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Major histocompatibility complex (MHC) is a transmembrane glycoprotein that plays an important role in immunological system. Human MHC molecule is usually called as human leukocyte antigen (HLA) and HLA molecules are grouped into class I and II. MHC class I molecule is a heterodimer of heavy chain called a chain, whose mass-weight is 45 kDa, and light chain called b2 micro globulin (b2m) with a mass-weight of 12 kDa. A complex of an HLA and a peptide derived from antigen is displayed on the surface of nucleated cell or platelet. HLA heavy chain has a quite large diversity and thousands of genes have been detected so far, and the number of known genes for HLA heavy chain is still increasing. Each individual has two or three kinds of HLA out of several thousands of HLA genes.Viral protein or cancer-related antigens are detected as foreign molecules in a cell and dissociated by proteasome into peptide fragments consisting of 6-15 amino residues.1) This peptide fragment is bound to HLA class I molecule and the complex is carried to the cell surface in antigen display cells. Cytotoxic T lymphocytes (CTL) expressing T cell receptor recognize the complex, which results in inducing the immunological response to attack the virus-infected or oncogenic cells to exclude virus or tumor. 2)Immunological response has attracted much recent attention because of its potential for immunotherapy. At present, several peptides are assumed to be effective for medical treatment of cancer, leukemia, hepatitis C, and applied to patients in a clinical trial as vaccine through the controlled vaccination.3-5) This peptide-vaccine therapy is expected to lead the regression of disease-related cells without damaging normal tissues. Since the combination of HLA genes has a large diversity, the selection of peptide adequate for each individual is one of the important issues to enhance the performance of immunotherapy.The discovery of a peptide inducing strong immunological response is a key factor in immunotherapy. Since the peptide firmly bound to HLA molecule will be effective as medical material, the search of potent peptide, usually called as epitope, is critically important. In this study, we focus on Wilms' tumor (WT1) protein.6) WT1 protein is encoded in b2 Wilms' tumor gene and overexpressed in leukemic and solid tumor cell. A 9-mer amino acid peptide encoded in WT1 protein was already known to work as an antigenic peptide for HLA-A*2402 molecule. Oka and his co-workers reported the experimental findings that the replacement of the second amino acid residue, which is considered to be deeply responsible for the peptide binding to HLA-A*2402, induced strong immunological response of WT1 specific CTLs compared to the natural peptide. A peptide containing a single amino residue mutation is currently applies for the clinical trial for a vaccination against solid tumor or leukemia. 7)Computer simulation enables us to visualize the interaction of proteins or the interaction of a chemical compound and its target protein. Molecular dynamics (MD) ...

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 μM and 250 μM, dose-response assays for determining IC 50 values, detergent counter screen assays, jumpdilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1 H NMR is shown for the purine-specific enzyme, while 19 F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its K m value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

Trichomoniasis is the most common nonviral sexually transmitted infection, affecting an estimated 275 million people worldwide. The causative agent is the parasitic protozoan Trichomonas vaginalis. Although the disease itself is typically mild, individuals with trichomonal infections have a higher susceptibility to more serious conditions. The emergence of parasite strains resistant to current therapies necessitates the need for novel treatment strategies. Since T. vaginalis is an obligate parasite that requires nucleoside salvage pathways, essential nucleoside ribohydrolase enzymes are promising new drug targets. Fragment screening and X-ray crystallography have enabled structure-guided design of inhibitors for two of these enyzmes. Linkage of enzymatic and antiprotozoal activity would be a transformative step toward designing novel, mechanism-based therapeutic agents. While a correlation with inhibition of purified enzyme would be mechanistically suggestive, a correlation with inhibition of in-cell enzyme activity would definitively establish this linkage. To demonstrate this linkage, we have translated our NMR-based activity assays that measure the activity of purified enzymes for use in T. vaginalis cells. The 19F NMR-based activity assay for the pyrimidine-specific enzyme translated directly to in-cell assays. However, the 1H NMR-based activity assay for the purine-specific enzyme required a switch from adenosine to guanosine substrate and the use of 13C-editing to resolve the substrate 1H signals from cell and growth media background signals. The in-cell NMR assays are robust and have been demonstrated to provide inhibition data on test compounds. The results described here represent the first direct measurement of enzyme activity in protozoan parasite cells.

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