Noriaki Hirayama

ASEDock-Docking Based on Alpha Spheres and Excluded Volumes

ASEDock is a novel docking program based on a shape similarity assessment between a concave portion (i.e., concavity) on a protein and the ligand. We have introduced two novel concepts into ASEDock. One is an ASE model, which is defined by the combination of alpha spheres generated at a concavity in a protein and the excluded volumes around the concavity. The other is an ASE score, which evaluates the shape similarity between the ligand and the ASE model. The ASE score selects and refines the initial pose by maximizing the overlap between the alpha spheres and the ligand, and minimizing the overlap between the excluded volume and the ligand. Because the ASE score makes good use of the Gaussian-type function for evaluating and optimizing the overlap between the ligand and the site model, it can pose a ligand onto the docking site relatively faster and more effectively than using potential energy functions. The posing stage through the use of the ASE score is followed by full atomistic energy minimization. Because the posing algorithm of ASEDock is free from any bias except for shape, it is a very robust docking method. A validation study using 59 high-quality X-ray structures of the complexes between drug-like molecules and the target proteins has demonstrated that ASEDock can faithfully reproduce experimentally determined docking modes of various druglike molecules in their target proteins. Almost 80% of the structures were reconstructed within the estimated experimental error. The success rate of approximately 98% was attained based on the docking criterion of the root-mean-square deviation (RMSD) of non-hydrogen atoms (< or = 2.0 A). The markedly high success of ASEDock in redocking experiments clearly indicates that the most important factor governing the docking process is shape complementarity.

A Novel Class of Prolyl Hydroxylase Inhibitors Induces Angiogenesis and Exerts Organ Protection Against Ischemia

Objective—Hypoxia inducible factor (HIF) plays a pivotal role in the adaptation to ischemic conditions. Its activity is modulated by an oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHD). Methods and Results—We discovered 2 unique compounds (TM6008 and TM6089), which inhibited PHD and stabilized HIF activity in vitro. Our docking simulation studies based on the 3-dimensional structure of human PHD2 disclosed that they preferentially bind to the active site of PHD. Whereas PHD inhibitors previously reported inhibit PHD activity via iron chelation, TM6089 does not share an iron chelating motif and is devoid of iron chelating activity. In vitro Matrigel assays and in vivo sponge assays demonstrated enhancement of angiogenesis by local administration of TM6008 and TM6089. Their oral administration stimulated HIF activity in various organs of transgenic rats expressing a hypoxia-responsive reporter vector. No acute toxicity was observed up to 2 weeks after a single oral dose of 2000 mg/kg for TM6008. Oral administration of TM6008 protected neurons in a model of cerebrovascular disease. The protection was associated with amelioration of apoptosis but independent of enhanced angiogenesis. Conclusions—The present study uncovered beneficial effects of novel PHD inhibitors preferentially binding to the active site of PHD.

Inhibition of Plasminogen Activator Inhibitor-1: Its Mechanism and Effectiveness on Coagulation and Fibrosis

Objective—Serine protease inhibitors (serpin) play a central role in various pathological processes including coagulation, fibrinolysis, malignancy, and inflammation. Inhibition of serpins may prove therapeutic. As yet, however, only very few small molecule serpin inhibitors have been reported. For the first time, we apply a new approach of virtual screening to discover novel, orally active, small molecule serpin inhibitors and report their effectiveness. Methods and Results—We focused on a clinically important serpin, plasminogen activator inhibitor-1 (PAI-1), whose crystal structure has been described. We identify novel, orally active molecules able to enter into the strand 4 position (s4A) of the A β-sheet of PAI-I as a mock compound. In vitro they specifically inhibit the PAI-1 activity and enhance fibrinolysis activity. In vivo the most effective molecule (TM5007) inhibits coagulation in 2 models: a rat arteriovenous (AV) shunt model and a mouse model of ferric chloride–induced testicular artery thrombosis. It also prevents the fibrotic process initiated by bleomycin in mouse lung. Conclusions—The present study demonstrates beneficial in vitro and in vivo effects of novel PAI-1 inhibitors. Our methodology proves to be a useful tool to obtain effective inhibitors of serpin activity.

