EPITOPE SCANNING USING PROTEIN SEMISYNTHESIS

DESCRIPTION: (Adapted from applicant's abstract) Recent advances in the chemoselective ligation of peptides has greatly increased the range of useful proteins that can be produced in this manner. The adaptation of this procedure to facilitate the internal insertion of designer synthetic peptides between recombinantly derived protein fragments will be studied using a particular carrier protein. This protein spontaneously assembles into ordered, soluble, 27 nm particles containing 180 subunits. This structure has been demonstrated to be a superior carrier for exogenous, pathogen derived epitopes. The chemoselective insertion of peptide cassettes between recombinant N- and C-terminal fragments of the particulate carrier will be investigated to determine the feasibility of this directional, three-piece ligation procedure for generating novel fusion proteins harboring synthetic sequences. The physical, chemical and immunological integrity of the semisynthetic particles will be examined. A critical comparison of these data with those acquired for the entirely recombinant equivalents will enable an assessment of the utility of this approach. This work is expected to yield a versatile method for the introduction of B- and T-cell sites as well as novel amino acids or other chemical entities into a proven carrier protein that dramatically enhances immune responses to inserted sequences and conjugated haptens. Rapid production of analytical quantities of hybrid proteins will enable epitope scanning to identify useful antibody reactivities. $ = TOTAL AWARD AMTS & NOT LIMITED TO PORTION OF PROJECT RELATED TO SUBJECT OF SEARCH SUBPROJECT $ = TOTAL AWARD AMOUNT DIVIDED BY NUMBER OF SUBPROJECTS SOURCE: CRISP FORMAT F FY 97 LAST UPDATE 04-07-98 1QUERY 1536 ID SEARCH 06/01/98 PAGE 321 --PROJECT NUMBER......1 R43 GM54503-01A2 INVESTIGATOR NAME/ADDRESS FY 97 PANKIEWICZ, KRYSZTOF W IRG/INTRAMURAL UNIT..ZRG3 CODON PHARMACEUTICALS, INC AWARD AMOUNT......... $95,000 200 PERRY PARKWAY GAITHERSBURG, MD 20877 PERFORMING ORGANIZATION: CODON, INC. TITLE INHIBITORS OF INOSINE MONOPHOSPHATE DEHYDROGENASE ABSTRACT: The long term objective of this research is to develop potent and selective inhibitors of Inosine Monophosphate Dehydrogenase for eventual clinical application. These targeted compounds are designed to be metabolically stable NAD analogues which should not affect other cellular dehydrogenases and should penetrate cell membranes. The specific affinity towards IMPDH (beta-specific) will be achieved by replacement of the nicotinamide riboside portion of NAD with conformationally restricted benzamide ribosides which are fixed in the "syn" range required for tight binding to IMPDH. These compounds will be also improved by introduction of fluorine at 2'-position of the adenosine moiety. Fluorination at the 2' position prevents the further conversion of NAD analogues to NADP like compounds. This is important for two reasons 1) The concentration of NAD analogue will not be depleted by conversion to a different analogue. 2) The inhibition of NADP requiring enzymes may prove deleterious to cells. The cells membrane penetration will be address by synthesis of their methylene mono- and di-thiophosphonates. Since oligo phosphorothioates are known to penetrate cell membranes, the cellular up-take of these cofactor type thiophosphonates should be dramatically improved. Methylene diphosphonate analogue of NAD, called beta-methylene TAD, has already been demonstrated to enter cells. Acute and chronic myelogenous leukemias are our particular targets for these compounds which are expected to inhibit cancerous cell growth as well as to induce cell differentiation into mature cells.