Our collaborators and we have successfully cloned and overexpressed human erythrocyte pyruvate kinase (R-PK). This reliable source of enzyme has allowed for the biological testing of new classes of molecules, and has subsequently enabled us to discover novel R-PK inhibitors that bind at micro molar (uM) concentrations. In conjunction with our collaborators, we have also successfully solved the X-ray crystal structure of human R-PK at a resolution of 2.7 A. This crystallization technique for the native structure will permit us to develop methods for co-crystallizing the newly discovered effectors with this enzyme, and thus facilitating structure-function studies (see preliminary results). Our approach to the generation of allosteric effectors of R-PK will integrate: Protein cloning and over-expression, X-ray crystallography, molecular modeling, organic synthesis, and biological studies.Therefore our specific aims include: (1) Comparison of human PK isozymes: cloning and structure determination: Ml-, M2-, and L-PK isozymes will be cloned for crystallographic structure solution and for comparison of allosteric binding sites between isozymes; (2) Structural determination of effector binding sites: we will co-crystallize newly identified inhibitors with R-PK to determine their binding sites and amino acid residue interactions. Refined Effector-Pyruvate Kinase (PK) crystal structures should also provide some information concerning which additional amino acids may be involved in regulating the allosteric transition of R-PK between the Tand R- States; (3) Structure based design of R-PK inhibitors: Initial studies will involve working with recently discovered lead compounds that are discussed in the proposal. Molecular modeling studies will employ fundamental drug design principles SAR, SAR, 3-D database searching, CoMFA, and HINT] to suggest modifications for improved binding of these compounds, as well as in the generation of novel allosteric effectors for synthesis. Development of new molecules will be directed by information from specific aims 1,2, and 4. An iterative process to improve compound binding and to increase potency will be developed as results are obtained and (4) Allosteric modulation of R-PK: Examination of R-PK- Effector kinetics to characterize the degree of allosteric function and effector/protein Structure activity relationships that arise. In summary, the goal of this proposal is to discover and employ new R-PK allosteric effectors that will also aid in unraveling the allosteric switch mechanism of this enzyme, and further the discovery of selective effector(s), which may translate into treatments for hypoxic diseases, including Alzheimer's disease. Another important aspect of this research may evolve from results obtained from specificity studies: the M2-PK isozyme is re-expressed in solid tumors, and any tested compounds that show specificity for this isozyme might lead to a new class of compounds for the treatment of cancer.
