Parking misc FK506 stuff that I want to
decode and try and understand a little later...
ncbi.nlm.nih.gov.
Curr Opin Biotechnol 1998 Oct;9(5):451-6
Recent advances in inducible gene expression systems.
Rossi FM, Blau HM
Department of Molecular Pharmacology, Stanford University School of Medicine, CA, USA. frossi@leland.stanford.edu
A means of controlling the level and timing of expression of specific genes in cultured cells or in animals would have broad applications. There has been recent progress in two very promising systems: problems due to the high background expression from tetracycline-responsive promoters have been solved by constructing tetracycline-sensitive transcriptional repressors; and new rapamycin analogues have been isolated that are capable of activating the FK506-inducible system but lack the cytostatic side effects of the original inducers.
Both systems now provide opportunities for expressing toxic genes, growth arrest genes, and therapeutic products in a regulated fashion previously not possible.
Publication Types:
Review
Review, tutorial
PMID: 9821271, UI: 99038712
enc-conference.org
Institute: ARIAD Pharmaceuticals, Inc. Cambridge, MA USA
Keywords:
Abstract:
A variety of high-affinity ligands are known for FKBP (FK506 binding protein). These ligands encompass natural products e.g. rapamycin and FK506 as well as lower molecular weight synthetic molecules. This high-affinity interaction has been used as the basis of a series of chemical inducers of dimerization which have found broad utility in biological and medical applications utilizing controlled protein-dimerization events, specifically in the area of regulated gene therapy. Using the three-dimensional structures of FKBP:ligand complexes, the ligand-protein interface has been redesigned to enhance selectivity over native complexes by incorporating FKBP "holes" and complementary ligand "bumps". One synthetic ligand
binds a F36V mutant of FKBP with an affinity of ~100pM. The X-ray structure of this complex shows a hydrophobic "hole" has opened at the site of the mutation which is partially occupied by the ethyl "bump" of the ligand (Proc. Natl. Acad. Sci. USA 95, 10437-10442, 1998). A series of ligand analogs has been prepared which display "bumps" of differing sizes and with differing stereochemistries in an effort to optimize the binding of the ligand to the "holed" protein. In the present study, NMR spectroscopy has been used to determine the solution structure of several of these analogs to correlate co-structural features with binding affinity. Interestingly, in several of these complexes we observe a switched binding mode where the "bump" no longer engages the engineered protein "hole", rather occupies a adjacent natural cavity on the surface of the protein, and the tetrahydropyrone (THP)-mimicking
group expected to occupy the cavity occupies the engineered "hole". As we show, the ligand binding mode is dictated by the stereochemistry of the "bump" substituent while relative
ligand affinities result from the combination of preferences for the "bump" and THP-mimic sidechains by both "hole" and cavity in either binding mode. The present study illustrates the
utility of NMR structure determination in studying a series of related compounds where differences in binding orientation make simple SAR analysis problematic.
This is an older one...for newbies
Stuart Schreiber Brings the Right Chemistry to Cell Biology
isinet.com
...
note to self...do query for inhibitors of calcineurin...
...
merck.com
Dr. Stephen A. Parent, Research Fellow, Department of Natural Products Discovery, P.O. Box 2000, Rahway,
NJ 07065
CONSERVED MOLECULAR INTERACTIONS MEDIATE THE BIOLOGICAL ACTIVITIES OF FK506 AND CsA IN T
CELLS AND IN YEAST
The immunosuppressive drugs FK506 and CsA bind conserved cytosolic receptors, FKBP12 and cyclophilin, respectively, and the drug-receptor complexes inhibit the Ca2+/calmodulin-dependent protein
phosphatase, calcineurin in T cells. Using genetic and biochemical approaches, we demonstrated that FK506-FKBP12 and CsA-cyclophilin complexes inhibit calcineurin in Saccharomyces cerevisiae. Two of the
genetic approaches involved isolating and characterizing mutants hypersensitive or resistant to FK506. Our research defines key physiological targets of FK506- and CsA-sensitive signaling pathways in yeast, reveals a high degree of mechanistic conservation with mammalian cells, and indicate that these model systems provide insights into the immunosuppressive and/or toxic properties of these drugs. |
