Inhibition of class A beta-lactamases by carbapenems: crystallographic observation of two conformations of meropenem in SHV-1

Carbapenem antibiotics are often the "last resort" in the treatment of infections caused by bacteria resistant to penicillins and cephalosporins. To understand why meropenem is resistant to hydrolysis by the SHV-1 class A beta-lactamase, the atomic structure of meropenem inactivated SHV-1 was solved to 1.05 A resolution. Two conformations of the Ser70 acylated intermediate are observed in the SHV-1-meropenem complex; the meropenem carbonyl oxygen atom of the acyl-enzyme is in the oxyanion hole in one conformation, while in the other conformation it is not. Although the structures of the SHV-1 apoenzyme and the SHV-1-meropenem complex are very similar (0.29 A rmsd for Calpha atoms), the orientation of the conserved Ser130 is different. Notably, the Ser130-OH group of the SHV-1-meropenem complex is directed toward Lys234Nz, while the Ser130-OH of the apo enzyme is oriented toward the Lys73 amino group. This altered position may affect proton transfer via Ser130 and the rate of hydrolysis. A most intriguing finding is the crystallographic detection of protonation of the Glu166 known to be involved in the deacylation mechanism. The critical deacylation water molecule has an additional hydrogen-bonding interaction with the OH group of meropenem's 6alpha-1 R-hydroxyethyl substituent. This interaction may weaken the nucleophilicity and/or change the direction of the lone pair of electrons of the water molecule and result in poor turnover of meropenem by the SHV-1 beta-lactamase. Using timed mass spectrometry, we further show that meropenem is covalently attached to SHV-1 beta-lactamase for at least 60 min. These observations explain key properties of meropenem's ability to resist hydrolysis by SHV-1 and lead to important insights regarding future carbapenem and beta-lactamase inhibitor design.     

Functionalised cyclopentadienyl zirconium compounds as potential anticancer drugs

New neutral and ionic functionalised zirconocene dichloride compounds have been isolated and characterised. The ionic zirconocene exhibits excellent cytotoxicity against a range of human tumour cell lines, which represents the first active anticancer zirconocene dichloride compound.     

Broadly tunable femtosecond parametric oscillator using a photonic crystal fiber

Pulses as short as 460 fs and a tuning range as wide as 200 nm around 1 microm have been achieved from a photonic-crystal-fiber-based parametric oscillator. The ring cavity with only 65 cm of photonic crystal fiber is synchronously pumped with a tunable passively mode-locked Yb-doped fiber laser. Widely extended tunability is achieved by using the modulation instability gain in normal dispersion as the result of high-order dispersion in the photonic crystal fiber.     

Luminol-dependent chemiluminescence analysis of human platelets

We describe a simple, sensitive, and reproducible assay system to measure the chemiluminescence (CL) produced by injecting arachidonic acid (AA) into a preparation of human platelets containing luminol.The CL appears to result from the metabolism of the AA by enzymes in human platelets, namely, cyclooxygenase, lipoxygenase, and possibly peroxidases. It is believed that when the AA is injected, free radicals and/or oxidizing agents are formed that react with the luminol producing an excited state and emitting blue light (425 nm).The enzymes can be inhibited by drugs to varying degrees. BW755C inhibits all CL at micromolar doses and it is known to inhibit both lipoxygenase and cyclooxygenase. Aspirin, indomethacin, and sulindac sulfide inhibit only cyclooxygenase and inhibit 35–65% of the light from an individual. This assay system can be used to screen certain drugs that are effective in inflammatory diseases. It could be used to determine whether the drugs would be effective in a given individual and also whether drugs have a long-term toxic effect in vivo on platelets. Further the assay is practical with a few milliters of blood.     

True fundamental solitons in a passively mode-locked short-cavity Cr(4+):YAG laser

We demonstrate a self-starting, passively mode-locked short-cavity Cr(4+):YAG laser that supports fundamental intracavity solitons over wide ranges of cavity group-velocity dispersion and pulse energies. The total dispersion and nonlinear effects are small enough that stable, N=1 soliton pulses are generated. Equally spaced multiple pulsing is also observed, with fundamental soliton behavior preserved. Regions of bistability exist where, at a constant cavity dispersion, the laser can produce transform-limited pulses of a different width and energy. The laser produces 200-fs pulses at approximately 0.9-, 1.8-, and 2.7-GHz repetition rates with a total of 82 mW of average output power.     

Phase locking and periodic evolution of solitons in passively mode-locked fiber lasers with a semiconductor saturable absorber

Passively mode-locked lasers with intracavity weakly birefringent fiber are theoretically analyzed based on two coupled complex one-dimensional Ginzburg-Landau equations. The model includes fiber birefringence, spectral filtering, saturable gain, and saturable loss. Phase-locked soliton solutions are found for small amounts of birefringence and several types of soliton with periodic polarization evolution for higher amounts of birefringence. Numerical simulations show qualitative agreement with experimental results.     

Considerations in compound database preparation--"hidden" impact on virtual screening results

Structure-based virtual screening (SBVS) utilizing docking algorithms has become an essential tool in the drug discovery process, and significant progress has been made in successfully applying the technique to a wide range of receptor targets. In silico validation of virtual screening protocols before application to a receptor target using a corporate or commercially available compound collection is key to establishing a successful process. Ultimately, retrieval of a set of active compounds from a database of inactives is required, and the metric of enrichment (E) is habitually used to discern the quality of separation of the two. Numerous reports have addressed the performance of docking algorithms with regard to the quality of binding mode prediction and the issue of postprocessing "hit lists" of docked ligands. However, the impact of ligand database preprocessing has yet to be examined in the context of virtual screening and prioritization of compounds for biological evaluation. We provide an insight into the implications of cheminformatic preprocessing of a validation database of compounds where multiple protonated, tautomeric, stereochemical, and conformational states have been enumerated. Several commonly used methods for the generation of ligand conformations and conformational ensembles are examined, paired with an exhaustive rigid-body algorithm for the docking of different "multimeric" compound representations to the ligand binding site of the human estrogen receptor alpha. Chemgauss, a shapegaussian scoring function with intrinsic chemical knowledge, was combined with PLP as a consensus-scoring scheme to rank output from the docking protocol and enrichment rates calculated for each screen. The overheads of CPU consumption and the effect on relative database size (disk requirement) for each of the protocols employed are considered. Assessment of these parameters indicates that SBVS enrichments are highly dependent on the initial cheminformatic treatment(s) used in database construction. The interplay of SMILES representations, stereochemical information, protonation state enumeration, and ligand conformation ensembles are critical in achieving optimum enrichment rates in such screening.     

Reizvorrichtungen, Thermostaten

Ohne Zusammenfassung     

MICROPARTICLES FOR STANDARDIZATION OF ELECTROPHORETIC DEVICES AND PROCESS CONTROL

An appreciation of the importance of particle surface charge as a determinant of the behavior of particle suspensions has stimulated interest in electrophoretic analyses of small particles. Currently available analytical particle electrophoresis equipment provide increasingly powerful techniques for probing the surface charge of small particles so that the potential of the application of the method may be realized in basic and applied research as well as in process development and control in manufacturing environments. Two recurrent issues in the application of the methods are the qualification of the instrument operation and the consistency of the reagents employed. Here we describe strategies for addressing these problems through the use of uniform detergent free, charge-stabilized polymer microspheres first as control particles for monitoring satisfactory instrument operation and secondly as sensitive ‘reporter” probes for detection of trace contaminants in the suspending media used for electrophoretie analysis.