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[Image: see text] Autotaxin (ATX) is a lysophospholipase D that is the main enzyme responsible for generating LPA in body fluids. Although ATX was isolated from a conditioned medium of melanoma cells, later it was discovered to play a critical role in vascular and neuronal development. ATX has also been implicated in primary brain tumor, fibrosis, and rheumatoid arthritis, as well as neurological diseases such as multiple sclerosis, Alzheimer’s disease, and neuropathic pain. As ATX and LPA levels are increased upon neuronal injury, a selective ATX inhibitor could provide a new approach to treat neuropathic pain. Herein we describe the discovery of a novel series of nonzinc binding reversible ATX inhibitors, particularly a potent, selective, orally bioavailable, brain-penetrable tool compound BIO-32546, as well as its synthesis, X-ray cocrystal structure, pharmacokinetics, and in vivo efficacy.

Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification.

Alkyne 40, 5-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methylpent-4-yn-2-ol (EC144), is a second generation inhibitor of heat shock protein 90 (Hsp90) and is substantially more potent in vitro and in vivo than the first generation inhibitor 14 (BIIB021) that completed phase II clinical trials. Alkyne 40 is more potent than 14 in an Hsp90α binding assay (IC(50) = 1.1 vs 5.1 nM) as well as in its ability to degrade Her-2 in MCF-7 cells (EC(50) = 14 vs 38 nM). In a mouse model of gastric tumors (N87), 40 stops tumor growth at 5 mg/kg and causes partial tumor regressions at 10 mg/kg (po, qd × 5). Under the same conditions, 14 stops tumor growth only at 120 mg/kg, and does not induce partial regressions. Thus, alkyne 40 is approximately 20-fold more efficacious than 14 in mice.

RORγt is a pivotal regulator of a pro-inflammatory gene expression program implicated in the pathology of several major human immune-mediated diseases. Evidence from mouse models demonstrates that genetic or pharmacological inhibition of RORγ activity can block the production of pathogenic cytokines, including IL-17, and convey therapeutic benefit. We have identified and developed a biaryl-carboxylamide series of RORγ inverse agonists via a structure based design approach. Co-crystal structures of compounds 16 and 48 supported the design approach and confirmed the key interactions with RORγ protein; the hydrogen bonding with His479 was key to the significant improvement in inverse agonist effect. The results have shown this is a class of potent and selective RORγ inverse agonists, with demonstrated oral bioavailability in rodents.

Anthocyanins are the red, blue and purple pigments responsible for the coloration of many plants. These pigments have been the subject of many studies due to their importance as a quality indicator in foods and as an important chemotaxonomic indicator for plants. Early work with anthocyanins employed paper chromatographic methods. More recently, high-performance liquid chromatography has been widely applied to the study of these pigments. The objective of this paper is to review the chromatographic methods that have been employed in the analysis of anthocyanins with emphasis on the more recent developments in high-performance liquid chromatographic analysis of anthocyanins as applied to food quality measurement.

The anthocyanin pigment profiles of commercial black currant, blackberry, black raspberry, elderberry, cherry, plum, grape, bilberry, and red cabbage products were characterized by high-performance liquid chromatography (HPLC)/photodiode array detection. Removal of acylated anthocyanins by alkaline hydrolysis and selective removal of anthocyanins on a reversed-phase cartridge with borate buffer were auxiliary techniques that proved helpful in making peak assignments. Both the retention properties on reversed-phase HPLC and the spectral properties by photodiode array detection were used to characterize the anthocyanins. Other properties including tinctoral strength, total anthocyanin concentration, browning, titratable acidity, and Hunter tristimulus values were also determined for these colorants.

A systematic procedure for separation and characterization of anthocyanins is described. Separation of pigments was achieved by high-performance liquid chromatography (HPLC) on a polymer reversed-phase column. Methods for preparation of an anthocyanin isolate free of other interfering phenolics were developed. Photodiode array detection was employed to determine the UV-visible spectral characteristics of the pigments. Derivatives of delphinidin (delphinidin, petunidin, malvidin) can be distinguished from derivatives of cyanidin (cyanidin, peonidin), which in turn can be distinguished from pelargonidin derivatives on the basis of their different UV-visible spectra. Acylation with cinnamic acids and differentiation between 3- and 3,5-glycosidation can also be determined from the UV-visible spectrum. Auxiliary sample preparation techniques useful for pigment characterization included alkaline hydrolysis of the anthocyanins for determination of acylation. Anthocyanins not containing an o-diphenolic system can be enriched on a C18 reversed-phase cartridge by elution with alkaline borate buffer. With a combination of these techniques, peak assignments for the anthocyanins from sources whose anthocyanin composition is known can be readily made.

Thirty-one samples of commercial cranberry juice drink and one sample of commercial cranberry juice concentrate were analyzed for nonvolatile acids and anthocyanidin profiles by liquid chromatography (LC). Ultraviolet-visible spectral measurements were used to measure pigment concentration, polymeric color, and percent polymeric color. Nineteen of the 31 samples analyzed were found to be adulterated. The adulterated samples exhibited nonvolatile organic acid profiles indicative of added malic and/or citric acid. Anthocyanidin profiles of the adulterated samples showed the presence of substantial quantities of delphinidin and malvidin, neither of which are present in cranberries in significant amounts. Grape skin extract is believed to be the added colorant.

Eight samples of cranberries (Vaccinium macrocarpon) representing the major varieties and principal commercial growing regions in the United States were processed into juice. Four of the 8 samples were concentrated to 50° Brix. Liquid chromatography (LC) was used to determine nonvolatile organic acid, anthocyanidin pigment, and sugar profiles. Ultraviolet-visible spectral methods were used to determine anthocyanin concentration, polymeric color, and percent polymeric color. Other data presented include stable isotope carbon ratios, degree Brix, pH, and Hunter color parameters. These data are presented to serve as an authentic data base for use in detection of adulterated cranberry juice.