Turning genes on and off using FKBP and FK-506

Natural products are utilized with increasing frequency as probes of signal transduction pathways. FK506, rapamycin, and cyclosporin A are microbial metabolites that block signaling systems in many cell types including T lymphocytes. This dissertation describes the synthesis of several nonnatural products that, like their naturally-occurring counterparts, are used to elucidate mechanisms of intracellular signal transmission.;Chapter One recounts the design and synthesis of a nonnatural product termed 506BD (for FKBP Binding Domain of FK506) that contains the common structural elements of FK506 and rapamycin. 506BD was synthesized and shown to be a high affinity ligand for the immunophilin FKBP, effectively inhibiting its enzymatic activity (K;Based on our improved understanding of signal transduction in the T cell we devised a system whereby a synthetic, lipophilic molecule is able to control the expression of a specific gene in T cells. These investigations are fully detailed in Chapter Two. A chimeric receptor comprised of the zeta chain of the T cell receptor fused to FKBP was constructed and transfected into T cells. A second construct was also transfected containing the cDNA for a reporter protein under the control of three NF-AT promoters. Administration of a symmetrical dimer of FK506 (known as FK1012) resulted in aggregation of the chimeric receptor and transcription of genes under the control of NF-AT promoters. In addition to its potential use as a general method for the study of signaling pathways, this work provides a blueprint for future therapeutic uses, especially in the field of gene therapy where the inability to control protein expression is currently a major limitation.