Galactokinase 1 is the source of elevated galactose-1-phosphate and cerebrosides are modestly reduced in a mouse model of classic galactosemia.

Classic galactosemia (CG) arises from loss-of-function mutations in the Galt gene, which codes for the enzyme galactose-1-phosphate uridylyltransferase (GALT), a central component in galactose metabolism. The neonatal fatality associated with CG can be prevented by galactose dietary restriction, but for decades it has been known that limiting galactose intake is not a cure and patients often have lasting complications. Even on a low-galactose diet, GALT's substrate galactose-1-phosphate (Gal1P) is elevated and one hypothesis is that elevated Gal1P is a driver of pathology. Here we show that Gal1P levels were elevated above wildtype (WT) in Galt mutant mice, while mice doubly mutant for Galt and the gene encoding galactokinase 1 (Galk1) had normal Gal1P levels. This indicates that GALK1 is necessary for the elevated Gal1P in CG. Another hypothesis to explain the pathology is that an inability to metabolize galactose leads to diminished or disrupted galactosylation of proteins or lipids. Our studies reveal that levels of a subset of cerebrosides-galactosylceramide 24:1, sulfatide 24:1, and glucosylceramide 24:1-were modestly decreased compared to WT. In contrast, gangliosides were unaltered. The observed reduction in these 24:1 cerebrosides may be relevant to the clinical pathology of CG, since the cerebroside galactosylceramide is an important structural component of myelin, the 24:1 species is the most abundant in myelin, and irregularities in white matter, of which myelin is a constituent, have been observed in patients with CG. Therefore, impaired cerebroside production may be a contributing factor to the brain damage that is a common clinical feature of the human disease.

Total synthesis of trifluorobutyryl-modified, globally protected sialyl Lewis x by a convergent [2+2] approach.

Structural and quantitative changes in the expression of sialic acid residues on the surface of eukaryotic cells profoundly influence a broad range of biological processes including inflammation, antigen recognition, microbial attachment, and tumor metastasis. Uptake and incorporation of sialic acid analogues in mammalian cells enable structure-function studies and perturbation of specific recognition events. Our group has recently shown that a trifluorobutyryl-modified sialic acid metabolite diminishes the adhesion of mammalian cells to E and P-selectin, presumably by leading to the expression of fluorinated sLex epitopes on cell surfaces, and interfering with the sLex-selectin interactions that are well known in mediating tumor cell migration.1 For studies directed towards understanding the molecular basis of this reduced adhesion, chemical synthesis of trifluorobutyrylated sialyl Lewis x (C4F3--sLex) was crucial. We have developed a highly efficient [2+2] approach for the assembly of C4F3-sLex on a preparative scale that contains versatile protective groups allowing the glycan to be surface immobilized or solubilized as needed for biophysical studies to investigate selectin interactions. This strategy can, in principle, be used for preparation of other N-modified sLex analogues.

Design, synthesis, and SAR of 2-dialkylamino-4-arylpyrimidines as potent and selective corticotropin-releasing factor(1) (CRF(1)) receptor antagonists.

A series of 2-dialkylamino-4-phenylpyrimidines (7) was designed and synthesized as CRF(1) antagonists. SAR studies of this series resulted in the discovery of potent and selective antagonists 7b and 7n bearing a 4-(2,4,6-trisubstituted-phenyl) ring and a bulky 2-(N-bis(cyclopropane)methyl-N-propyl)amino group.

A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo.

The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been implicated in the development and progression of several human cancers and are attractive targets for cancer therapy. PHA-665752 was identified as a small molecule, ATP-competitive, active-site inhibitor of the catalytic activity of c-Met kinase (K(i) 4 nM). PHA-665752 also exhibited >50-fold selectivity for c-Met compared with a panel of diverse tyrosine and serine-threonine kinases. In cellular studies, PHA-665752 potently inhibited HGF-stimulated and constitutive c-Met phosphorylation, as well as HGF and c-Met-driven phenotypes such as cell growth (proliferation and survival), cell motility, invasion, and/or morphology of a variety of tumor cells. In addition, PHA-665752 inhibited HGF-stimulated or constitutive phosphorylation of mediators of downstream signal transduction of c-Met, including Gab-1, extracellular regulated kinase, Akt, signal transducer and activator of transcription 3, phospholipase C gamma, and focal adhesion kinase, in multiple tumor cell lines in a pattern correlating to the phenotypic response of a given tumor cell. In in vivo studies, a single dose of PHA-665752 inhibited c-Met phosphorylation in tumor xenografts for up to 12 h. Inhibition of c-Met phosphorylation was associated with dose-dependent tumor growth inhibition/growth delay over a repeated administration schedule at well-tolerated doses. Interestingly, potent cytoreductive activity was demonstrated in a gastric carcinoma xenograft model. Collectively, these results demonstrate the feasibility of selectively targeting c-Met with ATP-competitive small-molecules and suggest the therapeutic potential of targeting c-Met in human cancers.

Structure-based design and discovery of novel inhibitors of protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) are important in the regulation of signal transduction processes. Certain enzymes of this class are considered as potential therapeutic targets in the treatment of a variety of diseases such as diabetes, inflammation, and cancer. However, many PTP inhibitors identified to date are peptide-based and contain a highly charged phosphate-mimicking component. These compounds usually lack membrane permeability and this limits their utility in the inhibition of intracellular phosphatases. In the present study, we have used structure-based design and modeling techniques to explore catalytic-site directed, reversible inhibitors of PTPs. Employing a non-charged phosphate mimic and non-peptidyl structural components, we have successfully designed and synthesized a novel series of trifluoromethyl sulfonyl and trifluoromethyl sulfonamido compounds as PTP inhibitors. This is the first time that an uncharged phosphate mimic is reported in the literature for general, reversible, and substrate-competitive inhibition of PTPs. It is an important discovery because the finding may provide a paradigm for the development of phosphatase inhibitors that enter cells and modify signal transduction.