My group is currently working in five different areas of biomedical research: (1) structural immunology of Class I MHC presented breast tumor antigens; (2) structure-based design of a MUC1 glycopeptide breast cancer vaccine; (3) structural and kinetic studies probing the molecular mechanisms of cytochrome P450 allostery; (4) structural studies probing the molecular mechanisms of catalysis for the prokaryotic arylamine N-acetyl transferase enzymes; and (5) structure-based design of an oral antiplatelet agent. All of these projects utilize 'state-of-the-art' multidimensional heteronuclear NMR techniques.
Structural Immunology of Class I MHC Presented Breast Tumor Antigens
MHC Class I-presented tumor associated antigens (TAAs) derived from MAGE, HER-2/neu, MUC1, and p53 expressing breast tumors provide potential targets for CTL-based immunotherapy of breast cancers. The goal of the first project, 'structural immunology of Class I MHC presented breast tumor antigens' is to study the interaction of these low affinity breast TAAs bound within the groove of Class I MHC using an NMR-based methodology. The approach involves the assembly of unlabeled Class I MHC with isotopically-labeled breast TAAs, followed by the implementation of isotope-edited NMR experiments to probe the structure and dynamics of each breast TAA in the Class I cleft. Once the unique structural and dynamical features that account for the low affinity of each TAA has been identified, modification of these features to increase TAA-MHC complex stability will be undertaken, with the aim of increasing the strength of the associated CTL response against the parent tumor.
Structure-Based Design of a MUC1 Glycopeptide Breast Cancer Vaccine
Mucin 1 (MUC1), a glycopeptide TAA, provides an excellent target for the design of a breast cancer vaccine due to its overexpression in 90% of breast cancers. The goal of the second project, 'structure-based design of a MUC1 glycopeptide breast cancer vaccine', is to use a detailed molecular picture of the humoral and cellular immune recognition of MUC1 antigen to guide the redesign and optimization of a weakly immunogenic MUC1 vaccine candidate into a potent second-generation MUC1 glycopeptide vaccine useful in the treatment of MUC1 expressing breast tumors. The approach involves production of isotopically labeled MUC1 peptides and glycopeptides, followed by the implementation of isotope-edited NMR experiments to probe the structure and dynamics of these labeled peptides and glycopeptides in complex with B- and T-cell associated receptors and proteins (antibody and MHC).
Structural and Kinetic Studies Probing the Molecular Mechanisms of Cytochrome P450 Allostery
Quantitative prediction of CYP-dependent metabolism-based drug-drug interactions remains inadequate, even with isolated recombinant CYPs in vitro. In part, this results from poorly understood allosteric behavior, where the term 'allosteric' is used to refer to cases where non-hyperbolic [substrate] vs. velocity curves are obtained, and when kinetic or equilibrium parameters are altered by addition of a second ligand. It is critically important to establish useful structural models for conceptualizing these processes, and to identify parameters that will aid in quantitative prediction of allosteric drug interactions. Thus, the goal of the third project, "structural and kinetic studies probing the molecular mechanisms of Cytochrome P450 allostery', is to understand at the molecular level allosteric mechanisms of Cytochrome P450 (CYP) catalysis. The approach chosen for the study of P450 allostery takes full advantage of the panoply of modern homonuclear and isotope-edited NMR techniques (HSQC-edited SAR-by-NMR, diffusion-edited NMR, and NOE-pumping, etc.) developed for the purposes of drug discovery and screening within the biotechnology and pharmaceutical industries.
Structural Studies Probing the Molecular Mechanisms of Catalysis for the Prokaryotic Arylamine N-Acetyl Transferase Enzymes
Arylamine N-acetyltransferases (NATs) are polymorphic drug metabolizing enzymes found in eukaryotes and prokaryotes that transfer an acetyl group from the cofactor acetyl-CoA to the primary amine of arylamines and hydrazines. The role of the NATs in parokaryotes is of particular interest, since NAT has recently been found to be expressed in M. tuberculosis, and appears to have a role in isoniazid resistance. Knowledge of the molecular mechanism of the prokaryotic NAT enzymes, and of M. tuberculosis NAT in particular, could allow for the design of mechanism-based inhibitors that prevent the acetylation of front-line anti-tubercular agents such as isoniazid. Thus, the goal of the fourth project, "structural studies probing the molecular mechanisms of catalysis for the prokaryotic arylamine N-acetyl transferase enzymes ', is to use modern multidimensional NMR methods to investigate the solution structures, backbone dynamics, and catalytic mechanisms of various prokaryotic NAT enzymes (S. typhimurium, M. smegmatis and M. tuberculosis) both in their free and substrate bound forms. These studies will be taken in collaboration with Dr. Edith Sim, Department of Pharmacology, University of Oxford, who has recently solved the x-ray crystal structures of both the S. typhimurium and M. smegmatis NAT enzymes.
Structure-Based Design of an Oral Antiplatelet Agent
The adhesion of platelets to the wall of an injured artery is a fundamental initating step in arterial thrombosis that leads to heart attack and stroke. Key in this process is the binding of the platelets to activated receptor GP1b. The development of inhibitors that could block this interaction would therefore represent a significant advancement in cardiovascular medicine. Thus, the development of an oral peptide-based anti-platelet agent useful in the prevention of cardiac thromboses represents the goal of the fifth project, 'structure-based design of an oral antiplatelet agent'. As collaborators with Dr. Gerald J. Roth, M.D., VA Medical Hospital in Seattle WA, we will make use of available NMR spectroscopic techniques to assist in the screening of a phage display peptide library raised against the inactivated resting state of the GP1b receptor. Once several best peptide candidates have been identified, we will then undertake more detailed homonuclear and/or heteronuclear NMR studies to probe structural aspects of the interactions of these peptides with the resting state Gp1b receptor. These structural studies should be able provide two types of information: the conformation of the receptor-bound peptide, and a map of the contact interface between peptide and receptor. This information may ultimately prove useful in the structure-based design of an oral antiplatelet agent that specifically targets and stabilizes the resting state of the Gp1b receptor.