Prolyl isomerases cyclophilins

The preparation of the fluoroolefin amide isosteres is reviewed. The incorporation of the amide isosteres in peptidomimetics and the influence of that isosteric substitution on biological activity on inhibition of peptidyl prolyl isomerases cyclophilin (CyP) and Pini, dipeptidyl peptidase IV/CD26 (DPP IV) and thermolysin is described. In addition, select fiuoroolefination procedures which may have utility in the construction of fluoroolefin amide isosteres are illustrated. 

Disulfide bond formation within the individual propeptides precedes folding and trimers are then formed by association of the C-terminal propeptides." Disulfide bonds between the chains are then formed and this formation is most likely catalyzed by PDI." As triple helix formation proceeds, the rate-limiting step in this process is the cis—trans isomerization of peptidyl-Pro bonds. This process can be catalyzed by peptidyl-prolyl cis—trans isomerases (cyclophilins and FKBPs). This activity is required to convert the proline residues to the trans form required for triple helix formation." " " ... 

Siekierka, JJ., Hung, SH., Poe, M., Lin, CS., and Sigal, NH. (1989). A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct ftom cyclophilin. Nature 341, 755-757. 

Tacrolimus (FK 506) is an immunosuppressant macrolide antibiotic produced by Streptomyces tsukubaensis. It is not chemically related to cyclosporine, but their mechanisms of action are similar. Both drugs bind to cytoplasmic peptidyl-prolyl isomerases that are abundant in all tissues. While cyclosporine binds to cyclophilin, tacrolimus binds to the immunophilin FK-binding protein (FKBP). Both complexes inhibit calcineurin, which is necessary for the activation of the T-cell-specific transcription factor NF-AT. 

We will use the term PPI when the enzymatic activity as prolyl isomerase is concerned, and the terms cyclophilin and FKBP to discriminate the two known families of prolyl isomerases that are inhibited by CsA and by FK506, respectively. Cyclophilin and FKBP differ strongly in sequence specificity. The PPI activity of cyclophilin shows only a small dependence on the chemical nature of the amino acid Xaa that precedes proline in the assay peptide. The activity of FKBP, however, varies by three orders of magnitude when the same set of peptides is used in the PPI assays (Harrison and Stein, 1990 see Stein, this volume). 

The amino acid sequences of the cyclophilins remained highly conserved during evolution. This holds in particular for the proteins from eukaryotes. The cyclophilins from bovine thymus and from porcine kidney are identical in sequence (Takahashi et al., 1989), and the human and the bovine cyclophilins share 98% identical amino acids (Haendler et al., 1987). The homology between the mammalian cyclophilins and the cytosolic PPl from E. coli is about 25% (Hayano et al., 1991). The PPIs from porcine kidney and E. coli cytoplasm were used in most of the work on the function of prolyl isomerases as catalysts of protein folding that will be discussed herein. 

Proteins homologous to the cytosolic cyclophilins from vertebrates or the corresponding genes are found virtually in every organism and in every subcellular compartment. All members of this family that could be obtained in purified form had a high prolyl isomerase activity and were inhibited by CsA, albeit with different effectiveness. Notably, the affinity... 

Prolyl isomerases of the cyclophilin type show some properties that would be expected for a catalyst of cellular protein folding. Cyclophilins occur in all cellular compartments where folding reactions occur. The activity toward accessible prolyl bonds is high, and the specificity with regard to the chemical nature of residue Xaa is low. Additional experiments are clearly needed, however, to clarify the possible role of prolyl isomerases for the in vivo folding process of nascent proteins. 

The finding that both classes of prolyl isomerases, the cyclophilins and the FK506-binding proteins, are strongly inhibited by immunosuppres-... 

Note Added in Proof. The structure of free prolyl isomerases of the cyclophilin type was rehned at 1.63 A (Ke, 1992). Further work on the complex of human cyclophilin with a tetrapeptide showed that the peptide N-acetyl-Ala-Ala-Pro-Ala-amidomethylcoumarin... 

