Protein grafting of p53TAD onto a leucine zipper scaffold generates a potent HDM dual inhibitor.

Reactivation of the p53 pathway by a potential therapeutic antagonist, which inhibits HDM2 and HDMX, is an attractive strategy for drug development in oncology. Developing blockers towards conserved hydrophobic pockets of both HDMs has mainly focused on small synthetic compounds; however, this approach has proved challenging. Here we describe an approach to generate a potent HDM dual inhibitor, p53LZ2, by rational protein grafting of the p53 transactivation domain onto a homodimeric leucine zipper. p53LZ2 shows tight binding affinity to both HDMs compared with wild-type p53 in vitro. X-ray crystallographic, comparative modelling and small-angle X-ray scattering studies of p53LZ2-HDM complexes show butterfly-shaped structures. A cell-permeable TAT-p53LZ2 effectively inhibits the cancer cell growth in wild-type but not mutant p53 by arresting cell cycle and inducing apoptosis in vitro. Thus, p53LZ2, designed by rational grafting, shows a potential therapeutic approach against cancer.

Inhibitory effects of luteolin on titanium particle-induced osteolysis in a mouse model.

Wear particles liberated from the surfaces of an implanted prosthesis are associated with peri-implant osteolysis and subsequent aseptic loosening. In the latter wear particle-induced inflammation and osteoclastogenesis have been identified as critical factors, and their inhibition as important steps in the treatment of affected patients, such as those undergoing total hip replacement. In this study the ability of luteolin to inhibit both titanium (Ti) particle-induced osteoclastogenesis in vitro and osteolysis in a murine calvaria Ti particle-induced model of osteolysis was examined. The results showed that luteolin, a highly potent and efficient inhibitor of tumor necrosis factor α (TNF-α) and interleukin-6 expression, inhibited Ti particle-induced inflammatory cytokine release, osteoclastogenesis, and bone resorption in bone marrow macrophages. Microcomputed tomography and histological analyses showed that the Ti particles caused significant bone resorption and increased TRAP(+) multinuclear osteoclasts in the murine calvarial model of osteolysis, whereas this was not the case in the luteolin treatment group, in which osteolytic suppression was accompanied by a decrease in both TNF-α production and serum levels of the osteoclast marker the C-terminal telopeptide fragment of type I collagen. These results support the use of luteolin as a natural compound in the prevention and treatment of aseptic loosening after total replacement arthroplasty.

Structure-activity relationship of the 7-hydroxy benzimidazole analogs as glycogen synthase kinase 3ß inhibitor.

Design, synthesis and the GSK3ß inhibitory activities of the 7-hydroxy benzimidazole analogs are described. The solid-phase synthetic route was also developed for preparation of the analogs consisting of the novel ATP competitive scaffold. In addition, the structure-activity relationship of the 7-hydroxy benzimidazole analogs and their biological activities are reported.

A newly identified CG301269 improves lipid and glucose metabolism without body weight gain through activation of peroxisome proliferator-activated receptor alpha and gamma.

OBJECTIVE: Peroxisome proliferator-activated receptor (PPAR)-α/γ dual agonists have been developed to alleviate metabolic disorders. However, several PPARα/γ dual agonists are accompanied with unwanted side effects, including body weight gain, edema, and tissue failure. This study investigated the effects of a novel PPARα/γ dual agonist, CG301269, on metabolic disorders both in vitro and in vivo. RESEARCH DESIGN AND METHODS: Function of CG301269 as a PPARα/γ dual agonist was assessed in vitro by luciferase reporter assay, mammalian one-hybrid assay, and analyses of PPAR target genes. In vitro profiles on fatty acid oxidation and inflammatory responses were acquired by fatty acid oxidation assay and quantitative (q)RT-PCR of proinflammatory genes. In vivo effect of CG301269 was examined in db/db mice. Total body weight and various tissue weights were measured, and hepatic lipid profiles were analyzed. Systemic glucose and insulin tolerance were measured, and the in vivo effect of CG301269 on metabolic genes and proinflammatory genes was examined by qRT-PCR. RESULTS: CG301269 selectively stimulated the transcriptional activities of PPARα and PPARγ. CG301269 enhanced fatty acid oxidation in vitro and ameliorated insulin resistance and hyperlipidemia in vivo. In db/db mice, CG301269 reduced inflammatory responses and fatty liver, without body weight gain. CONCLUSIONS: We demonstrate that CG301269 exhibits beneficial effects on glucose and lipid metabolism by simultaneous activation of both PPARα and PPARγ. Our data suggest that CG301269 would be a potential lead compound against obesity and related metabolic disorders.

