Directed molecular screening for RecA ATPase inhibitors

The roles of bacterial RecA in the evolution and transmission of antibiotic resistance genes make it an attractive target for inhibition by small molecules. We report two complementary fluorescence-based ATPase assays that were used to screen for inhibitors of RecA. We elected to employ the ADP-linked variation of the assay, with a Z′ factor of 0.83 in 96-well microplates, to assess whether 18 select compounds could inhibit ATP hydrolyis by RecA. The compounds represented five sets of related inhibitor scaffolds, each of which had the potential to cross-inhibit RecA. Although nucleotide analogs, known inhibitors of GHL ATPases, and known protein kinase inhibitors were not active against RecA, we found that three suramin-like agents substantially inhibited RecA's ATPase activity

Cyclotron resonance of the quasi-two-dimensional electron gas at Hg1-xCdxTe grain boundaries

The magnetotransmission of a p-type Hg0.766Cd0.234Te bicrystal containing a single grain boundary with an inversion layer has been investigated in the submillimetre wavelength range. For the first time the cyclotron resonance lines belonging to the various electric subbands of a quasi-two-dimensional carrier system at a grain boundary could be detected. The measured cyclotron masses and the subband densities determined from Shubnikov-de Haas experiments are compared with theoretical predictions and it is found that the data can be explained very well within the framework of a triangular well approximation model which allows for non-parabolic effects

A complementary pair of rapid molecular screening assays for RecA activities

The bacterial RecA protein has been implicated in the evolution of antibiotic resistance in pathogens, which is an escalating problem worldwide. The discovery of small molecules that can selectively modulate RecA’s activities can be exploited to tease apart its roles in the de novo development and transmission of antibiotic resistance genes. Toward the goal of discovering small-molecule ligands that can prevent either assembly of an active RecA-DNA filament or its subsequent ATP-dependent motor activities, we report the design and initial validation of a pair of rapid and robust screening assays suitable for the identification of inhbitors of RecA activities. One assay is based on established methods for monitoring ATPase enzyme activity and the second is a novel assay for RecA-DNA filament assembly using fluorescence polarization. Taken together, the assay results reveal complementary sets of agents that can either selectively suppress only the ATP-driven motor activities of the RecA-DNA filament or prevent assembly of active RecA-DNA filaments altogether. The screening assays can be readily configured for use in future automated HTS projects to discover potent inhibitors that may be developed into novel adjuvants for antibiotic chemotherapy that moderate the development and transmission of antibiotic resistance genes and increase the antibiotic therapeutic index

Probing the structure of RecA–DNA filaments. Advantages of a fluorescent guanine analog

The RecA protein of Escherichia coli plays a crucial roles in DNA recombination and repair, as well as various aspects of bacterial pathogenicity. The formation of a RecA-ATP-ssDNA complex initiates all RecA activities and yet a complete structural and mechanistic description of this filament has remained elusive. An analysis of RecA-DNA interactions was performed using fluorescently labeled oligonucleotides. A direct comparison was made between fluorescein and several fluorescent nucleosides. The fluorescent guanine analog 6-methylisoxanthopterin (6MI) demonstrated significant advantages over the other fluorophores and represents an important new tool for characterizing RecA-DNA interactions

High-Throughput Screening for RecA Inhibitors Using a Transcreener Adenosine 5′- O -Diphosphate Assay

The activities of the bacterial RecA protein are involved in the de novo development and transmission of antibiotic resistance genes, thus allowing bacteria to overcome the metabolic stress induced by antibacterial agents. RecA is ubiquitous and highly conserved among bacteria, but has only distant homologs in human cells. Together, this evidence points to RecA as a novel and attractive antibacterial drug target. All known RecA functions require the formation of a complex formed by multiple adenosine 5′-O-triphosphate (ATP)-bound RecA monomers on single-stranded DNA. In this complex, RecA hydrolyzes ATP. Although several methods for assessing RecA's ATPase activity have been reported, these assay conditions included relatively high concentrations of enzyme and ATP and thereby restricted the RecA conformational state. Herein, we describe the validation of commercial reagents (Transcreener® adenosine 5′-O-diphosphate [ADP]2 fluorescence polarization assay) for the high-throughput measurement of RecA's ATPase activity with lower concentrations of ATP and RecA. Under optimized conditions, ADP detection by the Transcreener reagent provided robust and reproducible activity data (Z′=0.92). Using the Transcreener assay, we screened 113,477 small molecules against purified RecA protein. In total, 177 small molecules were identified as confirmed hits, of which 79 were characterized by IC50 values ≤10 μM and 35 were active in bioassays with live bacteria. This set of compounds comprises previously unidentified scaffolds for RecA inhibition and represents tractable hit structures for efforts aimed at tuning RecA inhibitory activity in both biochemical and bacteriological assays

