A renaissance in pharmacy education at the University of North Carolina at Chapel Hill.

The UNC Eshelman School of Pharmacy is transforming its doctor of pharmacy program to emphasize active engagement of students in the classroom, foster scientific inquiry and innovation, and immerse students in patient care early in their education. The admissions process is also being reengineered.

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.

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 (1/k(off)) should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into k(off) involving a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase whose binding affinities vary by six orders of magnitude.

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.

Direct detection of structurally resolved dynamics in a multiconformation receptor-ligand complex.

Structure-based drug design relies on static protein structures despite significant evidence for the need to include protein dynamics as a serious consideration. In practice, dynamic motions are neglected because they are not understood well enough to model, a situation resulting from a lack of explicit experimental examples of dynamic receptor-ligand complexes. Here, we report high-resolution details of pronounced ~1 ms time scale motions of a receptor-small molecule complex using a combination of NMR and X-ray crystallography.

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.

Inhibitors of RecA activity discovered by high-throughput screening: cell-permeable small molecules attenuate the SOS response in Escherichia coli.

The phenomenon of antibiotic resistance has created a need for the development of novel antibiotic classes with nonclassical cellular targets. Unfortunately, target-based drug discovery against proteins considered essential for in vitro bacterial viability has yielded few new therapeutic classes of antibiotics. Targeting the large proportion of genes considered nonessential that have yet to be explored by high-throughput screening, for example, RecA, can complement these efforts.

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 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 the 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 inhibitors of RecA activities.

Inhibition of Escherichia coli RecA by rationally redesigned N-terminal helix.

Bacterial RecA promotes the development and transmission of antibiotic resistance genes by self-assembling into an ATP-hydrolyzing filamentous homopolymer on single-stranded DNA. We report the design of a 29mer peptide based on the RecA N-terminal domain involved in intermonomer contact that inhibits RecA filament assembly with an IC50 of 3 microM.

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.

Direct evaluation of a kinetic model for RecA-mediated DNA-strand exchange: the importance of nucleic acid dynamics and entropy during homologous genetic recombination.

The Escherichia coli RecA protein is the prototype of a class of proteins that play central roles in genomic repair and recombination in all organisms. The unresolved mechanistic strategy by which RecA aligns a single strand of DNA with a duplex DNA and mediates a DNA strand switch is central to understanding homologous recombination. We explored the mechanism of RecA-mediated DNA-strand exchange using oligonucleotide substrates with the intrinsic fluorophore 6-methylisoxanthopterin.

Origins of sequence selectivity in homologous genetic recombination: insights from rapid kinetic probing of RecA-mediated DNA strand exchange.

Despite intense effort over the past 30 years, the molecular determinants of sequence selectivity in RecA-mediated homologous recombination have remained elusive. Here, we describe when and how sequence homology is recognized between DNA strands during recombination in the context of a kinetic model for RecA-mediated DNA strand exchange. We characterized the transient intermediates of the reaction using pre-steady-state kinetic analysis of strand exchange using oligonucleotide substrates containing a single fluorescent G analog.

Intersubunit electrostatic complementarity in the RecA nucleoprotein filament regulates nucleotide substrate specificity and conformational activation.

The Escherichia coli RecA protein is the prototypical member of a family of molecular motors that transduces ATP binding and hydrolysis for mechanical function. While many general mechanistic features of RecA action are known, specific structural and functional insights into the molecular basis of RecA activation remain elusive. Toward a more complete understanding of the interdependence between ATP and DNA binding by RecA, we report the characterization of a mutant RecA protein wherein the aspartate residue at position 100 within the ATP binding site is replaced by arginine.

Conformationally selective binding of nucleotide analogues to Escherichia coli RecA: a ligand-based analysis of the RecA ATP binding site.

The roles of the RecA protein in the survival of bacteria and the evolution of resistance to antibiotics make it an attractive target for inhibition by small molecules. The activity of RecA is dependent on the formation of a nucleoprotein filament on single-stranded DNA that hydrolyzes ATP. We probed the nucleotide binding site of the active RecA protein using modified nucleotide triphosphates to discern key structural elements of the nucleotide and of the binding site that result in the activation of RecA for NTP hydrolysis.

Construction and evaluation of a kinetic scheme for RecA-mediated DNA strand exchange.

The Escherichia coli RecA protein is the prototype of a class of proteins playing a central role in genomic repair and recombination in all organisms. The unresolved mechanistic strategy by which RecA aligns a single strand of DNA with a duplex DNA and mediates a DNA strand switch is central to understanding its recombinational activities. Toward a molecular-level understanding of RecA-mediated DNA strand exchange, we explored its mechanism using oligonucleotide substrates and the intrinsic fluorescence of 6-methylisoxanthopterin (6MI).

A molecular target for suppression of the evolution of antibiotic resistance: inhibition of the Escherichia coli RecA protein by N(6)-(1-naphthyl)-ADP.

We report that N(6)-(1-naphthyl)-ADP inhibits the Escherichia coli RecA protein in vitro. A novel rapid screen identified it as a potent inhibitor of RecA nucleoprotein filament formation, and further characterization established it as an ATP-competitive inhibitor of RecA-catalyzed ATP hydrolysis. This and other inhibitors of RecA activities represent a new approach for understanding the molecular targets and pathways involved in the evolution of antibiotic resistance in bacteria.

Inhibition of the Escherichia coli RecA protein: zinc(II), copper(II) and mercury(II) trap RecA as inactive aggregates.

In bacteria, the RecA protein plays important roles in a number of DNA recombination and repair processes, including homologous recombination, SOS induction and recombinational DNA repair. We have explored the idea that the Escherichia coli RecA protein's functions could be controlled by small molecules. We investigated the 2:1 complex of zinc(II) with 1,4-dithio-l-threitol (l-DTT) that inhibits the E.

Direct evaluation of a mechanism for activation of the RecA nucleoprotein filament.

The RecA protein of Escherichia coli controls the SOS response for DNA damage tolerance and plays a crucial role in recombinational DNA repair. The formation of a RecA.ATP.

Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein.

The RecA protein of Escherichia coli plays important roles in homologous recombination, recombinational DNA repair, and SOS induction. Because its functions are conserved among the phylogenetic kingdoms, RecA investigations have provided a paradigm for understanding these biological processes. The RecA protein has been overproduced in E.

Elucidating a key intermediate in homologous DNA strand exchange: structural characterization of the RecA-triple-stranded DNA complex using fluorescence resonance energy transfer.

The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA-tsDNA complex), has been reported.

Synthesis and application of a chain-terminating dinucleotide mRNA cap analog.

[structure: see text] We describe the synthesis of a chain-terminating mRNA cap dinucleotide and its use in the in vitro transcription of homogeneously capped RNA. Computer modeling strongly indicates that RNA capped with the new compound will be a substrate for cap-dependent translation.