Structural basis of triclosan resistance.

Triclosan (5-chloro-2-(2,4-dichloro-phenoxy)-phenol, TCL) is a well known inhibitor against enoyl-acyl carrier protein reductase (ENR), an enzyme critical for cell-wall synthesis of bacteria. The inhibitory concentration at 50% inhibition (IC(50)) of TCL against the Escherichia coli ENR is 150nM for wild type (WT), 380, 470 and 68,500nM for Ala, Ser and Val mutants, respectively. To understand this high TCL resistance in the G93V mutant, we obtained the crystal structures of mutated ENRs complexed with TCL and NAD(+). The X-ray structural analysis along with the ab initio calculations and molecular dynamics simulations explains the serious consequence in the G93V mutant complex. The major interactions around TCL due to the aromatic(cation)-aromatic and hydrogen bonding interactions are found to be conserved both in WT and mutant complexes. Thus, the overall structural change of protein is minimal except that a flexible α-helical turn around TCL is slightly pushed away due to the presence of the bulky valine group. However, TCL shows substantial edge-to-face aromatic (π)-interactions with both the flexible R192-F203 region and the residues in the close vicinity of G93. The weakening of some edge-to-face aromatic interactions around TCL in the G93V mutant results in serious resistance to TCL. This understanding is beneficial to design new generation of antibiotics which will effectively act on the mutant ENRs.

A nonthiazolidinedione peroxisome proliferator-activated receptor α/γ dual agonist CG301360 alleviates insulin resistance and lipid dysregulation in db/db mice.

Activation of peroxisome proliferator-activated receptors (PPARs) have been implicated in the treatment of metabolic disorders with different mechanisms; PPARα agonists promote fatty acid oxidation and reduce hyperlipidemia, whereas PPARγ agonists regulate lipid redistribution from visceral fat to subcutaneous fat and enhance insulin sensitivity. To achieve combined benefits from activated PPARs on lipid metabolism and insulin sensitivity, a number of PPARα/γ dual agonists have been developed. However, several adverse effects such as weight gain and organ failure of PPARα/γ dual agonists have been reported. By use of virtual ligand screening, we identified and characterized a novel PPARα/γ dual agonist, (R)-1-(4-(2-(5-methyl-2-p-tolyloxazol-4-yl)ethoxy)benzyl)piperidine-2-carboxylic acid (CG301360), exhibiting the improvement in insulin sensitivity and lipid metabolism. CG301360 selectively stimulated transcriptional activities of PPARα and PPARγ and induced expression of their target genes in a PPARα- and PPARγ-dependent manner. In cultured cells, CG301360 enhanced fatty acid oxidation and glucose uptake and it reduced pro-inflammatory gene expression. In db/db mice, CG301360 also restored insulin sensitivity and lipid homeostasis. Collectively, these data suggest that CG301360 would be a novel PPARα/γ agonist, which might be a potential lead compound to develop against insulin resistance and hyperlipidemia.

Molecular mechanism for the regulation of human ACC2 through phosphorylation by AMPK.

Acetyl-CoA carboxylases (ACCs) have been highlighted as therapeutic targets for obesity and diabetes, as they play crucial roles in fatty acid metabolism. ACC activity is regulated through the short-term mechanism of inactivation by reversible phosphorylation. Here, we report the crystal structures of the biotin carboxylase (BC) domain of human ACC2 phosphorylated by AMP-activated protein kinase (AMPK). The phosphorylated Ser222 binds to the putative dimer interface of BC, disrupting polymerization and providing the molecular mechanism of inactivation by AMPK. We also determined the structure of the human BC domain in complex with soraphen A, a macrocyclic polyketide natural product. This structure shows that the compound binds to the binding site of phosphorylated Ser222, implying that its inhibition mechanism is the same as that of phosphorylation by AMPK.

