Structure based design of an in vivo active hydroxamic acid inhibitor of P. aeruginosa LpxC.

Lipid A is an essential component of the Gram negative outer membrane, which protects the bacterium from attack of many antibiotics. The Lipid A biosynthesis pathway is essential for Gram negative bacterial growth and is unique to these bacteria. The first committed step in Lipid A biosynthesis is catalysis by LpxC, a zinc dependent deacetylase. We show the design of an LpxC inhibitor utilizing a robust model which directed efficient design of picomolar inhibitors. Analysis of physiochemical properties drove design to focus on an optimal lipophilicity profile. Further structure based design took advantage of a conserved water network over the active site, and with the optimal lipophilicity profile, led to an improved LpxC inhibitor with in vivo activity against wild type Pseudomonas aeruginosa.

Perspective on Antibacterial Lead Identification Challenges and the Role of Hypothesis-Driven Strategies.

For the past three decades, the pharmaceutical industry has undertaken many diverse approaches to discover novel antibiotics, with limited success. We have witnessed and personally experienced many mistakes, hurdles, and dead ends that have derailed projects and discouraged scientists and business leaders. Of the many factors that affect the outcomes of screening campaigns, a lack of understanding of the properties that drive efflux and permeability requirements across species has been a major barrier for advancing hits to leads. Hits that possess bacterial spectrum have seldom also possessed druglike properties required for developability and safety. Persistence in solving these two key barriers is necessary for the reinvestment into discovering antibacterial agents. This perspective narrates our experience in antibacterial discovery-our lessons learned about antibacterial challenges as well as best practices for screening strategies. One of the tenets that guides us is that drug discovery is a hypothesis-driven science. Application of this principle, at all steps in the antibacterial discovery process, should improve decision making and possibly the odds of what has become, in recent decades, an increasingly challenging endeavor with dwindling success rates.

A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins.

Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD50 values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.

Characterization of d-boroAla as a novel broad-spectrum antibacterial agent targeting d-Ala-d-Ala ligase.

d-boroAla was previously characterized as an inhibitor of bacterial alanine racemase and d-Ala-d-Ala ligase enzymes (Biochemistry, 28, 1989, 3541). In this study, d-boroAla was identified and characterized as an antibacterial agent. d-boroAla has activity against both Gram-positive and Gram-negative organisms, with minimal inhibitory concentrations down to 8 µg / mL. A structure-function study on the alkyl side chain (NH(2) -CHR-B(OR')(2) ) revealed that d-boroAla is the most effective agent in a series including boroGly, d-boroHomoAla, and d-boroVal. l-boroAla was much less active, and N-acetylation completely abolished activity. An LC-MS / MS assay was used to demonstrate that d-boroAla exerts its antibacterial activity by inhibition of d-Ala-d-Ala ligase. d-boroAla is bactericidal at 1× minimal inhibitory concentration against Staphylococcus aureus and Bacillus subtilis, which each encode one copy of d-Ala-d-Ala ligase, and at 4× minimal inhibitory concentration against Escherichia coli and Salmonella enterica serovar Typhimurium, which each encode two copies of d-Ala-d-Ala ligase. d-boroAla demonstrated a frequency of resistance of 8 × 10(-8) at 4× minimal inhibitory concentration in S. aureus. These results demonstrate that d-boroAla has promising antibacterial activity and could serve as the lead agent in a new class of d-Ala-d-Ala ligase targeted antibacterial agents. This study also demonstrates d-boroAla as a possible probe for d-Ala-d-Ala ligase function.

Deletion of individual mRNA capping genes is unexpectedly not lethal to Candida albicans and results in modified mRNA cap structures.

Eukaryotic mRNAs are modified at the 5' end with a cap structure. In fungal cells, the formation of the mRNA cap structure is catalyzed by three enzymes: triphosphatase, guanylyltransferase, and methyltransferase. Fungal capping enzymes have been proposed to be good antifungal targets because they differ significantly from their human counterparts and the genes encoding these enzymes are essential in Saccharomyces cerevisiae. In the present study, Candida albicans null mutants were constructed for both the mRNA triphosphatase-encoding gene (CET1) and the mRNA methyltransferase encoding gene (CCM1), proving that these genes are not essential in C. albicans. Heterozygous deletions were generated, but no null mutants were isolated for the guanylyltransferase-encoding gene (CGT1), indicating that this gene probably is essential in C. albicans. Whereas these results indicate that Cet1p and Ccm1p are not ideal molecular targets for development of anticandidal drugs, they do raise questions about the capping of mRNA and translation initiation in this fungus. Southern blot analysis of genomic DNA indicates that there are not redundant genes for CET1 and CCM1 and analysis of mRNA cap structures indicate there are not alternative pathways compensating for the function of CET1 or CCM1 in the null mutants. Instead, it appears that C. albicans can survive with modified mRNA cap structures.