to be active in the B(II) bioassay, either as agonists or antagonists. Possible reasons for the prevalence of antagonists and for the low potency of the majority of them are discussed.

The atomic-resolution structure of a novel bacterial esterase.

BACKGROUND: A novel bacterial esterase that cleaves esters on halogenated cyclic compounds has been isolated from an Alcaligenes species. This esterase 713 is encoded by a 1062 base pair gene. The presence of a leader sequence of 27 amino acids suggests that this enzyme is exported from the cytosol. Esterase 713 has been over-expressed in Agrobacterium without this leader sequence. Its amino acid sequence shows no significant homology to any known protein sequence. RESULTS: The crystal structure of esterase 713 has been determined by multiple isomorphous replacement and refined to 1. 1 A resolution. The subunits of this dimeric enzyme comprise a single domain with an alpha/beta hydrolase fold. The catalytic triad has been identified as Ser206-His298-Glu230. The acidic residue of the catalytic triad (Glu230) is located on the beta6 strand of the alpha/beta hydrolase fold, whereas most other alpha/beta hydrolase enzymes have the acidic residue located on the beta7 strand. The oxyanion hole is formed by the mainchain nitrogens of Cys71 and Gln207 as identified by the binding of a substrate analogue, (S)-7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1, 4-benzodiazepine-2-acetic acid. Cys71 forms a disulphide bond with the neighbouring Cys72. CONCLUSIONS: Despite negligible sequence homology, esterase 713 has structural similarities to a number of other esterases and lipases. Residues of the oxyanion hole were confirmed by structural comparison with Rhizomucor miehei lipase. It is proposed that completion of a functional active site requires the formation of the disulphide bond between adjacent residues Cys71 and Cys72 on export of the esterase into the oxidising environment of the periplasmic space.

Crystal structure of the glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus.

The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the archaea shows low sequence identity (16-20%) with its eubacterial and eukaryotic counterparts. The crystal structure of the apo GAPDH from Sulfolobus solfataricus has been determined by multiple isomorphous replacement at 2.05 A resolution. The enzyme has several differences in secondary structure when compared with eubacterial GAPDHs, with an overall increase in the number of alpha-helices. There is a relocation of the active-site residues within the catalytic domain of the enzyme. The thermostability of the S. solfataricus enzyme can be attributed to a combination of an ion pair cluster and an intrasubunit disulphide bond.

Crystallization and preliminary x-ray diffraction studies of a novel bacterial esterase.

A novel bacterial esterase has been crystallized in two forms suitable for X-ray diffraction studies. Crystals have been obtained by vapour-phase diffusion at 290 K using ammonium sulfate as precipitant. The first crystals grew in space group C2 with unit-cell parameters a = 134.7, b = 55.8, c = 110.3 A, beta = 125.1 degrees. A monoclinic data set has been collected to 2.0 A resolution. Microseeding yielded a second crystal form which grew in space group P212121 with unit-cell parameters a = 57.1, b = 115.4, c = 130.4 A. Native data from these crystals have been collected to 1.6 A resolution. A molecular envelope has been determined using an uranyl acetate derivative for phase calculation.