Our Research

Outline of our target

• Control of the electronic structure of the iron atom in hemoprotein
  The iron in hemoglobin represents almost 70% of the total content in our body. The iron in hemoglobin is coupled with porphyrin, and functional integrity of hemoglobin comes from the symmetric molecular structure of porphyrin. It is expected that the electronic structure of heme iron could be regulated after rearrangement of the porphyrin structure. We have prepared new type of porphyrin isomers, porphycene and corrphycene. We found that the iron in these macrocycles exhibit anomalous reactivity toward exogenous ligand. 1-Methylimidazole coordinated to iron corrphycene in two steps whereas regular porphyrin exhibits a single-step binding of the ligand. The spin state of a 6-cooridnate azide complex of iron corrphycene is also anomalous. The spin-state of the azide complex is predominantly high-spin, in contrast to the low-spin state of the corresponding iron porphyrin. These observations demonstrate that rearrangement of the molecular shape of the porphyrin is a promising approach to control the reactivity of the iron atom in porphyrin.

• Molecular design of artificial oxygen carrier
  Myoglobin is oxygen carrying protein containing iron porphyrin as the prosthetic group. We prepared an artificial myoglobin reconstituted with synthetic porphyrin isomers such as corrphycene and hemiporphycene. Although the myoglobin with iron corrphycene exhibits an extremely low oxygen affinity, the oxygen carrying ability under physiological condition was enhanced by 5-fold. On the other hand, the oxygen affinity in the myoglobin bearing iron hemiporphycene is much higher than that of the native protein. These results combined together establish that the shape change of porphyrin macrocycle provides a useful guiding principle to design artificial blood substitute.

• Enzymatic reaction mechanism of HIV-1 protease
  HIV-1 protease is the most important enzyme for proliferation of HIV-1, a source virus of AIDS disease. Protease is therefore one of the target of anti-HIV-1 treatments. One of the characteristic features of the protease is its high polymorphism and adaptability. Nowadays, the appearance of drug-resistant virus causes a serious problem in clinical scene, and the genotypic and phenotypic assay prior to the medication by protease inhibitors becomes an important process in HIV therapy. We are trying to adapt the theoretical calculation for the evaluation of drug efficacy of several kinds of inhibitors for the mutated HIV virus, and are developing the computational technique to select the most preferable medicine for the individual patient.

• Monooxygenation mechanism by cytochrome P450
  CYP3A4 is a kind of cytochrome P450s and catalyzes 10,11-epoxidation to carbamazepine (CBZ), anti-convulsant drug. This is only one CBZ metabolic route by CYP3A4. However, potential energy of CBZ 10,11-epoxide is about 20 kcal/mol higher than that of hydroxy CBZ. To elucidate the reason why CYP3A4 produces epoxide only in CBZ metabolism, a mechanism of CBZ epoxidation by CYP3A4 was investigated by using density functional theory. It was found that the active site of CYP3A4 had geometry to catalyze epoxidation to CBZ and that CBZ 10,11-epoxidation was two-steps reaction. If hydroxylation mechanism of CBZ by CYP3A4 occurs, the activation energy of the first step reaction is higher than that of the rate-determining step of epoxidation of CBZ by CYP3A4. It is considered that the result explains the reason why 10,11-epoxidation of CBZ is only one CBZ metabolic route by CYP3A4.

Present Projects

1. Synthesis of non-planner corrphycene and analysis of the spin-state
   

   Heme-proteins show various spin-states, which significantly characterize their enzymatic activity. For instance, cytochrome c' and HRP exhibit an intermediate (S=3/2) and an admixed spin-state (S=5/2, 3/2), respectively. Because the S=3/2 character is essential for the enzymes, we are constructing the corrphycene having phenyl substituents to artificially control the spin state of the metallocore.

2. Synthesis and chemical modification of corrphycene
   

   Corrphycene is a kind of porphyrin isomer. No successful application of water-soluble iron corrphycene to hemoproteins has been reported. We are synthesizing a corrphycene with free acid chains to reconstitute myoglobin in aqueous medium. The functional analysis of the reconstituted myoglobin will be reported.

3. Functional analysis of mono-phenylporphyrins with carboxy or nitro groups
   

   We modify a part of protoporphyrin IX and synthesize mono-phenylporphyrin with carboxy groups or nitro groups. The porpose of this study is to investigate the bioinorganic characters of the synthesized porphyrins.

4. Synthesis of porphyrin isomer with sulfur atom and analysis of its physicochemical characters
   

   It have been reported that novel porphyrin derivatives or isomers are synthesized, and novel functions and characters of reconstituted heme-proteins with the porphyrin derivatives are examined. Heme is a porphyrin or porphyrin isomer in complex with Fe. In reconstituted heme-protein, native heme is replaced by modified heme. In our research, we synthesize a porphyrin isomer which has one sulfur atom and three nitrogen atoms in its center. We aim to clarify the characteristic of metal complex of the porphyrin isomer and the function of the alternate molecule.

5. Synthesis of porphyrin labeled by 13C for analysis of heme protein
   

   when we analyze a heme-protein by 1H-NMR,it is hard to determine a character of heme-protein. Thus, we aim to be able to analyze a heme-protein, using a porphrin by 13C. First, we synthesize 13C-labeled artificial porphyrin.

