Predicting Electrophoretic Mobility of Protein-Ligand Complexes for Ligands from DNA-Encoded Libraries of Small Molecules.

Selection of target-binding ligands from DNA-encoded libraries of small molecules (DELSMs) is a rapidly developing approach in drug-lead discovery. Methods of kinetic capillary electrophoresis (KCE) may facilitate highly efficient homogeneous selection of ligands from DELSMs. However, KCE methods require accurate prediction of electrophoretic mobilities of protein-ligand complexes. Such prediction, in turn, requires a theory that would be applicable to DNA tags of different structures used in different DELSMs. Here we present such a theory. It utilizes a model of a globular protein connected, through a single point (small molecule), to a linear DNA tag containing a combination of alternating double-stranded and single-stranded DNA (dsDNA and ssDNA) regions of varying lengths. The theory links the unknown electrophoretic mobility of protein-DNA complex with experimentally determined electrophoretic mobilities of the protein and DNA. Mobility prediction was initially tested by using a protein interacting with 18 ligands of various combinations of dsDNA and ssDNA regions, which mimicked different DELSMs. For all studied ligands, deviation of the predicted mobility from the experimentally determined value was within 11%. Finally, the prediction was tested for two proteins and two ligands with a DNA tag identical to those of DELSM manufactured by GlaxoSmithKline. Deviation between the predicted and experimentally determined mobilities did not exceed 5%. These results confirm the accuracy and robustness of our model, which makes KCE methods one step closer to their practical use in selection of drug leads, and diagnostic probes from DELSMs.

Prediction of protein-DNA complex mobility in gel-free capillary electrophoresis.

Selection of protein binders from highly diverse combinatorial libraries of DNA-encoded small molecules is a highly promising approach for discovery of small-molecule drug leads. Methods of kinetic capillary electrophoresis provide the high efficiency of partitioning required for such selection but require the knowledge of electrophoretic mobility of the protein-ligand complex. Here we present a theoretical approach for an accurate estimate of the electrophoretic mobility of such complexes. The model is based on a theory of the thin double layer and corresponding expressions used for the mobilities of a rod-like short oligonucleotide and a sphere-like globular protein. The model uses empirical values of mobilities of free protein, free ligand, and electroosmotic flow. The model was tested with a streptavidin-dsDNA complex linked through biotin (small molecule). The deviation of the prediction from the experimental mobility did not exceed 4%, thus confirming that not only is the model adequate but it is also accurate. This model will facilitate reliable use of KCE methods for selection of drug leads from libraries of DNA-encoded small molecules.

Skunk rabies in California (1992-2003)--implications for oral rabies vaccination.

Skunk-variant rabies is endemic in California (United States), and the development of oral vaccines and baits to vaccinate skunks is in progress. In 2003, the California Department of Public Health (CDPH) began to quantify the impacts of skunk-variant rabies and to assess the feasibility of using oral rabies vaccination (ORV) as a containment measure. The CDPH rabies case data for skunks were spatially depicted and analyzed using a geographic information system. Statewide, rabid skunks (1992-2003) primarily occurred in seven physiographic regions: Central Coast, North Coast, North Sierra, Sacramento Valley, San Francisco Bay and Delta, San Joaquin Valley, and South Sierra. Detailed analysis of rabid skunks in San Luis Obispo (SLO) and Santa Barbara (SB) counties showed that skunk rabies was endemic in the coastal plain of SLO County between 1992 and 2000, but only became epizootic in SB County during 2002. Despite the widespread distribution of striped skunks (Mephitis mephitis) throughout most of California, the skunk rabies variant has not been found in Los Angeles County since 1979. Results imply that future ORV campaigns for skunk-variant rabies in the Pacific Coastal Plain could deter spread from SLO into SB County, as well as deterring the reintroduction of skunk-variant rabies into southern California.

Raccoon (Procyon lotor) biomarker and rabies antibody response to varying oral rabies vaccine bait densities in northwestern Pennsylvania.

