Synthesis and Evaluation of Eight- and Four-membered Iminosugar Analogues as Inhibitors of Testicular Ceramide-specific Glucosyltransferase, Testicular β-Glucosidase 2, and other Glycosidases

Eight- and four-membered analogues of N-butyldeoxynojirimycin (NB-DNJ), a reversible male contraceptive in mice, were prepared and tested. A chiral pool approach was used for the synthesis of the target compounds. Key steps for the synthesis of the eight-membered analogues involve: ringclosing metathesis and Sharpless asymmetric dihydroxylation, and for the four-membered analogues: Sharpless epoxidation, epoxide ring opening (azide), and Mitsunobu reaction to form the four-membered ring. (3S,4R,5S,6R,7R)-1-Nonylazocane-3,4,5,6,7-pentaol (6), was moderately active against rat-derived ceramide-specific glucosyltransferase and four of the other eight-membered analogues were weakly active against rat-derived β-glucosidase 2. Among the four-membered analogues, ((2R,3s,4S)-3-hydroxy-1-nonylazetidine-2,4-diyl)dimethanol (25), displayed selective inhibitory activity against mouse-derived ceramide-specific glucosyltransferase and was about half as potent as NB-DNJ against the rat-derived enzyme. ((2S,4S)-3-Hydroxy-1-nonyl-azetidine-2,4-diyl)dimethanol (27) was found to be a selective inhibitor of β-glucosidase 2, with potency similar to NB-DNJ. Additional glycosidase assays were performed to identify potential other therapeutic applications. The eight-membered iminosugars exhibited specificity for almond-derived β-glucosidase and the 1-nonylazetidine 25 inhibited α-glucosidase (Saccharomyces cerevisiae) with an IC50 of 600 nM and β-glucosidase (almond) with an IC50 of 20 µM. Only N-nonyl derivatives were active, emphasizing the importance of a long lipophilic side chain for inhibitory activity of the analogues studied

Ack1 Mediated AKT/PKB Tyrosine 176 Phosphorylation Regulates Its Activation

The AKT/PKB kinase is a key signaling component of one of the most frequently activated pathways in cancer and is a major target of cancer drug development. Most studies have focused on its activation by Receptor Tyrosine Kinase (RTK) mediated Phosphatidylinositol-3-OH kinase (PI3K) activation or loss of Phosphatase and Tensin homolog (PTEN). We have uncovered that growth factors binding to RTKs lead to activation of a non-receptor tyrosine kinase, Ack1 (also known as ACK or TNK2), which directly phosphorylates AKT at an evolutionarily conserved tyrosine 176 in the kinase domain. Tyr176-phosphorylated AKT localizes to the plasma membrane and promotes Thr308/Ser473-phosphorylation leading to AKT activation. Mice expressing activated Ack1 specifically in the prostate exhibit AKT Tyr176-phosphorylation and develop murine prostatic intraepithelial neoplasia (mPINs). Further, expression levels of Tyr176-phosphorylated-AKT and Tyr284-phosphorylated-Ack1 were positively correlated with the severity of disease progression, and inversely correlated with the survival of breast cancer patients. Thus, RTK/Ack1/AKT pathway provides a novel target for drug discovery

Structural basis for the interaction of the fluorescence probe 8-anilino-1-naphthalene sulfonate (ANS) with the antibiotic target MurA

The extrinsic fluorescence dye 8-anilino-1-naphthalene sulfonate (ANS) is widely used for probing conformational changes in proteins, yet no detailed structure of ANS bound to any protein has been reported so far. ANS has been successfully used to monitor the induced-fit mechanism of MurA [UDPGlcNAc enolpyruvyltransferase (EC 2.5.1.7)], an essential enzyme for bacterial cell wall biosynthesis. We have solved the crystal structure of the ANS⋅MurA complex at 1.7-Å resolution. ANS binds at an originally solvent-exposed region near Pro-112 and induces a major restructuring of the loop Pro-112–Pro-121, such that a specific binding site emerges. The fluorescence probe is sandwiched between the strictly conserved residues Arg-91, Pro-112, and Gly-113. Substrate binding to MurA is accompanied by large movements especially of the loop and Arg-91, which explains why ANS is an excellent sensor of conformational changes during catalysis of this pharmaceutically important enzyme

X-ray crystallographic and solution state nuclear magnetic resonance spectroscopic investigations of NADP\u3csup\u3e+\u3c/sup\u3e binding to ferredoxin NADP reductase from Pseudomonas aeruginosa

The ferredoxin nicotinamide adenine dinucleotide phosphate reductase from Pseudomonas aeruginosa (pa-FPR) in complex with NADP+ has been characterized by X-ray crystallography and in solution by NMR spectroscopy. The structure of the complex revealed that pa-FPR harbors a preformed NADP + binding pocket where the cofactor binds with minimal structural perturbation of the enzyme. These findings were complemented by obtaining sequential backbone resonance assignments of this 29518 kDa enzyme, which enabled the study of the pa-FPR-NADP complex by monitoring chemical shift perturbations induced by addition of NADP+ or the inhibitor adenine dinucleotide phosphate (ADP) to pa-FPR. The results are consistent with a preformed NADP+ binding site and also demonstrate that the pa-FPR-NADP complex is largely stabilized by interactions between the protein and the 2′-P AMP portion of the cofactor. Analysis of the crystal structure also shows a vast network of interactions between the two cofaictors, FAD and NADP+, and the characteristic AFVEK258 C′-terminal extension that is typical of bacterial FPRs but is absent in their plastidic ferredoxin NADP+ reductase (FNR) counterparts. The conformations of NADP+ and FAD in pa-FPR place their respective nicotinamide and isoalloxazine rings 15 Å apart and separated by residues in the C′-terminal extension. The network of interactions among NADP +, FAD, and residues in the C-terminal extension indicate that the gross conformational rearrangement that would be necessary to place the nicotinamide and isoalloxazine rings parallel and adjacent to one another for direct hydride transfer between NADPH and FAD in pa-FPR is highly unlikely. This conclusion is supported by observations made in the NMR spectra of pa-FPR and the pa-FPR-NADP complex, which strongly suggest that residues in the C′-terminal sequence do not undergo conformational exchange in the presence or absence of NADP+. These findings are discussed in the context of a possible stepwise electron-proton-electron transfer of hydride in the oxidation of NADPH by FPR enzymes. © 2008 American Chemical Society

Crystallization and Preliminary X-Ray Diffraction Analysis of UDP-N-acetylglucosamine Enolpyruvyltransferase ofEnterobacter cloacae

Single crystals of UDP-N-acetylglucosamine enolpyruvyltransferase ofEnterobacter cloacaehave been grown by vapor diffusion using phosphate buffer as the precipitant. The crystals belong to the monoclinic space groupC2 witha= 86.9 Å,b= 155.9 Å,c= 83.8 Å, β = 91.6°. Assuming two monomers per asymmetric unit, the solvent content of these crystals is 63%. Flash-frozen crystals diffract to beyond 2 Å resolution