Novel Inhibitors of E. coli RecA ATPase Activity

The bacterial RecA protein has been implicated as a bacterial drug target not as an antimicrobial target, but as an adjuvant target with the potential to suppress the mechanism by which bacteria gain drug resistance. In order to identify small molecules that inhibit RecA/ssDNA nucleoprotein filament formation, we have adapted the phosphomolybdate-blue ATPase assay for high throughput screening to determine RecA ATPase activity against a library of 33,600 compounds, which is a selected representation of diverse structure of 350,000. Four distinct chemotypes were represented among the 40 validated hits. SAR and further chemical synthesis is underway to optimize this set of inhibitors to be used as antimicrobial adjuvant agents

Economic crisis and the construction of a neo-liberal regulatory regime in Korea

A consistent theme of the literature on the ontology of the 1997 South Korean crisis is the key role played by regulatory failures and the growing weakness of the state. This paper seeks to briefly highlight both the insights and the limitations of this approach to understanding the crisis. Having done so, we shall set out the argument that the crisis created an opportunity for reformist Korean élites to advance their longstanding, but previously frustrated, project to create a comprehensive unambiguously neo-liberal regulatory regime. This paper will also seek to highlight the implications of our reading of the development of the Korean political economy for broader debates on economic liberalisation, crisis and the future of the developmental state

Evidence for dynamics in proteins as a mechanism for ligand dissociation

Signal transduction, regulatory processes, and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times, or koff, should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into koff for a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase that vary by six orders of magnitude in binding affinity. NMR relaxation dispersion experiments revealed a common set of residues near the binding site that undergo a concerted, millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with Ki and koff, suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although kconf,forward is faster than koff, use of the ligand series allowed for connections to be drawn between kinetic events on different timescales

Inhibitors of Streptococcus pneumoniae Surface Endonuclease EndA Discovered by High-Throughput Screening Using a PicoGreen Fluorescence Assay

The human commensal pathogen, Streptococcus pneumoniae, expresses a number of virulence factors that promote serious pneumococcal diseases, resulting in significant morbidity and mortality worldwide. These virulence factors may give S. pneumoniae the capacity to escape immune defenses, resist antimicrobial agents, or a combination of both. Virulence factors also present possible points of therapeutic intervention. The activities of the surface endonuclease, EndA, allow S. pneumoniae to establish invasive pneumococcal infection. EndA’s role in DNA uptake during transformation contributes to gene transfer and genetic diversitifcation. Moreover, EndA’s nuclease activity degrades the DNA backbone of neutrophil extracellular traps (NETs), allowing pneumococcus to escape host immune responses. Given its potential impact on pneumococcal pathogenicity, EndA is an attractive target for novel antimicrobial therapy. Herein, we describe the development of a high-throughput screening assay for the discovery of nuclease inhibitors. Nuclease-mediated digestion of double-stranded DNA was assessed using fluorescence intensity changes of the DNA dye ligand, PicoGreen. Under optimized conditions, the assay provided robust and reproducible activity data (Z'=0.87) and was used to screen 4727 small molecules against an imidazole-rescued variant of EndA. In total, 10 small molecules were confirmed as novel EndA inhibitors that may have utility as research tools for understanding pneumococcal pathogenesis, and ultimately drug discovery

Tamm Review: Management of mixed-severity fire regime forests in Oregon, Washington, and Northern California

Increasingly, objectives for forests with moderate- or mixed-severity fire regimes are to restore successionally diverse landscapes that are resistant and resilient to current and future stressors. Maintaining native species and characteristic processes requires this successional diversity, but methods to achieve it are poorly explained in the literature. In the Inland Pacific US, large, old, early seral trees were a key historical feature of many young and old forest successional patches, especially where fires frequently occurred. Large, old trees are naturally fire-tolerant, but today are often threatened by dense understory cohorts that create fuel ladders that alter likely post-fire successional pathways. Reducing these understories can contribute to resistance by creating conditions where canopy trees will survive disturbances and climatic stressors; these survivors are important seed sources, soil protectors, and critical habitat elements. Historical timber harvesting has skewed tree size and age class distributions, created hard edges, and altered native patch sizes. Manipulating these altered forests to promote development of larger patches of older, larger, and more widely-spaced trees with diverse understories will increase landscape resistance to severe fires, and enhance wildlife habitat for underrepresented conditions. Closed-canopy, multi-layered patches that develop in hot, dry summer environments are vulnerable to droughts, and they increase landscape vulnerability to insect outbreaks and severe wildfires. These same patches provide habitat for species such as the northern spotted owl, which has benefited from increased habitat area. Regional and local planning will be critical for gauging risks, evaluating trade-offs, and restoring dynamics that can support these and other species. The goal will be to manage for heterogeneous landscapes that include variably-sized patches of (1) young, middle-aged, and old, closed canopy forests growing in upper montane, northerly aspect, and valley bottom settings, (2) a similar diversity of open-canopy, fire-tolerant patches growing on ridgetops, southerly aspects, and lower montane settings, and (3) significant montane chaparral and grassland areas. Tools to achieve this goal include managed wildfire, prescribed burning, and variable density thinning at small to large scales. Specifics on ‘‘how much and where?” will vary according to physiographic, topographic and historical templates, and regulatory requirements, and be determined by means of a socio-ecological process