A novel inhibitor of plasminogen activator inhibitor-1 provides antithrombotic benefits devoid of bleeding effect in nonhuman primates.

Inhibition of plasminogen activator inhibitor (PAI)-1 is useful to treat several disorders including thrombosis. An inhibitor of PAI-1 (TM5275) was newly identified by an extensive study of structure-activity relationship based on a lead compound (TM5007) which was obtained through virtual screening by docking simulations. Its antithrombotic efficacy and adverse effects were tested in vivo in rats and nonhuman primates (cynomolgus monkey). TM5275, administered orally in rats (1 to 10 mg/kg), has an antithrombotic effect equivalent to that of ticlopidine (500 mg/kg) in an arterialvenous shunt thrombosis model and to that of clopidogrel (3 mg/kg) in a ferric chloride-treated carotid artery thrombosis model. TM5275 does not modify activated partial thromboplastin time and prothrombin time or platelet activity and does not prolong bleeding time. Combined with tissue plasminogen activator, TM5275 improves the latters therapeutic efficacy and reduces its adverse effect. Administered to a monkey model of photochemical induced arterial thrombosis, TM5275 (10 mg/kg) has the same antithrombotic effect as clopidogrel (10 mg/kg), without enhanced bleeding. This study documents the antithrombotic benefits of a novel, more powerful, PAI-1 inhibitor in rats and, for the first time, in nonhuman primates. These effects are obtained without adverse effect on bleeding time.

Dissection and optimization of immune effector functions of humanized anti-ganglioside GM2 monoclonal antibody

A mouse/human chimeric monoclonal antibody (MAb) KM966, specific for the cell-surface tumor antigen ganglioside GM2, was humanized by the complementarity determining regions (CDRs) grafting method. Not only the amino acid residues in the CDRs but also several in the framework regions (FRs) were changed from the human to the murine residues. A humanized variant, huKM796H/Lm-28, containing eight and five amino acid alterations in variable light (VL) and variable heavy (VH) FRs, respectively, showed a 9-fold reduction in complement-dependent cytotoxicity (CDC) compared to the chimeric KM966, despite tight antigen binding and potent antibody-dependent cellular cytotoxicity (ADCC). Several additional variants were subsequently constructed to improve the CDC of the antibody. One of the variants, designated KM8969, which differs by three amino acids, exhibited a CDC within 3-fold of the chimeric KM966. In addition, humanized KM8969 bound GM2 antigen 1.25-fold more tightly than the chimeric KM966 and showed 5-fold higher ADCC than the chimeric KM966. These results clearly show that the humanized KM8969, having the optimized immune effector functions and theoretically minimal immunogenicity, is an ideal candidate to test the effectiveness of anti-GM2 MAb in human cancer therapy. Taken together, the results obtained here indicate that the ADCC and CDC of an antibody can be dissected independently via engineering of the antibody variable region.

Synthesis and cytotoxicity of the acyclic (E)- and (Z)-dienediyne systems related to neocarzinostatine chromophore

Abstract Synthesis of the title compounds could be achieved by featuring the Pd-catalyzed coupling reaction of stereo-defined (E)-and (Z)-enol triflates with an acetylene derivative. It was found that the acyclic (Z)-dienediyne system obviously exhibits in vitro cytotoxicity against P388 murine leukemia stronger than that for the corresponding (E)-isomer.