6. Clarifications of drug-resistant mechanisms against HIV-1 protease inhibitors
   

   HIV-1 has higher mutation rate than other viruses. Therefore, the drug-resistance against anti-HIV-1 drugs is caused more frequently. This is a serious problem of AIDS therapy. Although the amino acids related to the drug-resistance are identified, the atomistic-level resistant mechanisms are not clarified. We execute molecular dynamics simulations of the complexes of mutant proteases and inhibitors, and attempt to identify the resistant mechanism. We expect that further investigation helps not only the development of new potent HIV-1 protease inhibitors but also selection of the drugs in AIDS therapy.

7. Development of software program predictiong a binding mode of a ligand against the target protein
   

   The significance of Structure-Based Drug Design (SBDD) is increasing in the development of new drugs. However, in many cases when drugs are developed using SBDD, it is not clear what kind of ligands is bound to a target protein and where the binding site of ligands is. Therefore, the establishment of a computer program which correctly determines the binding site and the binding mode of ligands is essential for the success of SBDD. In this study, we will develop software program predicting the binding site, the binding mode, and the binding affinity of ligands against a target protein.

8. Evaluation of the cytochrome P450 affinity to ligands
   

   Cytochrome P450 is a heme-containing enzyme that is responsible for the metabolism of a lot of known drugs. Large number of genetic polymorphisms have been described for CYP2C9 and 2D6 etc. Some of which can either result in rapid or very poor metabolism. The goal of this research is to predict metabolic ability of the enzyme protein in silico by using the method such as docking simulation or computer analysis, and to measure binding energy in vitro by the biochemical approach.

9. Theoretical investigation on the binding affinity between HLA class I and the epitopes from a variety of viruses
   

   When a cell is infected with virus, 8-11mer peptides (epitopes) of the virus are produced from processing by proteasome, etc. Human leukocyte Antigen (HLA) class I molecule binds to those epitopes and present them at cell surface. When the presented epitope is recognized by Cytotoxic T lymphocytes (CTLs) , the infected cell is removed. The purpose of our investigation is to assess the affinity between HLA molecule and the epitopes both in vitro and in silico.

10. Biochemical and structural analysis of HIV-1 subtypeC protease
    

    HIV-1 subtype C is a majority of HIV-1 strains which are prevarent in the world, and mainly emerges in developing countries. However, no detail research have been carried out about HIV-1 subtype C, compared with subtype B variants which mainly emerge in developed countries. Hence, we plan to express clinically-derived subtype C protease and to examine thermodynamic affinity with the commercial protease inhibitors by isothermal titration calorimetry. Furthermore, we aim to execute molecular dynamics simulations to clarify drug-resistant mechanism in subtype C variants. We wish to use the results as the basic information to design novel drugs.

11. Theoretical modeling and analysis of lipid microdomains: molecular dynamics simulations of lipid bilayers including cholesterols and glycolipids
    

    Biological membranes consist of various types of lipids, and the differences of the lipid composition correspond to providing the optimal surrounding condition for specific enzymatic function. Recently, it has been revealed that cholesterols, sphingolipids, and glycolipids form more ordered, less fluid bilayers, termed microdomain, surrounded by other more fluid phospholipids. But how this heterogeneity is regulated and its functional implications remain unclear. To theoretically reveal the physical features of lipid microdomains, we have been developing new programs for modeling lipid microdomains. Utilizing these programs, we created several microdomain models, and have been performing molecular dynamics simulations of these microdomains.

12. Development of novel anti-AIDS drugs targeting HIV-1 RNase H
    

    For patients of AIDS, drug therapy called HAART is adapted. However, not a few numbers of cases using existing anti-drugs causes drug-resistant viruses. What we need is a novel anti-AIDS drug that acts at a different site of HIV-1. So, we focused on RNase H domain at reverse transcriptase. First, we design new compounds which hopefully inhibit the RNase H activity and perform some docking simulations. Then validate the results and carry out an organic synthesis of best scored compounds.

13. Synthesis of novel HIV-1 protease inhibitor
    

    Some viral proteins, such as reverse transcriptase, protease, integrase, play important role on proliferation of HIV. Hence, compounds which inhibit functions of these proteins, becomes an anti-HIV drug. An example of anti-HIV drug is inhibitors of HIV-1 protease. Darunavir, which is one of the protease inhibitor, has been recently developed. Darunavir is modified from Amprenavir, which is also the protease inhibitor, and forms stabler complex than Amprenavir. Darunavir shows high effects toward not only untreated patients but also patients in which protease confers drug resistance against some other protease inhibitors. In this study, on the basis of the previous computational study, we synthesize novel protease inhibitors modified from Lopinavir.

14. Ab initio prediction of tertiary structures of proteins
    

    A tertiary structure of a protein is one of the most important factors to design a drug. However, there are many proteins whose tertiary structure is unknown, although the amino acid sequences of them have been identified. Thus, the development of method to predict tertiary structures of target proteins on the basis of only amino acid sequences, has been noticed.One of the prediction method is ab initio method, which do not use empirical rules. In this research, we perform ab initio prediction of tertiary structures of proteins with molecular dynamics simulations.

15. Design of high affinity antibody against cancer
    

    The aim of this study is to develop antibody-drug against cancer which is highly effective and has no side effect by using computer simulation. We observe hydrogen bond, hydrophobic bond, binding energy and structural change of antibody before and after binding antigen, and then predict the amino acid sequence which enhances the antibody's affinity. Our goal is to establish the system for quick selection of antibody which has optimal amino acid sequence against antigen.

Top Page