Structural characterization of a soluble amyloid beta-peptide oligomer.

Alzheimer's disease (AD) is a neurodegenerative disorder that is linked to the presence of amyloid beta-peptides that can form insoluble fibrils or soluble oligomeric assemblies. Soluble forms are present in the brains and tissues of Alzheimer's patients, and their presence correlates with disease progression. Long-lived soluble forms can be generated in vitro by using small amounts of aliphatic hydrocarbon chains of detergents or fatty acids in preparations of amyloid beta-peptides. Using NMR, we have characterized soluble oligomers of Abeta preglobulomer and globulomer that are stable and alter synaptic activity. The NMR data indicate that these soluble forms have a mixed parallel and antiparallel beta-sheet structure that is different from fibrils which contain only parallel beta-sheets. Using the structural data, we engineered a disulfide bond into the soluble Abeta globulomer to give a "new" soluble antigen that is stable, homogeneous, and binds with the same affinity to selective antibodies as the parent wt globulomer.

Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists.

Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.

A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC). This assay gives an excellent signal window for measuring FAAH activity and, as a continuous assay, inherently offers improved sensitivity and accuracy over previously reported endpoint assays. The assay was validated using a panel of known FAAH inhibitors and purified recombinant human FAAH, then converted to a 384 well format and used to screen a large library of compounds (>600,000 compounds) to identify FAAH inhibitors. This screen identified numerous novel FAAH inhibitors of diverse chemotypes. These hits confirmed using a native FAAH substrate, anandamide, and had very similar rank order potency to that obtained using the D-AMC substrate. Collectively these data demonstrate that D-AMC can be successfully used to rapidly and effectively identify novel FAAH inhibitors for potential therapeutic use.

Structure of MurF from Streptococcus pneumoniae co-crystallized with a small molecule inhibitor exhibits interdomain closure.

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.

Transforming the work of early-stage drug discovery through bioprocess informatics.

Drug discovery has historically advanced by synergy and chance. These are proving insufficient to meet the needs of the marketplace and the demands of modern medicine. We describe our strategic approaches to building and employing flexible informatics tools to transform and improve the workflows and efficiencies of the early-stages of target development in drug discovery. We contrast our approach to strategies that have recently evolved at startup biotechnology companies who use similar technological approaches to drug development but who are less encumbered by precedent and history.

Activation of TRPA1 channels by the fatty acid amide hydrolase inhibitor 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597).

As a member of the transient receptor potential (TRP) ion channel superfamily, the ligand-gated ion channel TRPA1 has been implicated in nociceptive function and pain states. The endogenous ligands that activate TRPA1 remain unknown. However, various agonists have been identified, including environmental irritants (e.g., acrolein) and ingredients of pungent natural products [e.g., allyl isothiocyanate (ITC), cinnamaldehyde, allicin, and gingerol]. In general, these agents are either highly reactive, nonselective, or not potent or efficacious, significantly limiting their utilities in the study of TRPA1 channel properties and biological functions. In a search for novel TRPA1 agonists, we identified 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597), a potent and systemically active inhibitor of fatty acid amide hydrolase (FAAH). This enzyme is responsible for anandamide degradation and therefore has been pursued as an antinociceptive and antiepileptic drug target. Using Ca(2+) influx assays and patch-clamp techniques, we demonstrated that URB597 could activate heterologously expressed human and rat TRPA1 channels, whereas two other FAAH inhibitors (i.e., URB532 and Compound 7) had no effect. When applied to inside-out membrane patches expressing rat TRPA1, URB597 elicited single-channel activities with a unitary conductance of 40 pS. Furthermore, URB597 activated TRPA1 channels endogenously expressed in a population of rat dorsal root ganglion neurons that also responded to ITC. In contrast to its effect on TRPA1, URB597 inhibited TRPM8 and had no effects on TRPV1 or TRPV4. Thus, we conclude that URB597 is a novel agonist of TRPA1 and probably activates the channel through a direct gating mechanism.

From bacterial genomes to novel antibacterial agents: discovery, characterization, and antibacterial activity of compounds that bind to HI0065 (YjeE) from Haemophilus influenzae.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics-driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.