Structure-based virtual screening approach to the discovery of novel inhibitors of factor-inhibiting HIF-1: identification of new chelating groups for the active-site ferrous ion.

The inhibitors of factor-inhibiting HIF-1 (FIH1) have been shown to be useful as therapeutics for the treatment of anemia. We have been able to identify eight novel FIH1 inhibitors with IC(50) values ranging from 30 to 80microM by means of the virtual screening with docking simulations under consideration of the effects of ligand solvation in the scoring function. The newly identified inhibitors are structurally diverse and have various chelating groups for the active-site ferrous ion including sulfonamide, carboxylate, N-benzo[1,2,5]oxadiazol-4-yl amide, and 2-[1,2,4]triazolo[3,4-b]][1,3,4]thiadiazol-3-yl-quinoline moieties. Each of these four structural classes has not been reported as FIH1 inhibitor, and therefore can be considered for further development by structure-activity relationship or denovo design methods. The interactions with the amino acid residues responsible for the stabilizations of the inhibitors in the active site are addressed in detail.

Electrical conductivity-defect structure correlation of variable-valence and fixed-valence acceptor-doped BaTiO(3) in quenched state.

Insulation resistance degradation of dielectric BaTiO(3) is expected to be closely correlated to its defect structure frozen in from elevated processing temperatures. For BaTiO(3), respectively doped with variable-valence (Mn(Ti)) and fixed-valence acceptors (Al(Ti)), their defect structures were frozen in by quenching at different equilibrium oxygen activities in the range of -18 < log a(O(2))< or = 0 at 1000 and 900 degrees C, respectively, and their electrical conductivities were measured against temperature in the range of 200 < or =T/K < or = 494 by impedance spectroscopy. Frozen-in defect structures were calculated and compared with the conductivity as measured in the quenched state. A close correlation has been confirmed between the bulk conductivity as measured in the quenched state and the frozen-in defect structure as calculated. The effects of variable- and fixed-valence acceptor impurities on the defect structure and electrical conductivity in the quenched state are highlighted in the light of hole trapping, and the charge transport behavior in the quenched state is discussed.

Synthesis and structural analysis of 6-aminobicyclo[2.2.1]heptane-2-carboxylic acid as a conformationally constrained gamma-turn mimic.

An efficient asymmetric synthesis of 6-aminobicyclo[2.2.1]heptane-2-carboxylic acid as a novel gamma-turn mimic has been achieved. Structural analysis of the gamma-amino acid derivative was carried out using (1)H NMR spectroscopy and intramolecular hydrogen bonding between side chain amides confirmed the turn structure, which had been predicted by Ab initio computational study.

Design and synthesis of 7-hydroxy-1H-benzoimidazole derivatives as novel inhibitors of glycogen synthase kinase-3beta.

A hydroxy functional group was introduced as the hydrogen bond donor and acceptor at the hinge region of protein kinase in order to develop novel ATP-competitive inhibitors. Several derivatives of 7-hydroxyl-1H-benzoimidazole were designed as inhibitors of glycogen synthase kinase-3beta with the help of ab initio calculations and a docking study. Enzymatic assay and an X-ray complex study showed that these designed compounds were highly potent ATP-competitive inhibitors.

A predictive method for subsequent avascular necrosis of the femoral head (AVNFH) by observation of bleeding from the cannulated screw used for fixation of intracapsular femoral neck fractures.