Chiral Space Formed by (+)‐(1S)‐1,1′‐Binaphthalene‐2,2′‐diyl Phosphate: Recognition of Aliphatic L‐α‐Amino Acids

(+)-(1S)-1,1′-Binaphthalene-2,2′-diyl hydrogen phosphate (bnppa) is one of the useful optical selectors. To disclose the molecular mechanism by which bnppa recognizes aliphatic L-α-amino acids and separates them by fractional crystallization, X-ray analyses of bnppa and of its salts with L-alanine, L-valine, L-norvaline, and L-norleucine have been undertaken. All the amino acids adopt energetically favorable conformations in the crystal structures. The conformations and the packing patterns of bnppa in these crystal structures are very similar. The bnppa molecules are packed in a specific way to form hydrophobic and hydrophilic layers that are well separated. Between bnppa molecules, at the interface of these hydrophobic and hydrophilic layers, a space with chirality is formed. This space, designated as chiral space, recognizes the optically active amino acids. The packing of bnppa is mainly governed by intermolecular CH⋅⋅⋅π interactions between naphthalene moieties. The chiral space is responsible for the molecular recognition by bnppa allowing fractional crystallization of the L-α-amino acids.

A novel small molecule, N-(4-(2-pyridyl)(1,3-thiazol-2-yl))-2-(2,4,6-trimethylphenoxy) acetamide, selectively protects against oxidative stress-induced cell death by activating the Nrf2–ARE pathway: Therapeutic implications for ALS

Antioxidant defense is crucial in restoring cellular redox homeostasis. Recent findings have suggested that oxidative stress plays pivotal roles in the pathogenesis of many neurodegenerative diseases. Thus, an anti-oxidative stress remedy might be a promising means for the treatment of such disorders. In this study, we employed a novel ligand-based virtual screening system and identified a novel small molecule, N-(4-(2-pyridyl)(1,3-thiazol-2-yl))-2-(2,4,6-trimethylphenoxy) acetamide (CPN-9), which selectively suppressed oxidative stress-induced cell death in a cell-type-independent manner. CPN-9 upregulates NF-E2-related factor 2 (Nrf2), a key transcriptional regulator of the expression of phase II detoxification enzymes and antioxidant proteins, and Nrf2-regulated factors such as heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase modifier subunit (GCLM). The CPN-9-mediated upregulation of HO-1, NQO1, and GCLM was abolished by Nrf2 knockdown. Moreover, the antioxidant N-acetylcysteine reduced the protective effect of CPN-9 against oxidative stress-induced cell death with concomitant diminishing of Nrf2 nuclear translocation. These results indicate that CPN-9 exerts its activity via the reactive oxygen species-dependent activation of the Nrf2 signaling pathway in cultured cells. It is noteworthy that the postonset systemic administration of CPN-9 to a transgenic ALS mouse model carrying the H46R mutation in the human Cu/Zn superoxide dismutase (SOD1) gene sustained motor functions and delayed disease progression after onset. Collectively, CPN-9 is a novel Nrf2 activator and a neuroprotective candidate for the treatment of neurodegenerative diseases, including ALS.

The structure of tetronolide, the aglycone of antitumor antibiotic tetrocarcin

Abstract X-ray analysis has determined the structure of tetronolide, which has a unique spiro γ-lactone group.

Use of amino acid composition to predict epitope residues of individual antibodies

We identified specific amino acid propensities at the interfaces of antigen-antibody interactions in non-redundant qualified antigen-antibody complex structures from Protein Data Bank. Propensities were expressed by the frequency of each of the 20 x 20 standard amino acid pairs that appeared at the interfaces of the complexes and were named the antibody-specific epitope propensity (ASEP) index. Using this index, we developed a novel method of predicting epitope residues for individual antibodies by narrowing down candidate epitope residues which was predicted by the conventional method. The 74 benchmarked antigens were used in ASEP prediction. The efficiency of this method was assessed using the leave-one-out approach. On elimination of residues with ASEP indices in the lowest 10% of all measured, true positives were enriched for 49 antigens. On subsequent elimination of residues with ASEP indices in the lowest 50%, true positives were enriched for 40 of the 74 antigens assessed. The ASEP index is the first benchmark proposed to predict epitope residues for an individual antibody. Used in combination with mutation experiments, this index has the potential to markedly increase the success ratio of epitope analysis.