Distribution of oral rabies vaccine baits has been used as a strategy for managing rabies in the United States since the 1990s. Since that time, efforts have been made to improve baiting strategies with a focus on bait density to maximize both efficiency and cost effectiveness. An optimal rabies management strategy includes a vaccine bait preferred by the target species that is distributed at the minimal density needed to achieve population immunity to prevent rabies spread. The purpose of our pilot study was to examine the effect of 75, 150, and 300 baits/km2 vaccine bait densities on rabies virus neutralizing antibody (RVNA) seroprevalence in raccoons (Procyon lotor). Raboral V-RG® fishmeal polymer baits (Merial Inc. (now a part of Boehringer Ingelheim), Athens, Georgia) contain a tetracycline biomarker that was used to estimate bait consumption as another measure of intervention impact. Our results suggest that raccoon RVNA response increases as bait density increases, but the effect may not be sufficient to justify the cost except in the case of contingency actions or an epizootic. Non-target species, especially opossums (Didelphis virginianus) in certain areas, should be considered when determining an appropriate bait density to ensure sufficient baits are available for consumption by the target species.

Sex differences in the acquisition of a radial maze task in the CD-1 mouse.

The purpose of the present study was to investigate spatial processing performance in male and female CD-1 mice. A substantial literature supports the existence of significant sex differences in both human and rodent models of learning and memory. The nature of these differences is dependent upon the parameters of the task, species and strain of animal. In the present study, male and female CD-1 mice were trained for 3 days to perform a 4/8 spatial memory task in an eight-arm radial maze and then tested for a total of 5 days. On the final day of radial maze testing, male CD-1 mice committed marginally significantly fewer reference memory (RM) and significantly fewer working memory (WM) errors on the radial maze task than female CD-1 mice. In addition, female mice obtained significantly fewer rewards during the final two testing sessions. The present data provide the first evidence for sex differences in radial maze learning in the CD-1 mouse, a strain known for its estrogen insensitivity. Consistent with the majority of literature that supports sex differences in spatial processing in rodents, female CD-1 mice acquired significantly fewer rewards than male CD-1 mice during an eight-arm radial maze task.

Tridimensional personality traits of college student marijuana users.

The present study examined the possibility that marijuana use among college students might be associated with particular personality traits. College student volunteers (N = 176) were administered the Tridimensional Personality Questionnaire and a drug-use questionnaire. Analysis of variance showed that the mean score on Novelty Seeking was significantly higher and on Persistence significantly lower among lifetime marijuana users than among nonmarijuana using peers. Programs for prevention among college students may be enhanced by approaches tailored to individuals scoring high on Novelty Seeking and low on Persistence.

Influence of oral rabies vaccine bait density on rabies seroprevalence in wild raccoons.

The effect of different oral rabies vaccine (ORV) bait densities (75, 150, and 300 baits/km(2)) on the seroprevalence of rabies virus neutralizing antibodies (RVNAs) in raccoons (Procyon lotor) was assessed at a 15% seroprevalence difference threshold in rural areas of northeast Ohio. Results (n=588 raccoons) indicated that seropositivity for RVNAs was associated with both bait density and bait campaign frequency. Associations were not detected for raccoon gender, age, or macro-habitat. The odds of being seropositive were greater for raccoons originating from 300 bait/km(2) treatment areas relative to those coming from the 75 bait/km(2) areas (odds ratio [OR]=4.4, probability [P]<0.001, 95% confidence interval [CI]=2.4-7.9), while accounting for cumulative ORV campaigns. No statistical advantage in seroprevalence was detected when comparing 150-75 baits/km(2). These results indicate that a relatively extreme bait density when evenly distributed may be necessary to obtain a significant increase in seroprevalence. Higher bait densities may be more appropriate and less costly to address focused outbreaks than labor intensive trap-vaccinate-release and local population reduction campaigns. Finally, dramatic increases in seroprevalence of RVNA were not observed in raccoons between sequential, semi-annual campaigns, yet cumulative ORV campaigns were associated with gradual increases in seroprevalence.