Gregory J. Wiederrecht

Mechanism of action of rapamycin: New insights into the regulation of G1-phase progression in eukaryotic cells

The immunosuppressant drug, rapamycin (RAP), is a potent inhibitor of IL-2-dependent T-cell proliferation. The antiproliferative effect of RAP is mediated through the formation of an active complex with its cytosolic receptor protein, FKBP12. The molecular target of the FKBP12.RAP complex is a putative lipid kinase termed the mammalian Target Of Rapamycin (mTOR). This review will discuss recent findings suggesting that mTOR is a novel regulator of G1- to S-phase progression in eukaryotic cells.

Isolation of a cDNA encoding a novel human FK506-binding protein homolog containing leucine zipper and tetratricopeptide repeat motifs

Reduced-stringency PCR was used to isolate a cDNA encoding a novel human FK506-binding protein (FKBP) homolog. The encoded 38-kDa protein (FKBPr38) contains at its N-terminus a domain that is 33% identical to FKBP12. FKBPr38 is a member of a subclass of immunophilins, whose other members include FKBP52 and CyP40 (cyclophilin 40), that contain a three-unit tetratricopeptide repeat (TPR). In addition, FKBPr38 contains a consensus leucine-zipper repeat. The presence of the TPR domain and leucine zipper suggest that FKBPr38 may form homo-multimers or interact with other, as yet unidentified, proteins.

C32-O-imidazol-2-yl-methyl ether derivatives of the immunosuppressant ascomycin with improved therapeutic potential

A series of C32-O-aralkyl ether derivatives of the FK-506 related macrolide ascomycin have been prepared based on an earlier reported C32-O-cinnamyl ether design. In the present study, the nature of the aryl tethering group was varied in an attempt to improve oral activity. An imidazol-2-yl-methyl tether was found to be superior among those investigated and has resulted in an ascomycin analog, L-733,725, with in vivo immunosuppressive activity comparable to FK-506 but with an improved therapeutic index.

FKBP, the binding protein for the immunosuppressive drug, FK-506, is not an inhibitor of protein kinase C activity

Recently, the amino acid sequence of a 12 Kd endogenous protein inhibitor of protein kinase C (PKC-I 2) has been shown to be identical to that of the 12 KDa receptor for the immunosuppressive drug, FK-506. In view of this observation we examined the effects of recombinant and native human FKBP on protein kinase C (PKC) activity. FKBP, at molar concentrations up to 1900-fold over that of PKC, failed to inhibit PKC phosphorylation of histone H1 and failed to block the auto-phosphorylation of PKC. Interestingly, FKBP is phosphorylated by PKC in these reactions. The phosphorylation of FKBP by PKC appears to be specific since the catalytic subunit of cAMP-dependent protein kinase fails to phosphorylate the binding protein. Our results fail to support a role for FKBP as an inhibitor of protein kinase C.

Potent immunosuppressive C32-O-arylethyl ether derivatives of ascomycin with reduced toxicity.

The synthesis of C32-O-arylethyl ether derivatives of ascomycin that possess equivalent immunosuppressant activity but reduced toxicity, compared to FK-506, is described.

C32-O-phenalkyl ether derivatives of the immunosuppressant ascomycin: a tether length study

A tether length study of C32-O-phenalkyl ether derivatives of ascomycin was conducted wherein it was determined that a 2-carbon tether provides optimum in vitro immunosuppressive activity. Oxygen-bearing substituents along the 2-carbon tether can further increase the potency of this design.

YEAST IMMUNOPHILINS : PURIFICATION AND ASSAY OF YEAST FKBP12

Publisher Summary The chapter describes the purification and functional assay of yFKBP12 from Saccharomyces cerevisiae (S. cerevisiae) and the expression and purification of recombinant yFKBP12. FKBP12 can be assayed in three ways: by peptidylprolyl isomerase (PPIase) activity, by direct binding of the immunosuppressive ligand FK506, and by inhibition of calcineurin phosphatase activity by FK506 yFKBPI2 complexes. The chapter describes purification of FKBP 12 from S. cerevisiae, expression and purification of recombinant yeast FKBP12, peptidylprolyl isomerase activity of yeast FKBP12 and FKBP13, and calcineurin phosphatase inhibition by yFKBP 12 drug complex. The immunophilins constitute a class of ubiquitously expressed proteins named for their ability to bind the immunosuppressive drugs cyclosporin A (CsA), FK506 (Prograf), and rapamycin (RAPA). The genes for five CsA-binding proteins and three FK506/RAPA-binding proteins have so far been characterized in S. cerevisiae . The protein products of these genes possess properties essentially identical to those of the mammalian equivalents and bind either CsA or FK506/RAPA with high affinity and possess peptidylprolyl cis-trans -isomerase (PPIase, EC 5.2.1.8) activity.