The search for a common mechanism of action cf the immunosuppressive drugs cyclosporin (CsA) (7)and FK-506 (8)highlights another possibility in which the drug was I discovaed by screening in cellular or in vivo models but the exact mechanism or site of I action is unknown (157). Using the active mol-I ecules, cyclophilin (CyP) and the FK binding protein (FKBP)were identified as the specific receptors for cyclosporin and FK-506, respec-I lively. These binding proteins were discovered to be distinct and inhibitor-specific cis-trans peptidyl-prolyl isomerases that catalyze the... 

Prolyl isomerases belong to three structurally diverse families the cyclophilins, the FK506 binding proteins, and the parvulins. Usually, they are abbreviated to Cyp, FKBP and Parv, followed by the molecular mass and, sometimes, an indicator for the subcellular localization. Cypl8cy thus denotes the cytoplasmic cyclophilin with a molecular mass of 18 kDa. This cyclophilin is also often referred to as CypA. 

In 1989 the original prolyl isomerase was found to be identical with cyclophilin (Cyp), a high-affinity receptor for the immunosuppressive drug cyclosporin A (CsA) (Handschumacher et al., 1984 Fischer et al., 1989 Takahashi et al., 1989). Cyclophilins are ubiquitous proteins that are present in all organisms and all subcellular compartments. In yeast, there are seven different forms of cyclophilin, which are found in the cytosol, the mitochondria, the endoplasmic reticulum, and the nucleus. Yeast cells with disruptions in all these cyclophilins are viable, but show several growth defects (Dolinski et al., 1997). In the Drosophila melanogaster... 

The trigger factor of E. coli also contains a FKBP domain. This prolyl isomerase locates to the ribosome of bacteria and is an excellent catalyst of protein folding. It is described in Section IX. Additional proteins with cyclophilin- or FKBP-related domains have been described (Anderson etal., 1993 Fischer, 1994 Galatand Metcalfe, 1995 Heitman, 1997 Galat and Riviere, 1998). 

The prolyl isomerases catalyze isomerizations only at prolyl bonds and not at nonprolyl peptide bonds. The refolding of the P39A variant of RNase Tl, which is limited in rate by the very slow trans —> cis reisomerization of the Tyr38-Ala39 bond (see Section IV.B), is not catalyzed by cyclophilins, FKBPs, or parvulins. These enzymes are also unable to catalyze amide bond isomerizations in the proline-free model peptide Ala-Ala-Tyr-Ala-Ala (Scholz etal., 1998b). 

In contrast, the refolding of E. coli cyclophilin CypA is not autocatalytic. This protein refolds in two reactions. The molecules with correct prolines refold very rapidly (with a time constant of about 1.5 ms), and the molecules with incorrect prolines refold slowly (with a time constant of 25 s) in a proline-limited reaction. This reaction, however, cannot be accelerated in an autocatalytic fashion by increasing CypA concentration. It could also not be catalyzed by adding native CypA or other prolyl isomerases (Ikura et al., 2000), probably because the prolines of CypA itself are inaccessible for catalysis during folding. 

Bacillus subtilis contains only two prolyl isomerases—PPIB, which is a cytosolic cyclophilin, and trigger factor. Disruption of the corresponding genes individually or in combination had no effect on viability of... 

The periplasm of E. coli contains prolyl isomerases of all three families. PPIA is a member of the cyclophilin family (Liu and Walsh, 1990 Hayano et al., 1991), FkpA or FKBP26 (Horne and Young, 1995) contains a FKBP12-like domain, and SurA (Rouviere and Gross, 1996) and PpiD (Dartigalongue and Raina, 1998) show parvulin domains. SurA is a soluble protein, whereas PpiD is attached to the inner membrane with its catalytic domain exposed to the periplasmic space. 

---