Crystallographic fragment screening in academic cancer drug discovery.

Fragment-based drug discovery (FBDD) has brought several drugs to the clinic, notably to target proteins once considered to be challenging, or even undruggable. Screening in FBDD relies upon observing and/or measuring weak (millimolar-scale) binding events using biophysical techniques or crystallographic fragment screening. This latter structural approach provides no information about binding affinity but can reveal binding mode and atomic detail on protein-fragment interactions to accelerate hit-to-lead development. In recent years, high-throughput platforms have been developed at synchrotron facilities to screen thousands of fragment-soaked crystals. However, using accessible manual techniques it is possible to run informative, smaller-scale screens within an academic lab setting. This chapter describes general protocols for home laboratory-scale fragment screening, from fragment soaking through to structure solution and, where appropriate, signposts to background, protocols or alternatives elsewhere.

FragLites-Minimal, Halogenated Fragments Displaying Pharmacophore Doublets. An Efficient Approach to Druggability Assessment and Hit Generation.

Identifying ligand binding sites on proteins is a critical step in target-based drug discovery. Current approaches to this require resource-intensive screening of large libraries of lead-like or fragment molecules. Here, we describe an efficient and effective experimental approach to mapping interaction sites using a set of halogenated compounds expressing paired hydrogen-bonding motifs, termed FragLites. The FragLites identify productive drug-like interactions, which are identified sensitively and unambiguously by X-ray crystallography, exploiting the anomalous scattering of the halogen substituent. This mapping of protein interaction surfaces provides an assessment of druggability and can identify efficient start points for the de novo design of hit molecules incorporating the interacting motifs. The approach is illustrated by mapping cyclin-dependent kinase 2, which successfully identifies orthosteric and allosteric sites. The hits were rapidly elaborated to develop efficient lead-like molecules. Hence, the approach provides a new method of identifying ligand sites, assessing tractability and discovering new leads.

Antimicrobial effect and mode of action of terpeneless cold-pressed Valencia orange essential oil on methicillin-resistant Staphylococcus aureus.

AIMS: The objectives of this study were to evaluate the antistaphylococcal effect and elucidate the mechanism of action of orange essential oil against antibiotic-resistant Staphylococcus aureus strains. METHODS AND RESULTS: The inhibitory effect of commercial orange essential oil (EO) against six Staph. aureus strains was tested using disc diffusion and agar dilution methods. The mechanism of EO action on MRSA was analysed by transcriptional profiling. Morphological changes of EO-treated Staph. aureus were examined using transmission electron microscopy. Results showed that 0·1% of terpeneless cold-pressed Valencia orange oil (CPV) induced the cell wall stress stimulon consistent with the inhibition of cell wall synthesis. Transmission electron microscopic observation revealed cell lysis and suggested a cell wall lysis-related mechanism of CPV. CONCLUSIONS: CPV inhibits the growth of Staph. aureus, causes gene expression changes consistent with the inhibition of cell wall synthesis, and triggers cell lysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiple antibiotics resistance is becoming a serious problem in the management of Staph. aureus infections. In this study, the altered expression of cell wall-associated genes and subsequent cell lysis in MRSA caused by CPV suggest that it may be a potential antimicrobial agent to control antibiotic-resistant Staph. aureus.

Control of Listeria monocytogenes by lauric arginate on frankfurters formulated with or without lactate/diacetate.

Listeria monocytogenes (Lm) is a food safety concern that can be associated with ready-to-eat (RTE) meat and poultry products because of its persistence in the processing environment. Listeriosis has a fatality rate of 28% in immuno-compromised individuals. RTE meats receive a lethal heat treatment but may become contaminated by Lm after this treatment. Federal regulators and manufacturers of RTE meats are working to find additional ways to control postprocess contamination by Lm in RTE meats. This research was initiated to validate combinations of antimicrobials that would produce an immediate lethality of at least 1 log of Lm on artificially contaminated frankfurters, and also suppress Lm growth to less than 2 logs throughout the extended shelf life at refrigerated temperatures (4 degrees C). Based on our studies, 22-ppm lauric arginate (LAE, ethyl-N-dodecanoyl-L-arginate hydrochloride) gave more than a 1-log reduction of Lm surface inoculated onto frankfurters within 12 h. The combination of either 1.8%/0.13% or 2.1%/0.15% potassium lactate/sodium diacetate (L/D) in combination with 22 ppm LAE caused more than a 2-log reduction at 12 h. Storage studies revealed that complementary interactions of L/D and LAE also met the 2nd requirement. This combination initially reduced Lm by 2 logs and suppressed growth to less than 2 logs even at the end of the 156-d storage life for frankfurters. These results confirmed that the combination of L/D with LAE as a postprocessing-prepackaging application could be useful in complying with the USDA's Alternative 1 that requires validation for the control of Lm on RTE frankfurters.

Catalase protection of neuronal survival in vitro is not directed to the accumulation of peroxides in the culture medium.

Walicke et al. (1986, J. Neurosci. 6, 1114-1121) have shown that catalase can replace the pyruvate requirement for survival of CNS neurons cultured in vitro. Since presently the only known function of catalase is the enzymatic degradation of hydrogen peroxide to water and oxygen, the simplest interpretation of the ability of catalase to support neuronal survival would be that catalase removes from the culture medium hydrogen peroxide. To test this hypothesis 8-day embryonic chick forebrain cells were cultured for 24 hr in a modified Eagle's Basal Medium with the serum-free supplement N1 (HEBM/N1) in the presence or absence of Phenol Red, 20 micrograms/ml catalase, 1 mM pyruvate, and/or 25 mM N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES) on a polyornithine-laminin substratum. The various media were then assayed for peroxide content using the potassium iodide method described by Wang and Nixon (1978, In Vitro 14, 714-722). The present data reveal that (1) HEBM/N1 normally contains approximately 50 microM peroxides, little of which is hydrogen peroxide, (2) the organic peroxide levels accumulating in this medium are not reduced by either catalase or pyruvate, and (3) medium modifications can reduce to no longer detectable levels the peroxides accumulating in the medium, but catalase or pyruvate is still required for neuronal survival. We conclude that catalase must exert its survival-promoting action at levels other than peroxides accumulating in the culture medium.