Fragment-Based Discovery of a Novel, Brain Penetrant, Orally Active HDAC2 Inhibitor.

Fragment-based ligand discovery was successfully applied to histone deacetylase HDAC2. In addition to the anticipated hydroxamic acid- and benzamide-based fragment screening hits, a low affinity (∼1 mM) α-amino-amide zinc binding fragment was identified, as well as fragments binding to other regions of the catalytic site. This alternative zinc-binding fragment was further optimized, guided by the structural information from protein-ligand complex X-ray structures, into a sub-µM, brain penetrant, HDAC2 inhibitor (17) capable of modulating histone acetylation levels in vivo.

Novel Tetrafunctional Probes Identify Target Receptors and Binding Sites of Small-Molecule Drugs from Living Systems.

Significant advancement of chemoproteomics has contributed to uncovering the mechanism of action (MoA) of small-molecule drugs by characterizing drug-protein interactions in living systems. However, cell-membrane proteins such as G protein-coupled receptors (GPCRs) and ion channels, due to their low abundance and unique biophysical properties associated with multiple transmembrane domains, can present challenges for proteome-wide mapping of drug-receptor interactions. Herein, we describe the development of novel tetrafunctional probes, consisting of (1) a ligand of interest, (2) 2-aryl-5-carboxytetrazole (ACT) as a photoreactive group, (3) a hydrazine-labile cleavable linker, and (4) biotin for enrichment. In live cell labeling studies, we demonstrated that the ACT-based probe showed superior reactivity and selectivity for labeling on-target GPCR by mass spectrometry analysis compared with control probes including diazirine-based probes. By leveraging ACT-based cleavable probes, we further identified a set of representative ionotropic receptors, targeted by CNS drugs, with remarkable selectivity and precise binding site information from mouse brain slices. We anticipate that the robust chemoproteomic platform using the ACT-based cleavable probe coupled with phenotypic screening should promote identification of pharmacologically relevant target receptors of drug candidates and ultimately development of first-in-class drugs with novel MoA.

B(C6F5)3-Catalyzed Hydrodesulfurization Using Hydrosilanes--Metal-Free Reduction of Sulfides.

B(C6F5)3-catalyzed hydrodesulfurization of carbon-sulfur bonds was achieved using triethylsilane as the reducing agent. The corresponding products were obtained in good yields under mild reaction conditions. This protocol could be applied to the reduction of sulfides, including benzyl and alkyl sulfides and dithianes, with high chemoselectivities.

Three-dimensional structure database of natural metabolites (3DMET): a novel database of curated 3D structures.

A database of 3D structures of natural metabolites has been developed called 3DMET. During the process of structure conversion from 2D to 3D, we found many structures were misconverted at chiral atoms and bonds. Several popular converters were tested in regard to their conversion accuracy. For verification, three canonical strings were also tested. No procedure could satisfactorily cover all the structures of the natural products. The misconverted structures had to be corrected manually. However, a nonnegligible number of mistakes were also observed even after manual curation, so a self-checking system was developed and introduced to our work flow. Thus, the 3D structures in our 3DMET database were evaluated in two steps: automatically and manually. The current version includes most of the natural products of the KEGG COMPOUND collection [ http://www.genome.jp/kegg/compound/ ] and is searchable by string, value range, and substructure. 3DMET can be accessed via http://www.3dmet.dna.affrc.go.jp/ , which also has detailed manuals.

Phosphorylation of cytohesin-1 by Fyn is required for initiation of myelination and the extent of myelination during development.

Schwann cells respond to cues from axons by transforming their cellular morphology and forming myelin. We demonstrated that the guanine nucleotide exchange factor (GEF) cytohesin-1 promoted myelination by activating the small guanosine triphosphatase (GTPase) Arf6. In mice, ablating cytohesin-1 delayed myelination and diminished the amount of myelin produced. We determined that the Src-family kinase Fyn phosphorylated tyrosine 382 (Y(382)) of cytohesin-1, and we generated transgenic mice that expressed a Schwann cell-specific phosphorylation mutant of cytohesin-1 (Y382F) that could not be targeted by Fyn. During development, these transgenic mice displayed delayed myelination compared to that of wild-type mice, as well as a decrease in the amount of myelin produced, similar to that observed in cytohesin-1⁻/⁻ mice. These findings demonstrate that phosphorylation of cytohesin-1 by Fyn is required for full myelination and suggest that tyrosine phosphorylation of GEFs may be a mechanism to activate small GTPases engaged in cell morphogenesis.

Structural analysis of human glutamine:fructose-6-phosphate amidotransferase, a key regulator in type 2 diabetes.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is a rate-limiting enzyme in the hexoamine biosynthetic pathway and plays an important role in type 2 diabetes. We now report the first structures of the isomerase domain of the human GFAT in the presence of cyclic glucose-6-phosphate and linear glucosamine-6-phosphate. The C-terminal tail including the active site displays a rigid conformation, similar to the corresponding Escherichia coli enzyme. The diversity of the CF helix near the active site suggests the helix is a major target for drug design. Our study provides insights into the development of therapeutic drugs for type 2 diabetes.