OBJECTIVES: To examine the validity of bleeding from the drill holes used for cannulated screw placement as a method for predicting any subsequent avascular necrosis of the femoral head (AVNFH) after the fixation of intracapsular femoral neck fractures. DESIGN: Retrospective study. SETTING: University hospital. PARTICIPANTS: Forty-four patients (mean age, 51 years; range, 18-76 years) whose femoral neck fractures had been fixed with cannulated screws from March 1999 to January 2001 were enrolled in this study. The fractures were classified according to Garden and included 11 type I, 5 type II, 17 type III, and 11 type IV. The average delay between injury and surgery was 52 hours (< or =24 hours, 26; > or =24 hours, 18; range 7 to 504 hours). The follow-up period was more than 25 months (range, 25-57 months). INTERVENTION: 7.0 mm cannulated screws were used for fracture fixation. Three and 4 screws were used for fixation in 35 and 9 cases, respectively. MAIN OUTCOME MEASUREMENTS: The presence or absence of blood drainage from the holes of the proximal cannulated screws was determined by an independent observer and defined as bleeding or no bleeding throughout a 5 minute observation period. According to those findings, patients were classified into 2 groups: the bleeding group (38 cases), and the nonbleeding group (6 cases). The validity of the relationship between the 2 groups and the development of AVNFH was evaluated according to the sensitivity, specificity, positive predictive value, and negative predictive value. A chi test was used for univariate analysis of the relationship between the related factors with the development of AVNFH. RESULTS: The mean follow-up was 39 months (range, 25-57 months). AVNFH developed in 7 cases (16%). One patient of 38 in the bleeding group (2.6%) and all 6 patients in the nonbleeding group (100%) developed AVNFH. The sensitivity was 86%, specificity 100%, positive predictive value 100%, and negative predictive value 97%. Age (P < 0.734), sex (P < 0.587), the type of the fracture (P < 0.356), procedure interval (P < 0.398), the reduction status of the fracture site (P < 0.3849), the positions of the fixed screws (P < 0.2137), and the existence of osteoporosis (P < 0.4347) were not related to the development of AVNFH. CONCLUSION: It seems that bleeding from the holes of proximal cannulated screws is a simple and accurate perfusion assessment technique for predicting the development of AVNFH after a femoral neck fracture. Given that assumption, primary arthroplasty might be an appropriate choice as a treatment method in a nonbleeding-group patient whose treatment choice is ambivalent or who might not be able to undergo additional surgery should he or she develop a subsequent AVNFH after internal fixation of femoral neck fracture.

Structural chemoproteomics and drug discovery.

Our laboratories have developed several technologies to accelerate drug discovery process on the basis of structural chemoproteomics. They include SPS technology for the efficient determination of protein structures, SCP technology for the rapid lead generation and SDF technology for the productive lead optimization. Using these technologies, we could determine many 3D structures of target proteins bound with biologically active chemicals including the structure of phosphodiesterase 5/Viagra complex and obtain highly potent compounds in animal models of obesity, diabetes, cancer and inflammation. In this paper, we will discuss concepts and applications of structural chemoproteomics for drug discovery.

Molecular basis for the local conformational rearrangement of human phosphoserine phosphatase.

Human phosphoserine phosphatase (HPSP) regulates the levels of glycine and d-serine, the putative co-agonists for the glycine site of the NMDA receptor in the brain. Here, we describe the first crystal structures of the HPSP in complexes with the competitive inhibitor 2-amino-3-phosphonopropionic acid (AP3) at 2.5 A, and the phosphate ion (Pi) and the product uncompetitive inhibitor l-serine (HPSP.l-Ser.Pi) at 2.8 A. The complex structures reveal that the open-closed environmental change of the active site, generated by local rearrangement of the alpha-helical bundle domain, is important to substrate recognition and hydrolysis. The maximal extent of this structural rearrangement is shown to be about 13 A at the L4 loop and about 25 degrees at the helix alpha3. Both the structural change and mutagenesis data suggest that Arg-65 and Glu-29 play an important role in the binding of the substrate. Interestingly, the AP3 binding mode turns out to be significantly different from that of the natural substrate, phospho-l-serine, and the HPSP.l-Ser.Pi structure provides a structural basis for the feedback control mechanism of serine. These analyses allow us to provide a clear model for the mechanism of HPSP and a framework for structure-based drug development.