Hyperglycemia upregulates translation of the fibroblast growth factor 2 mRNA in mouse aorta via internal ribosome entry site.

Fibroblast growth factor 2 (FGF-2) is normally synthesized at low levels but is elevated in various pathophysiological conditions including diabetes-associated vascular diseases. FGF-2 expression is regulated translationally through an internal ribosome entry site (IRES) located in its mRNA, which allows a nonclassical cap-independent translation. We addressed the pathophysiological regulation of the IRES in vivo by using a streptozotocin-induced hyperglycemic model known to suppress markedly overall translation. Evaluation of FGF-2 IRES-dependent translation was performed with transgenic mice expressing dual luciferase bicistronic mRNA containing the FGF-2 IRES. FGF-2 IRES-dependent reporter activity increased 240% of control in the diabetic aorta although the reporter mRNA levels significantly decreased. Expression of endogenous FGF-2 protein in the aorta closely correlated with the IRES activity but not with FGF-2 mRNA levels. Moreover, the biosynthesis of endogenous FGF-2 protein was stimulated in an IRES-dependent manner by high glucose that significantly suppressed global protein synthesis in aortic smooth muscle cells from the transgenic mice. These results suggest that IRES-dependent translational regulation could play a pathological role in FGF-2 expression in vivo, especially in the cardiovascular consequences of diabetes.

Antidiuretic effects of a novel nonpeptide vasopressin V(2)-receptor agonist, OPC-51803, administered orally to dogs.

We elucidated the pharmacological properties of a novel nonpeptide vasopressin V(2)-receptor agonist, OPC-51803 ((5R)-2-[1-(2-chloro-4-(1-pyrrolidinyl)benzoyl-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]-N-isopropylacetamide), via both in vitro binding experiments incorporating canine kidney and platelet membrane fractions and in vivo experiments that would determine the compound's antidiuretic effects after oral administration to water-loaded dogs. OPC-51803 displaced [(3)H]arginine vasopressin (AVP) binding to canine V(2) and V(1a) receptors, as determined by resulting K(i) values of 15.2 +/- 0.6 nM (n = 4) and 653 +/- 146 nM (n = 4), respectively. These data indicate that OPC-51803 was about 43 times more selective for V(2) receptors than for V(1a) receptors. Antidiuretic studies showed that orally administered doses of OPC-51803 (0.03 to 0.3 mg x kg(-1)) decreased urine volume and increased urinary osmolality in a dose-dependent manner in water-loaded dogs. Intravenous OPC-51803 infusions (0.3 and 3 microg x kg(-1) x min(-1)) did not affect renal or systemic hemodynamics in anesthetized dogs. Since these results confirm that OPC-51803 shows antidiuretic action in dogs, the compound may be useful for treating AVP-deficient pathophysiological states.

Effects of OPC-51803, a novel, nonpeptide vasopressin V2-receptor agonist, on micturition frequency in Brattleboro and aged rats.

We assessed the effects of OPC-51803 ((5R)-2-[1-(2-chloro-4-(1-pyrrolidinyl)benzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]-N-isopropylacetamide), a nonpeptide vasopressin V(2)-receptor agonist, on micturition frequency in female homozygous Brattleboro rats (strain carries hereditary diabetes insipidus) and aged male Sprague-Dawley rats with polyuria. Female homozygous Brattleboro rats exhibited more diuresis and a larger micturition frequency over a 24-h period than did the heterozygous controls. In Brattleboro rats, an oral administration of OPC-51803 at 0.03 and 0.3 mg/kg significantly decreased urinary frequency and was accompanied by decreased urine volume. However, little effect was seen in the mean and maximal micturition volume. Aged male Sprague-Dawley rats (25-month-old) showed a significant increase in urine volume throughout a 0- to 24-h period compared with mature (6-month-old) rats. Orally administered OPC-51803 at 0.3 mg/kg decreased not only urine volume but also urinary frequency in aged rats. Furthermore, OPC-51803 prolonged the time prior to the first micturition. Therefore, OPC-51803 decreased micturition frequency in both rat species by reducing urine outflow. This suggests that the compound will be useful for treating micturition disorders that result in frequent micturition, such as that from polyuria, nocturnal polyuria, and some kinds of urinary incontinence.

Characterization of orally active nonpeptide vasopressin V(2) receptor agonist. Synthesis and biological evaluation of both the (5R)- and (5S)-enantioisomers of 2-[1-(2-Chloro-4-pyrrolidin-1-yl-benzoyl)-2,3,4,5-tetrahydro-1H-1-benzazepin- 5-yl]-N-isopropylacetamide.

The synthesis and evaluation of both the (R)- and (S)-enantioisomers about the 5-position on a tetrahydro-1H-1-benzazepine derivative were described. The absolute configuration of the (R,R)-isomer (10) was determined by X-ray crystallographic analysis. After evaluation of both enantiomers (compounds R-2, S-2) for binding affinity, cAMP accumulation, and an in vivo study using Brattleboro rats, R-2 showed more potent activity as a V(2) receptor agonist than S-2.